abstracts by category · impact of abo-mismatch on transfusion requirements in bone marrow...

Abstracts by CategoryCategory oral Presentation number * Poster Presentation number

regenerative medicine 1-4 159-175, 194-195

immunotherapy and Dendritic Cells 5-8 29-50, 176-179

legal & regulatory affairs 9, 11 56-59, 182

laboratory Practices 10, 12 51-55, 180-181, 183

mesenchymal stem Cells 13-17 60-97, 184-188

Hematopoietic stem Cells 18-22 118-158, 192-193

Cell and gene therapy or Cellular gene transfer 23-25 106-117, 191

translational Process Development 26-28 98-105, 189-190 *Please refer to the Educational Program for the full schedule of Oral Abstract Presentations

Poster Session 1 Posters 29-106, 176-190

MOndAy MAy 24th5:15pm – 6:15pmFreedom & Independence Ballroom

Poster Session 2 Posters 107-175, 191-195

tuESdAy MAy 25th5:15pm – 6:15pmFreedom & Independence Ballroom

16thISCTAnnual Meeting

may 23-26, 2010

isCt | www.celltherapysociety.org | t: 604.874.4366 | F: 604.874.43784

Author Last name Author First name Abstract title Abstract

name Oral/Poster Session

Akimova tatiana DEMETHYLATION OF THE FOXP3 LOCUS IS KEY TO PREDICTING AND MODULATING SUPPRESSIVE FUNCTION OF HUMAN TREGS 29 Poster Session 1

Al-Qirim tariq PHARMACOLOGICAL EVALUATION OF NOVEL INDOLE-2-CARBOXAMIDES AS POTENT LIPID-LOWERING AGENTS IN TRITON-WR1339-INDUCED HYPERLIPIDEMIC RATS 60 Poster Session 1

Amarnath Shoba ENGINEERED HUMAN REGULATORY T CELLS EXPRESSING LENTIVIRAL PDL1 UNDER CELL FATE CONTROL PREVENT LETHAL XENOGENEIC GVHD 30 Poster Session 1

Andriolo Gabriella VALIDATION OF GOOD MANUFACTURING PRACTICE (GMP) COMPLIANT QUALITY CONTROLS OF CD133+ CELLS FOR THE TREATMENT OF END STAGE LIVER DISEASE. 189 Poster Session 1

Annerén Cecilia PERFUSION CULTURE OF T LYMPHOCYTES IN THE WAVE BIOREACTOR™ SYSTEM 2/10 98 Poster Session 1

Antony Paul NAIVE TUMOR-SPECIFIC CD4+ T CELLS DIFFERENTIATED IN VIVO ERADICATE ESTABLISHED MELANOMA IN LYMPHOPENIC HOSTS 176 Poster Session 1

Aqui nicoleEARLY ADOPTIVE TRANSFERS OF ANTI-CD3/ANTI-CD28 COSTIMULATED AUTOLOGOUS T CELLS AFTER AUTOSCT FOR MYELOMA: SIGNIFICANTLY ENHANCED POST-TRANSPLANT HUMORAL AND CELLULAR IMMUNE RECONSTITUTION

5Oral Abstracts Session 2 – Immunotherapy and dendritic Cells

Astori GiuseppeTHE SWISS MULTICENTER INTRACORONARY STEM CELLS STUDY IN ACUTE MYOCARDIAL INFARCTION (SWISS-AMI): A CELL-BASED CLINICAL PROTOCOL FOR PATIENTS WITH ACUTE MYOCARDIAL INFARCTION. STUDY DESIGN AND PRELIMINARY DATA

159 Poster Session 2

Bae Jae-sungINTRACEREBRAL TRANSPLANTATION OF BM-MSCS REDUCES AMYLOID-BETA DEPOSITION AND RESCUES MEMORY DEFICITS IN ALZHEIMER’S DISEASE MICE BY MODULATION OF IMMUNE RESPONSES

61 Poster Session 1

Baum Christopher APPROACHES TO AVOID ADVERSE EVENTS IN THE GENETIC MODIFICATION OF HEMATOPOIETIC CELLS 191 Poster Session 2

Beres Wendy FLOW CYTOMETRIC METHOD FOR DETECTION OF IN VIVO RED CELL BOUND IGG 51 Poster Session 1

Berneman Zwi INDUCTION OF COMPLETE AND MOLECULAR REMISSIONS IN ACUTE MYELOID LEUKEMIA BY WILMS’ TUMOR 1 ANTIGEN-TARGETED DENDRITIC CELL VACCINATION 6

Oral Abstracts Session 2 – Immunotherapy and dendritic Cells

Bersenev Alexey EFFICIENT GENERATION OF HUMAN NK CELLS FROM CORD BLOOD CD34+ HEMATOPOIETIC PROGENITORS 31 Poster Session 1

Blaydes Ingersoll Susan DIFFERENTIAL CYTOTOXIC RESPONSE ELICITED BY UCB AND PBMC AGAINST OVARIAN CANCER CELLS 32 Poster Session 1

Boucher Shayne STEMPRO® LIPOMAX™, A DEFINED XENOFREE LIPID-RICH SUPPLEMENT FOR PRIMARY AND STEM CELL RESEARCH AND THERAPY APPLICATIONS 62 Poster Session 1

Bowersock Joscelyn TEMOZOLAMIDE RESISTANT γγ T CELL-BASED IMMUNOTHERAPY FOR GLIOBLASTOMA MULTIFORME 106 Poster Session 1

n/A n/A ABSTRACT WITHDRAWN. 179 Poster Session 1

Bracke Madelon EXPANSION OF HUMAN MESENCHYMAL STEM CELLS USING A MICROCARRIERS-BASED CULTIVATION SYSTEM 99 Poster Session 1

Bullough Jeremy ESTABLISHING A CORD BLOOD SAMPLE REPOSITORY FOR A PERSONALIZED MEDICINE INITIATIVE 118 Poster Session 2

Carrasco-yalán Antonio PARAMETERS THAT IMPACT ON TNC AND CD34+ CELL RECOVERY FOR UMBILICAL CORD BLOOD BANKING 119 Poster Session 2

Chase Lucas EXPANSION OF HUMAN MESENCHYMAL STEM CELLS IN A NOVEL SERUM-FREE AND XENO-FREE CULTURE SYSTEM 63 Poster Session 1

Chelluri Lakshmi PRELIMINARY REPORT ON IMMUNOMODULATION OF MESENCHYMAL STEM CELLS IN PATIENTS WITH TUBERCULOSIS INFECTION 184 Poster Session 1

Chen Mai LACK OF BETA-ARRESTIN-1 IN BONE MARROW STEM CELLS LEADS TO IMPAIRED OUTCOME AFTER MYCARDIAL INJURY DUE TO DEFECTIVE PROLIFERATIVE ACTIVITY 18 Oral Abstracts Session 5 –

hematopoietic Stem Cells

Chung yun Shin ANTIGEN SPECIFIC HUMAN ANTIBODY PRODUCTION IN HUMANIZED NOD/SCID IL2R GAMMA C NULL (NSG) MOUSE MODEL, TRANSPLANTED HUMAN FETAL TISSUES WITH CD34+ CELLS 120 Poster Session 2

Ciavarella Sabino HUMAN UMBILICAL CORD PERIVASCULAR CELLS: ROLE OF SPECIFIC GENES IN THEIR DIFFERENTIATION TO OSTEOBLASTS 64 Poster Session 1

Clarke dominic MAXIMIZING STABILITY OF HUMAN STEM CELLS THROUGH OPTIMIZED BIOPRESERVATION SOLUTIONS 160 Poster Session 2

Coutu daniel IDENTIFICATION OF THE MESENCHYMAL STEM CELL BONE NICHE USING NOVEL CELL SURFACE MARKERS: CAN FGF RECEPTORS DO THE TRICK? 185 Poster Session 1

Poster & Oral Abstract Author Index


may 23-26, 2010

isCt | www.celltherapysociety.org | t: 604.874.4366 | F: 604.874.4378 5



danieli Patrizia SOLUBLE FACTORS RELEASED BY HUMAN MESENCHYMAL STEM CELLS OF FETAL ORIGIN LEAD TO CARDIOMYOCYTE PROTECTION THROUGH THE INHIBITION OF PRO-APOPTOTIC SIGNALING 13 Oral Abstracts Session 4 –

Mesenchymal Stem Cells

danieli Patrizia THE CARDIOPROTECTIVE PARACRINE EFFECTS EXERTED BY HUMAN MESENCHYMAL STEM CELLS ARE NEGATIVELY INFLUENCED BY DONOR AGE 14 Oral Abstracts Session 4 –


de Santis Gil IMPACT OF ABO-MISMATCH ON TRANSFUSION REQUIREMENTS IN BONE MARROW TRANPLANTATION 121 Poster Session 2

de Santis Gil EFFECT OF WASHING CELL SUSPENSIONS ON ADVERSE REACTIONS ASSOCIATED WITH INFUSION OF CRYOPRESERVED HEMATOPOIETIC STEM CELL 122 Poster Session 2

de Santis Gil EXPRESSION OF ADHESION MOLECULES ON CD34 CELLS FROM MOBILIZED PERIPHERAL BLOOD, UMBILICAL CORD BLOOD AND BONE MARROW 123 Poster Session 2

deans Robert TRANSCRIPTOME PROFILING AFTER TRANSPLANTATION OF HUMAN ADHERENT ADULT PROGENITOR CELLS INTO RAT MODELS OF ISCHEMIC CNS INJURY 65 Poster Session 1

denman CeceleROBUST AND SUSTAINED EX VIVO EXPANSION OF NK CELLS WITH ARTIFICIAL APCS BEARING MEMBRANE-BOUND IL-21 IS ASSOCIATED WITH A DISTINCT PHENOTYPE AND TRANSCRIPTIONAL PROFILE.

33 Poster Session 1

deutsch Varda FIBRONECTIN AND STRESS GROWTH FACTORS ENHANCE EX VIVO EXPANSION AND MATURATION OF MEGAKARYOCYTE AND HEMATOPOIETIC PROGENITORS FROM UMBILICAL CORD BLOOD 124 Poster Session 2

do Byung-RokA COMPARATIVE STUDY OF EFFECT OF THE OXYGEN MICROENVIRONMENT ON IN VITRO THE ANGIOGENESIS OF HUMAN ADIPOSE TISSUE- OR UMBILICAL CORD-DERIVED STROMAL PROGENITOR CELLS

66 Poster Session 1

do Byung-Rok ENHANCEMENT OF MYOGENIC DIFFERENTIATION OF HUMAN ADIPOSE TISSUE DERIVED STROMAL CELLS IN VITRO 161 Poster Session 2

do Byung-Rok PROLIFERATION AND CHARACTERIZATION OF HUMAN UMBILICAL CORD DERIVED STROMAL PROGENITOR CELLS UNDER CULTURE CONDITION 162 Poster Session 2

dodd Megan ENGINEERING OF hMSC WITH LENTIVIRUS EXPRESSING HUMAN FIX FOR THE TREATMENT OF HEMOPHILIA B 107 Poster Session 2

dornsife RonnaAN AUTOMATED UMBILICAL CORD BLOOD UNIT (CBU) WASH PROCEDURE USING THE SEPAX® CELL PROCESSING SYSTEM PROVIDES HIGH QUALITY POST-THAW YIELDS FROM CRYOPRESERVED CBU


Egeler Oliver AUTOMATION OF A RAPID SEVEN-DAY COLONY-FORMING CELL ASSAY FOR MEASURING HEMATOPOIETIC PROGENITORS IN CORD BLOOD USING METHOCULT® EXPRESS 126 Poster Session 2

Eshel Rinat THE POTENTIAL USE OF MESENCHYMAL PROGENITOR CELLS FROM ADIPOSE TISSUE FOR CLINCAL APPLICATIONS 67 Poster Session 1

Fellowes Vicki PRE-CLINICAL DEVELOPMENT OF ALLOGENEIC TUMOR DERIVED LYMPHOCYTE (TDL) IMMUNOTHERAPY PRODUCT 190 Poster Session 1

Figliola Matthew CD19-SPECIFIC T CELLS SELECTIVELY EXPANDED ON ARTIFICIAL ANTIGEN PRESENTING CELLS ARE RESISTANT TO IMMUNOSUPPRESSIVE EFFECTS OF TACROLIMUS 108 Poster Session 2

Filby Caitlin STEM CELL LINES FROM CORD BLOOD, BONE MARROW AND PLACENTA SHOW DIFFERENCES IN GENE EXPRESSION AND FUNCTION 68 Poster Session 1

Filby Caitlin FORMATION OF ENDODERM AND LUNG EPITHELIAL CELLS FROM CORD BLOOD-DERIVED STEM CELLS 69 Poster Session 1

Foussat Arnaud REGULATORY TR1 CELL THERAPY OF CHRONIC INFLAMMATORY DISEASES 34 Poster Session 1

François MoïraA SINGLE INJECTION OF ALLOGENEIC MESENCHYMAL STROMAL CELLS IS SUFFICIENT TO RESCUE LETHALLY IRRADIATED MICE BY PROMOTING REGENERATION OF THE GASTROINTESTINAL EPITHELIUM AND RESTORING ENDOGENOUS HAEMATOPOIESIS.

70 Poster Session 1

Fu Cheng Cheng MESENCHYMAL STEM CELLS THERAPY FOR REFRACTORY EXTENSIVE CGVHD 71 Poster Session 1

Garlie nina ASSESSMENT OF TIL FOR RESIDUAL MELANOMA USING FLOW CYTOMETRY. 52 Poster Session 1

Geffner LuisTRANSPLANT OF AUTOLOGOUS BONE MARROW STEM CELLS TO PARKINSON’S DISEASE PATIENTS VIA MULTIPLE MINIMALLY INVASIVE ROUTES IS SAFE AND MAY IMPROVE THEIR QUALITY OF LIFE: PRELIMINARY RESULTS


Glaser Lawrence NOVEL USE OF MATURE ENUCLEATED RED BLOOD CELLS AS A VIRAL TRAP/SINK FOR HIV AND OTHER MAMMALIAN VIRUSES. 127 Poster Session 2

Godfrin yann L-ASPARAGINASE LOADED INSIDE RED BLOOD CELLS AS A NEW CELL BASED MEDICINAL PRODUCT 26

Oral Abstracts Session 7 – translational Process development





Godfrin yann IMMUNOTHERAPY USING RED BLOOD CELLS: ANTIGEN AND ADJUVANT DELIVERY SYSTEM 35 Poster Session 1

Grynspan Frida IMMUNOLOGICAL PROFILE OF CRITICAL LIMB ISCHEMIA PATIENTS FOLLOWING INTRAMUSCULAR ADMINISTRATION OF PLACENTA ADHERENT STROMAL CELLS 15 Oral Abstracts Session 4 –


Grynspan Frida THE OPTIMAL MESENCHYMAL CELL PRODUCT: IS BONE MARROW THE ANSWER? 72 Poster Session 1

Grynspan Frida IMMUNOMODULATION PROPERTIES OF PLACENTA DERIVED ADHERENT STROMAL CELLS. 73 Poster Session 1

Gul-uludag hilal HIGHLY EFFICIENT LABELLING OF HEMATOPOIETIC STEM/PROGENITOR CELLS USING MAGNETIC CARBON NANOTUBES: IMPLICATIONS FOR STEM CELL TRACKING 19 Oral Abstracts Session 5 –


Gul-uludag hilal ULTRASOUND TREATMENT ENHANCES PROLIFERATION OF HEMATOPOIETIC STEM/PROGENITOR CELLS: IMPLICATIONS FOR CLINICAL TRANSPLANTATION, GENE AND CELLULAR THERAPIES 128 Poster Session 2

Gunetti Monica BONE MARROW MESENCHYMAL STEM CELL THERAPY FOR URINARY INCONTINENCE: IN VITRO STUDIES 74 Poster Session 1

Gunetti Monica CELL THERAPY FOR AMI TREATMENT USING EX-VIVO EXPANDED BM CD34 POSITIVE CELLS 164 Poster Session 2

ha JueunCO-CULTURE OF HUMAN UMBILICAL CORD BLOOD DERIVED MESENCHYMAL STEM CELLS REDUCES ENDOTOXIN-INDUCED APOPTOSIS OF ALVEOLAR EPITHELIAL CELLS AND INFLAMMATORY REACTION OF ALVEOLAR MACROPHAGES IN VITRO

75 Poster Session 1

habibullah Chittoor SAFETY AND EFFICACY OF HUMAN FETAL LIVER DERIVED STEM CELL TRANSPLANTATION FOR THE MANAGEMENT OF DECOMPENSATED LIVER CIRRHOSIS- 2 YEAR FOLLOW UP STUDY 165 Poster Session 2

haley Rebecca CREATING A MULTI-USER CGMP CELLULAR PROCESSING FACILITY TO ENABLE TRANSLATIONAL RESEARCH IN THE ACADEMIC ENVIRONMENT 100 Poster Session 1

haller Jodi AMNIOTIC FLUID-DERIVED STEM CELLS GENETICALLY ENGINEERED FOR CELL-BASED THERAPY 109 Poster Session 2

heimbach Baron FUNCTIONAL CHARACTERISTICS OF HUMAN ADULT BONE MARROW-DERIVED SOMATIC CELLS: A UNIQUE POPULATION OF MESENCHYMAL SUPPORT CELLS 76 Poster Session 1

henderson Mary Beth REGULATORY CONSIDERATIONS FOR BIOLOGICS INCORPORATED INTO COMBINATION PRODUCTS 56 Poster Session 1

henon Philippe DIRECT INTRA-CARDIAC DELIVERY OF MOBILIZED BLOOD STEM CELLS IN PATIENTS WITH SEVERE MYOCARDIAL INFARCT: AN ALTERNATIVE FOR HEART TRANSPLANTATION? 129 Poster Session 2

hetland GeirA MEDICINAL MUSHROOM EXTRACT BASED ON AGARICUS BLAZEI MURILL INDUCES EXPRESSION OF CELL SURFACE MARKERS AND PRODUCTION OF CYTOKINES IN HUMAN MONOCYTE-DERIVED DENDRITIC CELLS (MDDC) IN VITRO

36 Poster Session 1

hildebrandt MartinAPHERESIS-RELATED ENRICHMENT OF CD26BRIGHT T LYMPHOCYTES IN HEMATOPOIETIC PROGENITOR CELL TRANSPLANTS IS PREDICTIVE FOR AN UNFAVOURABLE OUTCOME IN AUTOLOGOUS TRANSPLANTATION


Imam Asiya SAFETY BEGINS AT THE SOURCE- THE IMPORTANCE OF RAW MATERIAL SELECTION AND CONTROL 57 Poster Session 1

Irwin Stuart BIOBURDEN REDUCTION IN AUTOLOGOUS ISLET TRANSPLANTATION 53 Poster Session 1

Ishige Ikuo COMPARATIVE STUDY OF MESENCHYMAL STEM CELLS DERIVED FRON ARTERIAL, AND VENOUS, AND WHARTON’S JELLY EXPLANTS OF HUMAN UMBILICAL CORD 77 Poster Session 1

Jain deepak SMOOTH MUSCLE CELLS DERIVED FROM HUMAN ADIPOSE TISSUE FOR USE IN UROLOGIC TISSUE ENGINEERING 1 Oral Abstracts Session 1 –

Regenerative Medicine

Janssen William VIABILITY OF COLLECTED CD34+ CELLS FOLLOWING FREEZING AND THAWING DIMINISHES WITH INCREASING PERIOD OF G-CSF MOBILIZATION 20 Oral Abstracts Session 5 –


Janssen WilliamMINOR ABO MISMATCHED ALLOGRAFTS FROM DONORS WITH HIGH ISOHEMAGGLUTININ TITERS DO NOT PRODUCE MORE INFUSION REACTIONS OR LOSS OF RECIPIENT ERYTHROCYTE MASS THAN DO ABO MATCHED GRAFTS OR GRAFTS FROM DONORS WITH LOWER TITERS


Jin hee KyungBM-MSCS PREVENT THE LOSS OF NIEMANN-PICK TYPE C MOUSE PURKINJE NEURONS BY CORRECTING ABNORMAL SPHINGOLIPID METABOLISM AND INCREASING SPHINGOSINE-1-PHOSPHATE

78 Poster Session 1

Johnson Kevin REGENERATIVE MEDICINE CASE STUDY: MAKING THE TRANSITION FROM RESEARCH INSTITUTE TO TRANSLATIONAL RESEARCH AND DEVELOPMENT ORGANIZATION 9

Oral Abstracts Session 3 - Legal & Regulatory Affairs/Laboratory Practices

Keever-taylor Carolyn CHARACTERISTICS OF CD34-ENRICHED PRODUCTS PROCESSED AT MULTIPLE CENTERS USING THE CLINIMACS SYSTEM - BMT CTN 0303 21 Oral Abstracts Session 5 –


Kelley Rusty BIOACTIVE RENAL CELLS AUGMENT KIDNEY FUNCTION IN A RODENT MODEL OF CHRONIC KIDNEY DISEASE 2 Oral Abstracts Session 1 –


Kelley Linda REDUCTION OF INCOMING BIOBURDEN IN TISSUE PRODUCTS INTENDED FOR CELL ISOLATION AND CULTURE EXPANSIOIN 10




may 23-26, 2010




Kelley Linda DEVELOPMENT OF A XENO-FREE MICROCARRIER-BASED HUMAN MESENCHYMAL STEM CELL EXPANSION SYSTEM 101 Poster Session 1

Kelley Linda DEVELOPMENT AND IMPLEMENTATION OF A STRAIGHTFORWARD REAGENT AND VENDOR QUALIFICATION PROGRAM BASED ON IMPACT SCORE 102 Poster Session 1

Kelley Linda LABOR EFFICIENCY MANAGEMENT USING SIMULATION SOFTWARE LEADS TO SIGNIFICANT COST SAVINGS IN A HEMATOPOIETIC STEM CELL LABORATORY 132 Poster Session 2

Kim dal-Soo IL-8 MEDIATED HUCB-MSCS MIGRATION TOWARD U87-MG, HUMAN GLIOMA CELL. 79 Poster Session 1

Kim MiJung THE EFFECT OF VEGF ON THE MYOGENIC DIFFERENTIATION OF ADIPOSE TISSUE DERIVED STEM CELLS WITH IN VIVO GEL FORMING MPEG-PCL 166 Poster Session 2

Kim Sanghoon TROPHIC MOLECULES DERIVED FROM HUMAN MESENCHYMAL STEM CELLS ENHANCE SURVIVAL, FUNCTION, AND ANGIOGENESIS OF ISOLATED ISLETS FOLLOWING TRANSPLANTATION 188 Poster Session 1

Kim Seong Muk IRRADIATION ENHANCES THERAPEUTIC POTENTIAL OF MESENCHYMAL STEM CELL-BASED TRAIL GENE DELIVERY IN GLIOMA 110 Poster Session 2

Kim Sung JooSELF-SECRETED HUMAN INTERLUKIN-6 AND SOLUBLE INTERLIKIN-6 RECEPTOR ALPHA INITIATED THE JAK1/STAT3 AND PI3K/AKT SIGNALS AND INDUCED MAINTAIN STEMNESS DURING EX VIVO EXPANSION WITH HUMAN CORD BLOOD CD34+ AND AFT024 CELLS.


Kim Sung Joo ESTABLISHMENT OF COMPLETE RECONSTITUTED HUMAN LYMPHOCYTES AND PRODUCED ANTIGEN-SPECIFIC ANTIBODIES AFTER BUSULFAN TREATED OF NOD/SCID/IL2RγNULL MICE. 134 Poster Session 2

Kim Sung JooSUCCESSFUL ESTABLISHMENT OF HUMANIZED MICE MODEL HAVE FUNCTIONAL HUMAN HEMATOPOIETIC CELLS THROUGH TRANSPLANTED OF UMBILICAL CORD BLOOD CD34+ CELLS INTO BUSULFAN SOLUTION INJECTED NEONATAL NOD/SCID/IL2RγNULL MICE LIVER.


Klamer Guy EXAMINING THE EFFECTS OF GSK3γ INHIBITION ON T CELL ACTIVATION 37 Poster Session 1

Klein hans-Michael INSTEM TRIAL - INTRAOPERATIVE, AUTOLOGOUS CD133+ CELL ISOLATION AND TMLR-SUPPORTED TRANSPLANTATION DURING CABG 136 Poster Session 2

Ko Kap hyoun GSK-3γ INHIBITION INDUCES ENGRAFTMENT OF EX VIVO EXPANDED HEMATOPOIETIC STEM CELLS 137 Poster Session 2

Kolvenbach Ralf INTRAOPERATIVE STEM CELL TREATMENT IN PATIENTS SUFFERING FROM CRITICAL LIMB ISCHEMIA 167 Poster Session 2

Kotovuori Annika STEM CELL SURFACE GLYCOMICS TO IMPROVE THE QUALITY AND EFFICACY OF CELLULAR THERAPEUTICS 80 Poster Session 1

Kumar Vijay METHOD FOR EVALUATION OF TEFLON OVERWRAP BAGS USED TO PREVENT MICROBIAL SURFACE CONTAMINATION OF CRYOPRESERVED CORD BLOOD UNITS 138 Poster Session 2

Kumar Vijay EVALUATION OF CELL CONCENTRATE PREPARED FROM BONE MARROW ASPIRATE USING THE RES-Q™ 60 BMC SYSTEM 139 Poster Session 2

Kvalheim Gunnar T CELL RESPONSES IN PATIENTS VACCINATED WITH TELOMERASE (HTERT)-MRNA TRANSFECTED FAST DENDRITIC CELLS 38 Poster Session 1

Kvalheim Gunnar USE OF PLERIXAFOR IN COMBINATION WITH G-CSF AND CHEMOTHERAPY YIELD SUFFICIENT NUMBERS OF CD34+ CELLS TO OFFER “POOR MOBILIZERS” HIGH DOSE THERAPY 140 Poster Session 2

Laitinen Saara HYPOXIC CONDITION ENHANCES PROLIFERATION AND MAINTENANCE OF UMBILICAL CORD BLOOD MESENCHYMAL STEM CELLS IN XENOFREE MEDIUM 81 Poster Session 1

Lakshmipathy uma EFFICIENT GENE DELIVERY PLATFORM FOR NEURAL STEM AND PROGENITOR CELLS 111 Poster Session 2

Lanitis Evripidis OVARIAN CANCER CELLS UBIQUITOUSLY EXPRESSED HER-2 AND CAN BE DISTINGUISHED FROM NORMAL OVARY BY GENETICALLY REDIRECTED T CELLS 112 Poster Session 2

Larson Christopher CGMP VALIDATION OF A FLOW CYTOMETRY IDENTITY ASSAY FOR GMP-PRODUCED STEM CELLS 54 Poster Session 1

Lee In Ja ANALYTICAL METHOD VALIDATION OF FLOW CYTOMETRY-BASED IDENTIFICATION AND PURITY TEST FOR HUCB-MSCS 82 Poster Session 1

Lee Shing-Mou SURGICAL GRAFTS FOR REPAIRING CHONDRAL DEFECTS 194 Poster Session 2

Li Junzhi CHARACTERIZATION OF CELL CONCENTRATES PREPARED AT POINT OF CARE FROM BONE MARROW ASPIRATE USING THE MARROWXPRESS™ SYSTEM 168 Poster Session 2

Lim Ok-jae CHARACTERIZATION OF EX VIVO-EXPANDED ALLOGENEIC NATURAL KILLER CELLS FOR CLINICAL APPLICATION IN ANTI-TUMOR IMMUNOTHERAPY 39 Poster Session 1

Lipowska-Bhalla Grazyna A NOVEL SCREENING METHOD FOR THE IDENTIFICATION OF TUMOUR ANTIGEN SPECIFIC scFv’s FOR USE IN ADOPTIVE IMMUNE THERAPY OF CANCER 178 Poster Session 1

Loudovaris Maureen COMMERICALIZEd MAnuFACtuRInG PROCESS OF CVAC™, A dEndRItIC CELL VACCInE, FOR A PhASE II tRIAL In AdVAnCEd OVARIAn PAtIEntS 40 Poster Session 1





Maas MaryLEVELS OF CIRCULATING PSA AND HLA-DR- MONOCYTES IN PROSTATE CANCER PATIENTS VACCINATED WITH ALLOGENEIC PROSTATE CANCER CELLS (APCC) WITH OR WITHOUT AUTOLOGOUS DENDRITIC CELLS (DCS)

41 Poster Session 1

Machado Janaina EVALUATION OF INCIDENCE OF MICROBIOLOGICAL CONTAMINATION IN THE CORD BLOOD UNITS COLLECTED BY PRIVATE UMBILICAL CORD BLOOD BANK IN BRAZIL. 141 Poster Session 2

Maiti Sourindra ANALYZING INTEGRATIONS OF TRANSPOSONS TO GENERATE CLINICAL-GRADE TUMOR-SPECIFIC T CELLS 42 Poster Session 1

Majkowski Gemini ELECTRONIC SYSTEM FOR MANAGING THE DEVIATION PROCESS 183 Poster Session 1

Mareschi KatiaMULTIPOTENT MESENCHYMAL STEM CELLS FROM AMNIOTIC FLUID ORIGINATE NEURAL PRECURSORS WITH FUNCTIONAL VOLTAGE-GATED SODIUM CHANNELS AND SHOW HIGH IMMUNOMODULATION PROPRIETIES

83 Poster Session 1

Marquez-Curtis Leah COMPARISON OF PROPERTIES OF MESENCHYMAL STEM CELLS DERIVED FROM HUMAN BONE MARROW AND CORD BLOOD 84 Poster Session 1

Marquez-Curtis Leah CONTRASTING EFFECTS OF THE COMPLEMENT COMPONENTS C1Q AND C5A ON HSPC TRAFFICKING 142 Poster Session 2

Mayani hector COMPARATIVE IN VITRO ANALYSIS OF DIFFERENT CELL POPULATIONS FROM HUMAN UMBILICAL CORD BLOOD: IN SEARCH OF THE BEST OPTION FOR CELL EXPANSION. 143 Poster Session 2

Mazouz Chaya REGULATORY STUDY DESIGN CONSIDERATIONS FOR PLX-PAD, AN ALLOGENEIC CELL PRODUCT FOR THE TREATMENT OF CRITICAL LIMB ISCHEMIA (CLI) 11


McCarter donald POST-THAW STABILITY OF DILUTED/RECONSTITUTED UMBILICAL CORD BLOOD PRODUCTS 144 Poster Session 2

McKenna david CGMP PRODUCTION OF UMBILICAL CORD BLOOD-DERIVED T-REGULATORY CELLS IN SUPPORT OF A FIRST-IN-HUMAN CLINICAL TRIAL 43 Poster Session 1

McMannis John VALIDATION OF MILTENYI CRYOMACS FREEZING BAGS FOR HEMATOPOIETIC CELL PRODUCTS 145 Poster Session 2

McMannis John EVALUATION OF THE COBE 2991 AND THE SEPAX INSTRUMENT FOR VOLUME REDUCTION OF HEMATOPOIETIC PROGENITOR CELLS (HPC,APHERESIS) 146 Poster Session 2

Mcniece Ian EXPRESSION OF MICRORNA (MIR) IN CARDIAC STROMAL CELLS 3 Oral Abstracts Session 1 – Regenerative Medicine

Mcniece Ian CFU-F LEVELS IN BONE MARROW ASPIRATES OF CARDIAC PATIENTS TREATED WITH MESENCHYMAL STEM CELLS 85 Poster Session 1

Mcniece Ian GROWTH FACTOR STIMULATION OF CARDIAC STEM CELLS (CSCs) TO GENERATE CELLS WITH IN VIVO POTENTIAL 169 Poster Session 2

Melenhorst Jan BOTH NAïVE AND MEMORY T CELLS EXERT ALLOREACTIVITY ACROSS HLA BARRIERS 7Oral Abstracts Session 2 – Immunotherapy and dendritic Cells

Michalek Jaroslav OPTIMIZED MATURATION COCTAIL FOR CLINICAL GRADE DENDRITIC CELL VACCINATION 44 Poster Session 1

Miller timothy INTRAMUSCULAR DELIVERY OF ACRX-100 ENHANCES ANGIOGENESIS IN A RABBIT MODEL OF HIND LIMB ISCHEMIA 113 Poster Session 2

Montesinos Juan COMPARATIVE ANALYSIS OF MESENCHYMAL STROMAL CELLS FROM ADULT AND UMBILICAL CORD BLOOD. 86 Poster Session 1

nguyen thu Minh OVERVIEW OF REGULATIONS AND STANDARDS FOR STEM CELL-BASED THERAPIES: AN INTERNATIONAL PERSPECTIVE 58 Poster Session 1

O’Connor Colleen T-CELL THERAPY FOR OUT BRED CANINES WITH SPONTANEOUS B CELL NON HODGKIN LYMPHOMA 45 Poster Session 1

O’doherty una PLERIXAFOR MOBILIZATION LEADS TO A LOWER RATIO OF CD34+ CELLS TO TOTAL NUCLEATED CELLS WHICH RESULTS IN GREATER STORAGE COSTS 147 Poster Session 2

Ofir Racheli PLACENTA DERIVED MESENCHYMAL-LIKE STROMAL CELLS- NOT ALL CELLS WERE CREATED EQUAL 87 Poster Session 1

Otsuru Satoru TRANSPLANTATION OF MESENCHYMAL STROMAL CELLS (MSCs) INDUCES LINEAR BONE GROWTH BY STIMULATING GROWTH PLATE CHONDROCYTE PROLIFERATION 16 Oral Abstracts Session 4 –


Padley doug USE OF THE ENDOSAFE PTS FOR MEASURING ENDOTOXIN LEVELS IN PLATELET LYSATE 55 Poster Session 1

Page Kristin A NOVEL SCORING SYSTEM, THE CORD BLOOD APGAR, PREDICTS ENGRAFTMENT AFTER CORD BLOOD TRANSPLANTATION: OPTIMIZATION OF SELECTION OF CBUS FOR TRANSPLANTATION 27


Partanen Jukka VPU102, A NOVEL SINGLE MOLECULE MATRIX SUPPORTS UNDIFFERENTIATED GROWTH OF HUMAN ES AND IPS CELLS 4 Oral Abstracts Session 1 –




may 23-26, 2010




Pasquini Marcelo CELLuLAR thERAPy FOR REGEnERAtIVE MEdICInE: ACtIVIty In thE uS duRInG 2008 195 Poster Session 2

Pastore Joseph IntRAMyOCARdIAL dELIVERy OF ACRX-100 EnhAnCES CARdIAC FunCtIOn And InCREASES VASCuLOGEnESIS In A PORCInE MOdEL OF ChROnIC hEARt FAILuRE 23

Oral Abstracts Session 6 - Cell and Gene therapy/Cellular Gene transfer

Pattasseril Jacob hIGh ShEAR RAtES nEGAtIVELy AFFECt CELL VIABILIty And FInAL PROduCt QuALIty In tFF PROCESSInG OF LARGE SCALE thERAPEutIC CELL hARVEStS 170 Poster Session 2

Pieken Wolfgang huMAn AutOLOGOuS LIVER CELL tRAnSPLAntAtIOn FOR thE tREAtMEnt OF CIRRhOSIS 171 Poster Session 2

Poirot Laurent dEVELOPMEnt OF nOn VIRAL APPROAChES tO MEGAnuCLEASE EnGInEEREd CELLuLAR thERAPy FOR tREAtMEnt OF MOnOGEnEIC InhERItEd dISEASES 114 Poster Session 2

Porterfield nancy MICROEnVIROnMEnt OF WAR WOundS: COnCERnS FOR REGEnERAtIVE MEdICInE APPLICAtIOnS 17 Oral Abstracts Session 4 –


Powell daniel AutOLOGOuS WhOLE-tuMOR AntIGEn COMBInAtORIAL IMMunOthERAPy FOR RECuRREnt OVARIAn CAnCER 8

Oral Abstracts Session 2 – Immunotherapy and dendritic Cells

Powell Jr. daniel LARGE-SCALE CELL COnCEntRAtIOn And WAShInG uSInG A MOdIFIEd IntRAOPERAtIVE BLOOd RECOVERy dEVICE hAEMOnEtICS CELL SAVER 5 SyStEM. 103 Poster Session 1

Prata Karen Cd34+ CELLS FREQuEnCy And VIABILIty EVALuAtIOn OF hPC-C PRE-CRyOPRESERVAtIOn 148 Poster Session 2

Praught Melanie IMPACt OF AntICOAGuLAnt And tIME FROM COLLECtIOn tO PROCESSInG On CORd BLOOd CELL VIABILIty And COunt. 149 Poster Session 2

Putnam Amy EX VIVO EXPAndEd Cd4+Cd127LO/-Cd25+ POLyCLOnAL tREGS: PhASE I CLInICAL tRIAL FOR thE tREAtMEnt OF RECEnt OnSEt t1d 46 Poster Session 1

Pütsch Kathrin CLInICAL-SCALE ISOLAtIOn OF MESEnChyMAL StROMAL CELLS (MSC) FROM BOnE MARROW WIth thE CLInIMACS® PLuS InStRuMEnt And Cd271 MICROBEAdS. 88 Poster Session 1

Raab Matthew StEM CELL duROtAXIS - tRAFFICKInG tO RIGId FIBROtIC SCARS 89 Poster Session 1

Rae Michelle An InVEStIGAtIOn IntO thE KInEtICS OF MSC-InduCEd IMMunOSuPPRESSIOn duRInG thE ALLO-REACtIVE PhASE OF An MLR 186 Poster Session 1

Ragaglia VanessaChARACtERIStICS OF A nOn-hEMAtOPOIEtIC ALLOGEnEIC BOnE-MARROW dERIVEd CELL POPuLAtIOn thAt CAn BE MAnuFACtuREd tO PhARMACEutICAL StAndARdS And QuAntItIES.

90 Poster Session 1

Roquemore Elizabeth APPLICAtIOn OF A hIGh COntEnt IMAGInG SyStEM FOR ChARACtERIZAtIOn And QuALIty ASSuRAnCE OF StEM CELL dERIVEd PROduCtS. 104 Poster Session 1

Rosenau Emma ChARACtERIStICS OF thAWEd AutOLOGOuS uMBILICAL CORd BLOOd 150 Poster Session 2

Sadeghi Arian LARGE SCALE BIOREACtOR EXPAnSIOn OF tuMOR SPECIFIC t LyMPhOCytES FOR AdOPtIVE CELL thERAPy 47 Poster Session 1

Scholler John dECAdE-LOnG PERSIStEnCE OF AdOPtIVELy tRAnSFERREd AutOLOGOuS REdIRECtEd Cd4-ζ ChIMERIC RECEPtOR t CELLS In hIV+ Study SuBJECtS 24


Shin Jae-Won EnRIChMEnt OF PRIMItIVE hEMAtOPOIEtIC StEM CELLS WIth POLyPLOIdIZAtIOn OF PROLIFERAtInG CELLS By SMALL MOLECuLE InhIBItIOn OF MyOSIn 151 Poster Session 2

Shoulars Kevin dEVELOPMEnt OF A SEGMEnt-BASEd ALdEhydE dEhydROGEnASE ASSAy tO dEtERMInE uMBILICAL CORd BLOOd unIt (CBu) POtEnCy 152 Poster Session 2

Shroff Geeta huMAn EMBRyOnIC StEM CELL thERAPy In CEREBRAL PALSy: A 15 PAtIEnt CASE SERIES 91 Poster Session 1

Siqueira Rubens AutOLOGOuS BOnE MARROW dERIVEd StEM CELLS tRAnSPLAntAtIOn FOR REtInItIS PIGMEntOSA 193 Poster Session 2

Solmesky Leonardo ChARACtERIZAtIOn OF huMAn MESEnChyMAL StEM CELLS (hMSC) FROM BOnE MARROW In SERuM FREE COndItIOnS 92 Poster Session 1

Song degangROBuSt InVO AntI-tuMOR FunCtIOn By huMAn t CELLS EnGInEEREd tO EXPRESS A 41-BB COStIMuLAtORy ζ-FOLAtE RECEPtOR-SPECIFIC ChIMERIC IMMunE RECEPtORS FOR OVARIAn CAnCER


Speziale Robert EXPERIEnCE WIth dIVERSE CELL COnCEntRAtIOn, REtEntIOn, And WASh APPLICAtIOnS FOR uSE In StEM CELL PROCESSInG And CELL thERAPy PROCESSInG 153 Poster Session 2

Spiller Kara BIOACtIVE, SEMI-dEGRAdABLE hydROGELS FOR CARtILAGE tISSuE EnGInEERInG 172 Poster Session 2

Stadtmauer Edward PRIMEd EX-VIVO CO-StIMuLAtEd AutOLOGOuS t-CELLS And POSt-ASCt InFLuEnZA VACCInAtIOn FOR MuLtIPLE MyELOMA 48 Poster Session 1

Stephan Matthias thERAPEutIC CELL EnGInEERInG uSInG SuRFACE-COnJuGAtEd SynthEtIC nAnOPARtICLES 177 Poster Session 1





Stepp Wesley EFFECT OF WOUND MICROENVIRONMENT ON MESENCHYMAL STEM CELL PROLIFERATION AND DIFFERENTIATION. 93 Poster Session 1

Sun Jiali CONSTITUTIVELY ACTIVE AKT IN T CELLS IMPARTS RESISTANCE TO NEUROBLASTOMA MEDIATED SUPPRESSION 49 Poster Session 1

Sun Jessica QUALITY OF AUTOLOGOUS UMBILICAL CORD BLOOD UNITS INFUSED INTRAVENOUSLY IN CHILDREN WITH ACQUIRED NEUROLOGICAL CONDITIONS 154 Poster Session 2

tebas Pablo PROLONGED CONTROL OF VIREMIA AFTER TRANSFER OF AUTOLOGOUS CD4 T CELLS GENETICALLY MODIFIED WITH A LENTIVIRAL VECTOR EXPRESSING LONG ANTISENSE TO HIV ENV (VRX496) 25


thirumala Sreedhar GROWTH CHARACTERISTICS OF ENDOMETRIAL REGERATIVE CELLS (ERCS) CULTIVATED IN DIFFERENT MEDIA 94 Poster Session 1

thirumala Sreedhar A NEW CLOSED CONTAINER SYSTEM FOR CLINICAL CRYOPRESERVATION OF BIOMATERIALS 155 Poster Session 2

tiumina / Volchkov

Olesya / Stanislav

DEFINING THE BEST SOURCE OF HUMAN MULTIPOTENTIAL MESENCHYMAL STROMAL CELLS FROM CORD BLOOD, FAT TISSUE AND BONE MARROW. 187 Poster Session 1

tizon RichardA RETROSPECTIVE COHORT STUDY OF ETANERCEPT VERSUS CORTICOSTEROIDS FOR THE TREATMENT OF IDIOPATHIC PNEUMONIA SYNDROME AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION.

50 Poster Session 1

trickett Annette FACTORS AFFECTING THE MICROBIAL CONTAMINATION RATE OF CORD BLOOD COLLECTED FOR TRANSPLANTATION AT THE SYDNEY CORD BLOOD BANK (SCBB) 12


trickett Annette STRATEGIES TO MINIMISE THE RISK OF ERROR IN TRANSPLANT CONDITIONING PROTOCOLS 156 Poster Session 2

Verter Frances THE GROWTH OF CORD BLOOD USE 157 Poster Session 2

Wagey Ravenska ENHANCED CHONDROGENIC POTENTIAL AND IMMUNOSUPPRESSIVE ACTIVITY OF HUMAN MESENCHYMAL PROGENITOR CELLS CULTURED IN A NOVEL XENO-FREE MEDIUM 95 Poster Session 1

Wakeda takako FCGR3A-158V GENE TRANSDUCTION AUGMENT CD16 EXPRESSION AND ADCC ACTIVITY OF PERIPHERAL BLOOD MONONUCLEAR CELLS. 116 Poster Session 2

Wallace Shay QUANTITATIVE EX VIVO CHARACTERIZATION OF HUMAN RENAL CELL POPULATION DYNAMICS VIA HIGH-CONTENT IMAGE-BASED ANALYSIS (HCA) 28


Wanderi George GENE THERAPY AND THE BRAIN:IS AFRICA ETHICALLY, LEGALLY AND SOCIALLY READY? 59 Poster Session 1

Wang Bin FUNCTIONAL TENDON ENGINEERING AND ITS APPLICATION IN REPAIRING MONKEY TENDON DEFECT 173 Poster Session 2

Weinberg Rona TWO CASES OF ELEVATED LIVER FUNCTION TESTS (LFTS) FOLLOWING INFUSION OF CRYOPRESERVED HEMATOPOIETIC PROGENITOR CELL APHERESIS (HPC, APHERESIS) PRODUCTS 158 Poster Session 2

Wesa Amy HUMAN MAPC LABELED WITH A FLUORINE MRI TRACER RETAIN MULTI-LINEAGE DIFFERENTIATION POTENTIAL 96 Poster Session 1

Wofford Jonathan EVALUATION OF PROCESSING TECHNOLOGIES FOR UNRELATED UMBILICAL CORD BLOOD (UCB) 192 Poster Session 2

Wu Luke APOPTOSIS IN CRYOPRESERVED AUTOLOGOUS PBSCS AND DELAYED ENGRAFTMENT AFTER TRANSPLANTATION DESPITE SUFFICIENT CD34(+) CELLS 22 Oral Abstracts Session 5 –


yamaki yuni ESTABLISHMENT OF A CLINICALLY APPLICABLE METHOD FOR LYMPHOCYTE EXPANSION USING RECOMBINANT HUMAN FIBRONECTIN FRAGMENT (CH-296; RETRONECTIN) 105 Poster Session 1

yen Chun-Che THE VALIDATION STUDY OF MYCOPLASMA DETECTION METHODS FOR THE QUALITY CONTROL OF CELL THERAPEUTIC PRODUCTS IN HOSPITALS 180 Poster Session 1

yen Chun-Che THE VALIDATION STUDY OF GROWTH-BASED RAPID BACTERIAL DETECTION SYSTEM FOR THE QUALITY CONTROL OF CELL THERAPEUTIC PRODUCTS 181 Poster Session 1

yen Chun-Che FIVE-YEAR DEVELOPMENT OF REGULATION FOR CELL THERAPY IN TAIWAN - A REGULATORY MODEL OF CELL THERAPY IN HOSPITALS FOR SMALL COUNTRIES 182 Poster Session 1

yousefi Amy 3D-PRINTED SCAFFOLDS FOR TISSUE ENGINEERING AND CELL-BASED GENE THERAPY 174 Poster Session 2

n/A n/A ABSTRACT WITHDRAWN. 97 Poster Session 1

Zanette dalila INCREASED EXPRESSION OF SIDE POPULATION MARKERS IN HUMAN AMNIOTIC EPITHELIAL STEM CELLS CULTURED WITH A COMMERCIAL SERUM REPLACEMENT 175 Poster Session 2

Zhao yangbing PRE-CLINICAL EVALUATION OF AN OPTIMIZED RNA ELECTROPORATION SYSTEM FOR T LYMPHOCYTE BASED CANCER ADOPTIVE IMMUNOTHERAPY 117 Poster Session 2



may 23-26, 2010


Oral Abstracts

1SMOOth MuSCLE CELLS dERIVEd FROM huMAn AdIPOSE tISSuE FOR uSE In uROLOGIC tISSuE EnGInEERInGD. Jain, J. W. Ludlow, K. Guthrie, N. Sangha, C. Genheimer, J. Shokes, T. Burnette, D. Justewicz, R. M. Ilagan, B. J. Wagner, T. A. Bertram; Tengion Inc, Winston-Salem, NC.

approximately 10,000 cystectomies (surgical removal of the bladder) are performed annually in the us with bladder cancer being the leading indication. once the bladder is removed, a mechanism for urinary diversion must be constructed. While there are several surgical options for a urinary diversion they all involve the use of a gastrointestinal segment. However, use of gi tissue exposes gut mucosa to urine leading to multiple complications including gi and metabolic abnormalities. tengion is developing autologous regenerative products such as the neo-urinary Conduit™ (nuC) for urologic tissue engineering applications. tengion’s nuC is engineered to reconstruct the urinary tract as an alternative to using gastrointestinal tract segments. the nuC is produced by seeding smooth muscle cells (smC) on a biodegradable scaffold to form a nuC Construct. in a porcine model, the nuC regenerated an incontinent urinary diversion composed of native-like urinary tissue. early products for regenerating urinary tissue used smC isolated from urinary bladder (e.g., tengion’s autologous neo-bladder augment™). For nuC development, since most patients will have a cancerous bladder, the feasibility of sourcing autologous smC from adipose tissue was evaluated. Canine and porcine adipose tissue biopsies were digested with collagenase and the dissociated cells recovered by centrifugation. Cells were cultured and characterized using rt-PCr, FaCs, elisa and immunocytochemistry. isolated cells showed typical smC morphology and growth characteristics. Phenotypic characterization revealed a robust population expressing multiple smC markers involved in muscle contraction. endothelial, epithelial, and myofibroblast marker expression was not observed. adipose-derived cells expressed mCP-1 protein indicative of functional smC. seeding adipose-derived smC on the nuC scaffold resulted in a nuC Construct suitable for implantation in preclinical studies. these results support the hypothesis that adipose tissue can be used as a source of smC for autologous products to regenerate urinary tissue in patients requiring reconstruction of the lower urinary tract.

3EXPRESSIOn OF MICRORnA (MIR) In CARdIAC StROMAL CELLSY. Wang, S. Sivajothi, S. Landa, C. Joseph, I. McNiece; University of Miami, Miami, FL.

stromal cells play an important role in control of proliferation and differentiation of stem cells and are a key component of the stem cell niche. bone marrow (bm) stromal cells (also termed mesenchymal stem cells; msC) have been extensively studied and shown to control differentiation of hematopoietic stem cells (HsCs) in part through secreted growth factors. recent studies have demonstrated the presence of stromal cells in cardiac tissue however, the role of cardiac stromal cells (CstrC) is unclear. in this study we have compared human CstrCs to human bm-msCs and demonstrate that CstrCs have a similar morphology and surface marker expression as bm-msCs. to further characterize the CstrCs we performed micro array analysis of human CstrCs compared to human bm-msCs. the CstrCs expressed a distinct cytokine and cytokine receptor profile compared to bm-msC. in addition, a number of micro rnas were expressed at very high levels in CstrCs compared to bm-msC. the micro rna mir-206 was expressed at more than a thousand fold higher in CstrCs compared to bm-msCs. other cardiac- associated micrornas, including mir-1 and mir-133a, were expressed at 63 fold and 57 fold higher in CstrCs compared to bm-msC. these data demonstrate that cardiac derived stromal cells have a similar phenotype to bm-msC but have a distinct gene expression profile. Further the identification of cardiac associated micrornas provides insight into the role of CstrCs in control of proliferation and differentiation of cardiac stem cells.

2BIOACtIVE REnAL CELLS AuGMEnt KIdnEy FunCtIOn In A ROdEnt MOdEL OF ChROnIC KIdnEy dISEASER. Kelley, E. S. Werdin, A. T. Bruce, S. Choudhury, S. M. Wallace, P. Tatsumi-Ficht, E. A. Rivera, T. Spencer, H. S. Rapoport, R. M. Ilagan, B. J. Wagner, M. J. Jayo, T. A. Bertram, S. C. Presnell; Tengion Inc, Winston-Salem, NC.

the rising cost of established chronic kidney disease (CKD) management, the morbidity associated with dialysis, and the limited numbers of donor kidneys suitable for transplant provide impetus for developing new treatment paradigms for CKD. Previous studies demonstrated that ex vivo cell cultures propagated from whole kidney tissue (unFX) contained renal cells from all major compartments of the kidney and were capable of stimulating regeneration and stabilizing renal function after orthotopic transplantation in the rodent remnant kidney model of renal failure. also demonstrated previously was that a subpopulation of unFX (b2), enriched for specific renal epithelial cells, enhanced nearly all aspects of nephron function and outperformed unFX in all parameters. the present study further segregated unFX into component cell subpopulations, evaluated in vitro characteristics, and tested in vivo performance of specific subpopulations alone and in combination. the selected subpopulations had unique profiles encompassing phenotypic, genotypic, and functional characteristics and elicited distinct systemic outcomes after orthotopic transplantation. one such subpopulation (b4), selected for its enrichment of glomerular, vascular and endocrine cells, enhanced renal endocrine functions compared to unFX. interestingly, a controlled admixture of the b2/b4 cell populations provided the most comprehensive benefit, outperforming b2, b4, unFX, and four other cell prototypes across most parameters, providing statistically-significant improvements in survival, weight gain, systemic blood pressure, protein retention, and endocrine function when compared to untreated controls. Histological evaluation of kidney and bone marrow six months post-treatment confirmed that two treatments (b2 and the b2/b4 combination) initiated a tissue-level regenerative response in the kidney, reducing glomerulosclerosis and tubulointerstitial fibrosis throughout the renal parenchyma. taken together, these studies support continued investigation into alternative treatments for progressive CKD that employ specific, biologically-active renal cells in the stimulation of a regenerative response.

4VPu102, A nOVEL SInGLE MOLECuLE MAtRIX SuPPORtS undIFFEREntIAtEd GROWth OF huMAn ES And IPS CELLSJ. Partanen1, M. Mikkola1, K. Alfthan1, M. Tiittanen1, V. Pitkanen1, M. Lampinen1, T. Satomaa2, L. Valmu1, T. Otonkoski3; 1Finnish Red Cross Blood Service, Helsinki, FINLAND, 2Glykos Finland ltd, Helsinki, FINLAND, 3Stem Cell Center, University of Helsinki, Helsinki, FINLAND.

Culturing of human embryonic stem cell (hesC) and induced pluripotent cells (hiPsC) requires an extracellular matrix coating. matrigel, a mixture from murine matrix is the current golden standard but is not acceptable for potential clinical applications. We here report culturing of hesCs and hiPs on a novel matrix molecule VPu102.We have demonstrated using 4 hesC cell lines and 4 local hiPsC lines that VPu102 is compatible for culturing these cells. undifferentiation and pluripotency were demonstrated by the expression of relevant surface and mrna markers as well as by using an in vitro embryoid body assay. the cellular response for VPu102 and matrigel based culturing was compared by studying the mrna expression profiles of 84 genes related to differentiation.based on the results the VPu102 system enables fully-defined, animal-free culture system for human pluripotent stem cells.


Oral Abstracts

5EARLy AdOPtIVE tRAnSFERS OF AntI-Cd3/AntI-Cd28 COStIMuLAtEd AutOLOGOuS t CELLS AFtER AutOSCt FOR MyELOMA: SIGnIFICAntLy EnhAnCEd POSt-tRAnSPLAnt huMORAL And CELLuLAR IMMunE RECOnStItutIOnN. A. Aqui1, A. P. Rapoport2, E. A. Stadtmauer3, D. T. Vogl3, H. Fang2, S. J. Janofsky1, J. Storek4, A. Chew3, E. Veloso3, G. Akpek2, S. Yanovich2, M. T. Tan2, Y. Wu2, A. Cross2, S. Phillip2, H. Murphy3, R. Bagat3, Y. Liu4, B. L. Levine3, R. H. Vonderheide3, C. H. June3; 1Dept. of Pathology, Univ. of Pennsylvania, Philadelphia, PA, 2Marlene and Stewart Greenebaum Cancer Center, Univ. of Maryland, Baltimore, MD, 3Abramson Cancer Center of the Univ. of Pennsylvania, Philadelphia, PA, 4University of Calgary, Calgary, AB, CANADA.

Previously, we showed that adoptive transfer of in vivo, vaccine-primed and ex vivo (anti-CD3/anti-CD28) costimulated autologous t cells (CD3/CD28tC) after auto-sCt increased CD4 and CD8 levels and augmented vaccine-specific immune responses in patients with myeloma (mm). We have now completed a 2-arm clinical trial where advanced mm patients received auto-sCt followed by CD3/CD28tC at day +2 post-sCt. Patients also received pretransplant and posttransplant immunizations using a pneumococcal conjugate vaccine (PCV) only (arm b;n=26) or PCV plus an Hla-a2 restricted htert+survivin peptide tumor antigen vaccine and CmV peptide (arm a;n=28). after CD3/CD28tC infusion, the median CD3, CD4 and CD8 counts were 4198, 1545 and 2858 cells/ul at day +14 posttransplant. Higher CD3+ counts at day +14 and higher CD8+ counts at day +14 and day +60 were significantly associated with improved eFs. 7/17 arm a patients had tetramer responses to htert and/or survivin peptides at one or more timepoints after immunization. Highly protective antibody levels to 4/7 pneumococcal conjugate vaccine serotypes were induced at day +180 compared to enrollment. Compared to an historical cohort of 103 mm pts who were autografted without CD3/CD28tC, post-transplant antibody levels at day +180 were significantly higher in patients who received day +2 t-cell transfers. remarkably, a pattern of CD8 t-cell “reprogramming” was also observed after day +2 t-cell transfers such that CD8+/CD45ra+ t-cells at day +60 posttransplant was 75% vs 36% in a cohort of mm transplant pts who did not receive CD3/CD28tC (p< 0.0001). the log ratio of teffector/tregulatory cells was significantly higher among the patients who received early t-cell transfers (2.57 vs. 1.93, p = 0.007). these novel immune reconstitution data may have important implications for better myeloma control and, more broadly, for protection from infection and induction of clinically significant immune responses to cancer vaccines in the transplant setting.

7BOth nAïVE And MEMORy t CELLS EXERt ALLOREACtIVIty ACROSS hLA BARRIERSJ. J. Melenhorst, P. Scheinberg, D. R. Ambrozak, K. Keyvanfar, N. F. Hensel, D. C. Douek, A. J. Barrett; National Institutes of Health, Bethesda, MD.

t cell responses to allogeneic targets arise predominantly from the naïve pool. However, in man the risk of graft-versus-host disease (gvHD) is increased if the donor has circulating t cells recognizing multiple persistent Dna viruses, suggesting that memory t cells also contribute to the alloresponse. We used a flow cytometry-based proliferation & activation assay to assess the contribution of naïve and memory t cells to alloreactivity. alternatively, donor peripheral blood mononuclear cells (PbmC) were primed for 8 days with allogeneic, Hla mismatched PbmC (alloPbmC), after which the intracellular cytokine production (iCD) was examined upon re-challenge with autologous or alloPbmC. both naïve and memory t cells were able to mount a response to Hla-mismatched targets. the direct ex vivo alloreactivity of both t-cell subsets was tested by iCD where donor PbmC were stimulated allogeneic targets. this experiment showed that both naïve and memory t cells are rapidly recruited into the alloreactive t-cell pool. since a prominent role of memory t cells was apparent from our experiments we next tested the alloreactive potential of antigen-specific t cells. both ebV- and CmV-specific t cells displayed exquisite specificity for certain mismatched Hla alleles, indicating that Dna virus antigen-specific t cells cross-react with peptide-mHC complexes unrelated to the cognate antigens. sequencing of the t cell receptor-β chain of t cells demonstrated clonotypic identity of cells that responded to both viral and allogeneic pmHC stimulation. Collectively our data demonstrate that both naïve and memory t cells can mount an alloresponse, but that memory t cells are more rapidly and strongly recruited by alloantigen stimulation. these findings indicate that in man alloresponses are not confined to particular subsets of post-thymic t cells and that donor-derived viral antigen-specific t cells in an unrelated recipient may cross-react with peptide-mHC complexes against which they were not negatively selected.

6InduCtIOn OF COMPLEtE And MOLECuLAR REMISSIOnS In ACutE MyELOId LEuKEMIA By WILMS’ tuMOR 1 AntIGEn-tARGEtEd dEndRItIC CELL VACCInAtIOnZ. N. Berneman1, A. Van de Velde1, S. Anguille1, A. Van Driessche1, K. Vermeulen1, K. Pieters1, G. Nijs1, B. Stein1, E. Smits1, W. A. Schroyens1, A. P. Gadisseur1, I. Vrelust1, I. de Vries2, N. Cools1, V. F. Van Tendeloo1; 1University of Antwerp and Antwerp University Hospital, Edegem, BELGIUM, 2Radboud Universiteit, Nijmegen, NETHERLANDS.

active immunization using tumor antigen-loaded dendritic cells holds promise for the adjuvant treatment of cancer to eradicate or control residual disease, but so far most dendritic cell trials have been performed in end-stage cancer patients with high tumor load. Here, in a phase i/ii trial, we investigated the effect of autologous dendritic cell vaccination in 17 patients with acute myeloid leukemia in remission but at high risk of full relapse. the Wilms’ tumor 1 antigen (Wt1), a nearly universal tumor antigen, was chosen as an immunotherapeutic target because of its established role in leukemogenesis and superior immunogenic characteristics. two out of 3 patients who were in partial remission with morphologically demonstrable disease after chemotherapy were brought into complete remission following intradermal administration of Wt1 mrna-electroporated dendritic cells. in those 2 patients as well as in 7 other patients who were in complete remission but who had molecularly demonstrable residual disease, there was a return to normal of the aml-associated tumor marker following dendritic cell vaccination, compatible with the induction of molecular remission in 9/17 patients vaccinated thus far. immunomonitoring showed a significant increase in Wt1-specific CD8+ t cells and changes indicative of general immune stimulation, such as a significant increase post-vaccination of plasma levels of interleukin 2 and of Hla-Dr+ activated CD4+ t-cells. in conclusion, vaccination with Wt1 mrna-loaded dendritic cells elicits immunological and clinical responses in acute myeloid leukemia patients. Dendritic cell-based immunotherapy emerges as a feasible and effective strategy to control residual disease and prevent full relapse in acute myeloid leukemia.

8AutOLOGOuS WhOLE-tuMOR AntIGEn COMBInAtORIAL IMMunOthERAPy FOR RECuRREnt OVARIAn CAnCERD. J. Powell1, L. Kandalaft1, L. Smith1, J. Liao1, A. Hagemann1, J. Tanyi1, Q. Ye1, A. Best1, D. A. Torigian1, C. Chu1, S. A. Rubin1, E. A. Stadtmauer1, M. Bosch2, B. L. Levine1, C. H. June1, G. Coukos1; 1University of Pennsylvania, Philadelphia, PA, 2NorthWest Biotherapeutics, Bethesda, MD.

background: novel therapeutic strategies are warranted in recurrent ovarian cancer. We report the pilot application of combinatorial immunotherapy comprising dendritic cell (DC)-based autologous whole tumor antigen vaccination in combination with anti-angiogenesis therapy, followed by adoptive transfer of autologous vaccine-primed CD3/CD28-costimulated lymphocytes in patients with recurrent ovarian cancer.methods: Patients with recurrent progressive stage iii and iV ovarian cancer underwent priming with intravenous bevacizumab and oral metronomic cyclophosphamide, followed by vaccination with DCVax-l, an autologous DC preparation pulsed with autologous tumor lysate, plus bevacizumab. this was followed by lymphodepletion using high-dose outpatient cyclophosphamide and fludarabine (cy/flu) and transfer of autologous vaccine-primed, ex vivo CD3/CD28-costimulated peripheral blood t cells, in combination with DCVax-l vaccination.results: seven subjects have completed vaccination alone; three subsequently completed t cell transfer. Vaccination with DCVax-l following bev/cy produced few grade 1 toxicities and elicited tumor-specific t cell and humoral responses in two patients evaluated. Partial objective response was observed in two patients after completion of vaccine; two additional patients demonstrated stable disease. among the former, one subject progressed through bev/cy, but had objective response to DCVax-l. of the three patients receiving adoptive transfer of vaccine-primed, CD3/CD28-costimulated autologous t cells following outpatient cy/flu lymphodepletion, one patient, who exhibited no evidence of disease at end of study, experienced a durable reduction of CD4+FoXP3+ t regulatory cells, increased total lymphocyte counts and restoration of vaccine-induced antitumor immunity. stable disease was observed in a second subject. in the third subject, adoptive immunotherapy was not followed by restoration of vaccine-induced antitumor immunity and disease progression was observed. updates will be presented at the meeting.interpretation: the use of combinatorial cellular immunotherapy comprising bev/cy with DC vaccination with whole tumor antigen and adoptive immunotherapy using tumor antigen-primed t cells for the treatment of patients with recurrent ovarian cancer warrants further investigation.


may 23-26, 2010


Oral Abstracts

9REGEnERAtIVE MEdICInE CASE Study: MAKInG thE tRAnSItIOn FROM RESEARCh InStItutE tO tRAnSLAtIOnAL RESEARCh And dEVELOPMEnt ORGAnIZAtIOnK. B. Johnson, T. B. Clarkson; Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC.

at the Wake Forest institute for regenerative medicine (WFirm), one of the world’s largest freestanding facilities dedicated to regenerative medicine, leading experts in molecular biology, genetics, cell biology, physiology, pharmacology, biomaterials, and nanotechnology work collaboratively to solve the field’s toughest challenges: applying the principles of regenerative medicine to repair or replace diseased tissues and organs.in addition to the significant technical and scientific challenges that WFirm and others in regenerative medicine are undertaking, WFirm’s mission toward translational medicine and progressing technologies efficiently to patients provides another set of unique challenges. this embrace of translational medicine has lead to the development of various initiatives and infrastructure components within the institute to directly address the challenges associated with transitioning from an academic institute to a focused translational research and development (tr&D) organization.specific examples of WFirm initiatives and infrastructure components will be discussed in this presentation, including the following:• Organizational design and change management foundations used to develop initiatives and infrastructure to support the “r to tr&D” transition,• Mechanisms to assist the organizational evolution from a scientific, innovation- focused organizational to a more formal, process- and results-oriented organization without losing the stimulating, academic culture,• Educational initiatives surrounding regulatory and compliance requirements,• New methods and mechanisms for cross-functional collaboration,• Development of new cross functional team structures including representation in areas of various scientific disciplines as well as intellectual property, business development, preclinical and clinical development, and regulatory affairs,• New project evaluation and implementation mechanisms involving cross-functional development teams that combine scientific and technical expertise with operational, project management, and product development functions,• Upstream mechanisms for education about technology transfer and regulatory requirements to support both internal and external technology transfer, and• Unique business development and funding models, including mechanisms for utilizing public/private partnerships.

11REGuLAtORy Study dESIGn COnSIdERAtIOnS FOR PLX-PAd, An ALLOGEnEIC CELL PROduCt FOR thE tREAtMEnt OF CRItICAL LIMB ISChEMIA (CLI)C. Mazouz, F. Grynspan, R. Ofir; Pluristem therapeutics, Haifa, israel.

two phase i studies using placenta adherent stromal cells, termed PlX-PaD, intended for treatment of Cli, were designed to evaluate safety, including immunological profile associated with local administration.these open-label, dose-escalation studies are performed in parallel in the eu and u.s.. submissions took into consideration the different regulatory approaches toward allogeneic cell products and diverse guidelines for phase i studies. manufacturing and controls were designed to comply with the more strict requirements between both agencies; furthermore a qualified person (QP) has approved the gmP facility for manufacturing PlX-PaD for the eu.the design of the studies is similar, but not identical. Follow up period and dose escalation schedule differ following regulatory requirements and previous experience of the clinical sites. Clinical follow-up period for both studies is three months after treatment; however, in the eu, the patients are observed for 24 months versus 12 months in the u.s..altogether five dosing groups are evaluated, adding to a better understanding of the interaction between cell number and cell distribution. Furthermore, for the low dose group in the u.s., PlX-PaD is multiply injected during one session, and during two sessions (two week apart) for the higher dose group, whereas, in the eu, higher dose is administered in a single session using higher volume per injection.Performing two phase i studies in parallel, enrolling altogether a minimum of 21 patients, will enable Pluristem, to collect data to support the in vitro and pre-clinical observations that PlX-PaD cells are immunologically privileged and illustrate the safety profile following both single and recurrent im administration.manufacturing a cell based product for phase i studies, while complying with two regulatory authorities was a challenge, which forced the company to adhere very closely to available guidelines, and will allow a smooth transaction to manufacturing for the phase ii study.

10REduCtIOn OF InCOMInG BIOBuRdEn In tISSuE PROduCtS IntEndEd FOR CELL ISOLAtIOn And CuLtuRE EXPAnSIOnC. Johnston1, J. Morris1, A. M. Preslar1, J. Pierce1, J. T. Campanelli2, M. Winters3, N. Patterson3, L. L. Kelley1; 1University of Utah, Salt Lake City, UT, 2Q Therapeutics Inc., Salt Lake City, UT, 3Nelson Laboratories, Salt Lake City, UT.

most cellular therapy products are obtained sterile from the donor and further processed under asceptic conditions in the laboratory. However, exceptions exist when the tissue cannot be procured under sterile conditions (e.g. pancreas for islet isolation, tumor samples for cellular vaccine development and fetal-derived tissues). our laboratory isolates, purifies and expands glial-restricted progenitor (grP) cells from fetal tissue through a complex series of processing steps. the grP are undergoing development by Q therapeutics inc. for use as therapy for patients with amyotrophic lateral sclerosis (als). since the tissue source is recovered from the vaginal canal there is a high probability of contamination with normal flora including aerobic, anaerobic and fungal microorganisms. We undertook a study to i) determine the quantity and identity of microorganisms in the starting tissue; ii) evaluate the kill effect of antibiotics and antimycotics during tissue transport; and iii) assess the effect of processing steps on bioburden reduction. sterility testing of 19 consecutive tissues processed from august 2008-January 2010 showed that a substantial number were contaminated with microorganisms including yeast (17%), gram-positive (9%) and gram-negative (74%) bacteria. therefore, a spike and recovery study was performed by inoculating sterile tissue with 1.0-2.5 x 107 of C. albicans, e. coli and C. sporogenes. inoculated tissue was refrigerated for a minimum of 15 hours in transport solution containing antibiotic and antimycotic (to simulate shipping conditions) and then processed to a single cell suspension and immunopurified to isolate grPs. the bioburden count of each microorganism was reduced by 4-5 logs during refrigeration and by an additional 2-3 logs after tissue dissociation and grP purification, resulting in negligible CFu detection after the entire process. these data describe a methodological approach to successfully reduce the bioburden of incoming tissues by 4-8 logs, allowing subsequent cell culture and expansion under asceptic conditions.

12FACtORS AFFECtInG thE MICROBIAL COntAMInAtIOn RAtE OF CORd BLOOd COLLECtEd FOR tRAnSPLAntAtIOn At thE SydnEy CORd BLOOd BAnK (SCBB)P. Clark1,2, A. Trickett3,2, D. Stark4, M. Vowels1; 1Sydney Cord Blood Bank, Randwick, NSW, AUSTRALIA, 2University of New South Wales, Sydney, NSW, AUSTRALIA, 3BMT Network NSW, Darlinghurst, NSW, AUSTRALIA, 4Microbiology, SydPath, Darlinghurst, NSW, AUSTRALIA.

Collection and processing of cord blood (Cb) is associated with significant risk of exposure to microbial contamination and hence all relevant standards mandate microbial screening of the final product. this study aimed to determine the contamination rate and associated risk factors during 14 years of banking at the sCbb.Cb was collected using a validated closed system technique. Cb units were tested for contamination using bact/alert blood culture bottles (biomerieux). Four microbial screening methodologies were used with different combinations of inoculated bottles (adult vs paediatric) and associated sample volumes (10ml vs 1ml). all bottles were incubated for a minimum of 5 days.of 13,344 Cb screened, 537 (4.0%) tested positive for contamination, with bacteroides sp. (21.5%), staphylococcus sp. (20.6%), and Propionibacterium sp. (14.2%) the most common organisms isolated. the contamination rate reduced from 10% in 1997 to the current rate of 1.1% (2009).univariate analysis demonstrated that the following were associated with higher contamination rates: vaginal deliveries (5.4%) compared to caesarean (1.1%); Cb collection by obstetric staff (4.8%) compared to sCbb staff (3.5%); and cleaning the cord with 70% isopropanol (4.4%) compared to chlorhexidine-cetrimide (1.3%). in addition, the detection rate was influenced by the screening methodology: inoculation of 10ml of the plasma waste fraction into adult aerobic and anaerobic culture bottles yielded a detection rate of 5.6%, reducing to 1.5% when only 1ml of the final product was inoculated into a paediatric bottle. these factors were also shown to be significant for higher contamination rates in multivariate logistic regression analysis.this study demonstrates that microbial contamination rates of Cb collected for transplantation can be substantially reduced by use of effective antiseptics and by utilizing trained Cb collection staff. this data also indicates that testing only a sample of the final product may be suboptimal for sensitive detection of contaminating microbes.


Oral Abstracts

13SOLuBLE FACtORS RELEASEd By huMAn MESEnChyMAL StEM CELLS OF FEtAL ORIGIn LEAd tO CARdIOMyOCytE PROtECtIOn thROuGh thE InhIBItIOn OF PRO-APOPtOtIC SIGnALInGP. Danieli1, E. Cervio1, M. C. Ciuffreda1, F. Pisano1, M. Roccio2, M. Gnecchi1,3; 1Cardiology, Fondazione IRCCS Policlinico San Matteo, Pavia, ITALY, 2Gynecology, Fondazione IRCCS Policlinico San Matteo, Pavia, ITALY, 3University of Pavia, Pavia, ITALY.

background: adult mesenchymal stem cells (msC) repair infarcted hearts mainly through cytoprotective paracrine mechanisms. recently, msC of fetal origin have been isolated from human amniotic membrane (a-msC) but it is unknown if a-msC can mediate cytoprotection and which pathways are eventually involved.methods: a-msC were isolated from human term placenta. rat neonatal cardiomyocytes (H9c2) were exposed to 6 hours of hypoxia followed by 18 hours of reoxygenation in the presence of control medium (Ctrl-m) or conditioned medium from a-msC (a-msC-Cm). H9c2 viability was evaluated by mts assay. apoptosis was measured by tunel staining and by Caspase 3 activation (colorimetric assay and Western blot). saPK/JnK and p38 maPK activation was analyzed by Western blotting. We used rt-PCr to evaluate pro- and anti-apoptotic genes in H9c2 cells and known cytoprotective factors in a-msC.results: Compared with Ctrl-m, a-msC-Cm remarkably increased viability of H9c2 by 45% (p<0.001) and significantly reduced the number of tunel positive nuclei by 91% (p<0.001). Furthermore, both colorimetric and Western blot assay showed that a-msC-Cm prevented Caspase 3 cleavege (p= n.s. vs normoxia). the hypoxia/reoxygenation induced a marked activation of saPK/JnK and p38 maPK that was strongly limited by a-msC-Cm. Furthermore, the a-msC-Cm increased the expression of bcl-2 and stat3 and inhibited transcription of tnF-β and Fasl compared with Ctrl-m. Finally, we documented that a-msC express cytoprotective factors such as PDgF-β , bmP2, ePo, igF-1, FgF2 and VegF.Conclusions: We demonstrated that a-msC express several cytoprotective factors and that a-msC-Cm remarkably protects cardiac myocytes against hypoxia/reoxygenation damage, through the inhibition of saPK/JnK and p38 maPK pro-apoptotic pathways. a-msC-Cm also mediates the over-expression of anti-apoptotic genes bcl-2 and stat3 and the downregulation of pro-apoptotic factors tnF-β and Fasl. a-msC theraphy may represent a novel and powerful approach for cardioprotection in ischemic heart disease.

14

15IMMunOLOGICAL PROFILE OF CRItICAL LIMB ISChEMIA PAtIEntS FOLLOWInG IntRAMuSCuLAR AdMInIStRAtIOn OF PLACEntA AdhEREnt StROMAL CELLSF. Grynspan1, C. Mazouz1, E. M. Horwitz2, N. Netzer1, J. A. Schmidt-Luke3, P. Reinke4, H. Volk4; 1Pluristem Therapeutics Inc, Haifa, ISRAEL, 2The Children Hospital of Philadelphia and The University of Pennsylvania School of Medicine, Philadelphia, PA, 3Franziskus Hospital, Berlin, GERMANY, 4Charité - Universitaetsmedizin Berlin and The Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Berlin, GERMANY.

Cell therapy for critical limb ischemia (Cli) offers an opportunity for patients that have exhausted all currently available interventions. Pluristem therapeutics has initiated two Phase i dose escalation clinical trials, performed in parallel in the usa and germany, utilizing placenta adherent stromal cells (PlX-PaD). Five dosing groups are being evaluated reaching a maximum dose of 560x106 cells. the cells are injected intramuscularly (im) in a single session for the low dose group, and in two sessions two weeks apart for the high dose. Due to the allogeneic source of the PlX-PaD cells, it is imperative to evaluate each patient for both humoral and cellular responses to the allogeneic Hla type. We further test for different criteria of immunosuppression of lymphocytes and monocytes, and the level of pro and anti-inflammatory cytokines in the blood following ex-vivo stimulation of PbmCs. in addition, our patients are tested for endothelial cell activation and for the level of the immune responses to latent pathogens. Preliminary results of nine patients, reported no significant adverse effects related to PlX-PaD administration. three of the patients completed their three-month follow up and the data demonstrated a trend towards efficacy with a reduction in their rutherford Category, a measure of the severity of their limb ischemia. the immunological tests for the low and intermediate doses will be presented alongside correlations with parameters accumulated from the different placentas used for the production of the PlX-PaD batches used in the studies. our data is the first extensive immunological evaluation of any mesenchymal-like cell therapy in clinical trials of Cli. moreover, such data will have broad implications for mesenchymal-like cell therapy in other disorders.

thE CARdIOPROtECtIVE PARACRInE EFFECtS EXERtEd By huMAn MESEnChyMAL StEM CELLS ARE nEGAtIVELy InFLuEnCEd By dOnOR AGEP. Danieli1, E. Cervio1, M. C. Ciuffreda1, F. Pisano1, M. Roccio2, M. Gnecchi1,3; 1Cardiology, Fondazione IRCCS Policlinico San Matteo, Pavia, ITALY, 2Gynecology, Fondazione IRCCS Policlinico San Matteo, Pavia, ITALY, 3University of Pavia, Pavia, ITALY.

background: mesenchymal stem cells (msC) repair infarcted hearts mainly through the release of cytoprotective factors. We compared fetal msC (F-msC) with adult msC from young and elderly patients to establish if donor age influences their cardioprotective paracrine properties.methods: F-msC were isolated from human placenta while adult msC were isolated from bone marrow samples of young (ybm-msC; age < 65 years) or old (obm-msC; age > 65 ) donors. rat neonatal cardiomyocytes (H9c2) were exposed to hypoxia/reoxygenation (6/18 hours) in the presence of control medium (Ctrl-m) or conditioned medium from F-msC (F-Cm), ybm-msC (y-Cm) or obm-msC (o-Cm). H9c2 viability was evaluated by mts assay. apoptosis was measured by tunel staining and by Caspase 3 activation (colorimetric assay and Western blot). We used rt-PCr to verify the expression of known cytoprotective factors.results: the hypoxia/reoxygenation protocol reduced H9c2 viability by 55% compared with basal condition (p<0.001). F-Cm and y-Cm increased cell viability by 45% and 33% (p<0.017), respectively; o-Cm had no significant effect on H9c2 viability (p=n.s. vs Ctrl-m). Compared with Ctrl-m and o-Cm, F-Cm significantly reduced the number of tunel positive nuclei by 91% (p<0.001) and 89% (p<0.001), respectively. the y-Cm also reduced H9c2 apoptotic nuclei (- 67,5% vs Ctrl-m, p<0.01; - 64% vs o-Cm, p<0.01). in contrast, o-Cm did not prevent H9c2 apoptotic death (-11% vs Ctrl-m, p=n.s.). both colorimetric assay and Western blot analysis showed that Caspase-3 activation was prevented by F-Cm and y-Cm but not by o-Cm. F-msC expressed PDgF-β, bmP2, ePo, FgF2 and VegF at significantly higher level compared with obm-msC (p<0.05); VegF, FgF2 and HgF transcripts were significantly higher in ybm-msC than in obm-msC (p<0.05).Conclusions: msC can mediate cardiomyocyte protection through the release of soluble anti-apoptotic factors. However, we documented that donor age negatively influences the paracrine cytoprotective properties of adult msC.

16tRAnSPLAntAtIOn OF MESEnChyMAL StROMAL CELLS (MSCs) InduCES LInEAR BOnE GROWth By StIMuLAtInG GROWth PLAtE ChOndROCytE PROLIFERAtIOnS. Otsuru1, K. Shimono2, T. J. Hofmann1, M. Iwamoto2, E. M. Horwitz1; 1The Children’s Hospital of Philadelphia, Philadelphia, PA, 2Thomas Jefferson University, Philadelphia, PA.

mesenchymal stromal cells (msCs) are one of the most rapidly emerging cell therapies. We reported the first clinical trial of allogeneic msC infusions in a study of msC therapy for children with osteogenesis imperfecta (oi), a genetic disorder of collagen type i. the children showed an acute acceleration of growth following the cell therapy. Despite the striking clinical outcome, very low levels of donor msCs were detected in the patients’ bone suggesting that the mechanism underlying the clinical effect of msC therapy is unrelated to osteopoietic differentiation. We reasoned that since bone growth is initiated by chondrocytes in the growth plate (gP), msCs may be directly acting at gP. However, in our murine model, msCs do not engraft and differentiate to chondrocytes in the gP. moreover, freshly isolated primary chondrocytes co-cultured with msCs do not proliferate greater than controls suggesting that msCs do not secrete a soluble mediator that directly stimulates gP chondrocyte activity. We then considered that the growth may be the result of a complex metabolic pathway initiated by a msC-secreted soluble mediator. to test this hypothesis, we infused mice with msCs or buffer and obtained serum 7 days later. Chondrocytes assayed in media supplemented with serum taken from mice with msC infusion stimulated greater chondrocytes proliferation than controls (P<0.01). analysis of proliferating cell nuclear antigen (PCna) in the gP of mice infused with msCs revealed significantly more proliferating cells compared to controls (P=0.006). to determine whether our findings were applicable to oi, we transplanted msCs into oi mice. Four weeks after the transplantation, we observed a significantly increased lumbar vertebral length (P=0.0008) as well as total body weight (P=0.0025). our data indicate that the mechanism of msC-stimulated linear growth is the secretion of a soluble factor that initiates a metabolic pathway resulting in growth plate chondrocyte proliferation.


may 23-26, 2010


Oral Abstracts

17MICROEnVIROnMEnt OF WAR WOundS: COnCERnS FOR REGEnERAtIVE MEdICInE APPLICAtIOnSN. Porterfield, N. Crane, T. Davis, E. Elster, W. Stepp, D. Tadaki, T. Brown; Naval Medical Research Center, Silver Spring, MD.

mesenchymal stem cells (msC) have been used in many regeneration models and have been implicated as potentially useful in wound healing. studies have shown that msCs can regenerate muscle; however these models often involve sterile, surgical injuries. the traumatic extremity injuries seen in combat casualties are infected, have a large zone of injury, and also have a systemic component. We examined the micro-environment of these wounds if tools such as stem cell therapy are to be applied for the care of this patient population.in this study, operation iraqi Freedom (oiF) and operation enduring Freedom (oeF) patients with high-energy, penetrating acute wounds were monitored from arrival thorough 30 days post-closure. tissue biopsies were collected from within the wound bed during each debridement and effluent was collected before and after each debridement. rna was extracted from the tissue biopsies and the expression profile was quantitated using real time PCr for 190 inflammatory and wound healing genes. Wound effluent was analyzed for 22 different cytokines and chemokines using a multiplex bead-based assay.results:the environment of a wound plays a substantial role in the differentiation of a pluripotent msCs. the micro-environment of these wounds differed significantly between wounds that later developed heterotopic ossification (Ho), in which bone forms in soft tissue, and those that did not. as msCs have been a focus of developing stem cell therapies and are progenitors of osteoblasts, we investigated the influence of wound effluent on msC cultures. the result showed little to no mineralization (quantitative alizarin red staining) in the presence of non-Ho-forming effluent to gross mineralization in the presence of Ho-forming wound effluent. With these results in mind, caution should be taken in assessing candidates with traumatic acute wounds for stem cell therapies as a method of tissue regeneration.

19hIGhLy EFFICIEnt LABELInG OF hEMAtOPOIEtIC StEM/PROGEnItOR CELLS uSInG MAGnEtIC CARBOn nAnOtuBES: IMPLICAtIOnS FOR StEM CELL tRACKInGH. Gul-Uludag1,2, W. Lu2, P. Xu1, J. Chen1,3, J. Xing2,4; 1University of Alberta, Edmonton, AB, CANADA, 2IntelligentNano Inc., Edmonton, AB, CANADA, 3National Institute for Nanotechnology, Edmonton, AB, CANADA, 4Cross Cancer Institute, Edmonton, AB, CANADA.

background: labelling and dynamic tracking of hematopoietic stem/progenitor cells (HsPC) are critical to our understanding of their homing/engraftment into the bone marrow after transplantation, and therapeutic efficacy for future stem cell-based therapies. However, the methodology to real-time track HsPC in vivo is still lacking, which seriously restricts the optimal development of these cells for clinical use. recently, magnetic carbon nanotubes (mCnt) have received considerable interest due to their highly efficient magnetic-field driven biomolecule delivery into cells. nevertheless, the uptake or labelling efficiency of mCnt in HsPC has not been addressed or explored yet. Here, we investigated the uptake efficiency of fluorescein isothiocyanate-labelled mCnt (FitC-mCnt) into HsPC and their effect on HsPC cytotoxicity and differentiation. methods: CD34+ cells obtained from cord blood and leukepheresis product were exposed to the solutions of different concentrations of FitC-mCnt or mCnt alone (10, 20, 40 µg/ml) in the presence of magnetic field. the uptake efficiencies of these cells were determined using FaCs and confocal microscopy. moreover, HsPC viability after FitC-mCnt uptake was evaluated by trypan blue exclusion assay. to investigate their impact on differentiation of HsPC, a colony forming unit (CFu) assay of FitC-mCnt labelled HsPC was performed. results: We found that uptake of FitC-mCnt into HsPC reached up to 100% with the highest mean flourescence (mF). more importantly, efficient FitC-mCnt uptake has no adverse effect on the cell viability, cytotoxicity, and differentiation of HsPC, which was confirmed by CFu assay. Conclusions: the results reported here suggest the potential use of mCnt for in vivo tracking of HsPC or gene delivery into HsPC.

18LACK OF BEtA-ARREStIn-1 In BOnE MARROW StEM CELLS LEAdS tO IMPAIREd OutCOME AFtER MyOCARdIAL InJuRy duE tO dEFECtIVE PROLIFERAtIVE ACtIVItyM. Chen, H. Brinks, E. Gao, X. Shang, K. Peppel, P. Most, A. D. Eckhart, W. J. Koch; Thomas Jefferson University, Philadelphia, PA.

background: bone marrow (bm) hematopoietic stem cells (HsCs) show potential to transdifferentiate and regenerate myocardium after myocardial infarction (mi). HsC mobilization, egress from the bm and homing to the site of ischemic injury can be regulated by signals through g protein-coupled receptors (gPCrs). β-arrestin-1 (βarr1) is downstream regulator of gPCr desensitization and endocytosis that can initiate novel signaling pathways due to scaffolding capabilities. We hypothesized that βarr1 might modulate the regenerative capacity of HsCs and tested this in a mi model.methods: Whole bm mononuclear cell (bmnC) content and the population of HsCs assessed at baseline and in response to mi were studied in mice lacking βarr1 (βarr1-Ko) using flow cytometry for lin-, ckit+ and sca-1+ cells. the proliferative capacity of these cells was tested in a colony-forming unit assay. to assess regenerative capacity of these cells wild-type (Wt) mice were irradiated and subsequently reconstituted with control (Wt) or βarr1-Ko bm. reconstitution of the bm in chimeric animals was tested at 2 and 4 weeks. mice were then subjected to mi via coronary artery ligation and followed up for 15 weeks with in vivo echocardiographic and invasive hemodynamic measurements of cardiac function.results: Proliferation of βarr1-Ko HsCs was markedly reduced compared to control (45±16% of Wt). ckit/sca-1 positive cells was significantly decreased in chimeric mice 3 days after mi (βarr1-Ko 0.26±0.10% vs Wt 0.65±0.18%), but was not different at baseline (0.2±0.05% vs 0.24±0.07%). Functional assessment showed lower ejection fraction in βarr1-Ko chimeric mice (26±2.3%) 4 weeks after mi compared to control mice (34±3.4%). Hemodynamic measurements paralleled these findings.Conclusion: βarr1 appears to be a critical molecule for normal HsC proliferation and myocardial homing presumably via abnormal modulation of gPCr signals. importantly, the lack of βarr1 in bm leads to impaired functional recovery after mi.

20VIABILIty OF COLLECtEd Cd34+ CELLS FOLLOWInG FREEZInG And thAWInG dIMInIShES WIth InCREASInG PERIOd OF G-CSF MOBILIZAtIOnW. E. Janssen, U. Premraj, W. Bauer, R. C. Smilee, A. Ribickas; Moffitt Cancer Center, Tampa, FL.

the most common source of hematopoietic progenitor cells (HPC) for transplantation is from circulating blood, even though the primary residence for such cells in adult humans is in the bone marrow. it has been hypothesized that, in the unmobilized individual, the exit of HPC from marrow to blood is a disposal pathway wherein the outward migrating cells cannot return and must enter apoptosis. Whether this might be true of HPC exiting the marrow under mobilizing conditions has not been explored. We hypothesized that if exit from the marrow might trigger eventual cell death, even under mobilizing conditions, then we would expect to see reduced ability of HPC to survive cryopreservation and thawing with increased time under mobilizing conditions, namely days of g-CsF administration. in our facility, as a quality/safety test prior to releasing cryopreserved HPC products for use, we thaw a sample of each frozen product and determine thawed CD34+ cell viability by 7aaD exclusion. We have reviewed thawed CD34+ cell viability from 590 products, and analyzed as a function of the number of days of g-CsF administration at the time of cell collection. We have compared the apparent declining CD34+ viability over the days observed using analysis of variance, and have found the recoveries to be significantly different (p<0.0001). these results are consistent with the hypothesis that circulating HPC may be on a deletion pathway. the results also suggest that all effort should be made to collect HPC following the least number of days of g-CsF exposure.


Oral Abstracts

21ChARACtERIStICS OF Cd34-EnRIChEd PROduCtS PROCESSEd At MuLtIPLE CEntERS uSInG thE CLInIMACS SyStEM - BMt Ctn 0303C. Keever-Taylor1, S. M. Devine2, R. J. Soiffer3, S. L. Carter4, M. Pasquini1, P. Hari1, A. Stein5, H. M. Lazarus6, C. Linker7, S. Goldstein8, R. J. O’Reilly9; 1Medical College of Wisconsin, Milwaukee, WI, 2Ohio State University, Columbus, OH, 3Dana Farber Cancer Institute, Boston, MA, 4The EMMES Corporation, Rockville, MD, 5City of Hope, Duarte, CA, 6Case Western Reserve University, Cleveland, OH, 7University of California - San Francisco, San Francisco, CA, 8University of Pennsylvania, Philadelphia, PA, 9Memorial Sloan-Kettering Cancer Center, New York, NY.

eight centers participated in the nHlbi-sponsored blood and marrow transplant Clinical trials network (bmt Ctn) 0303 study to determine the effect of extensive t-cell depletion (tCD) on the outcome of Hla identical transplant for acute myeloid leukemia. tCD used the ClinimaCs system for CD34-enrichment. grafts needed to contain ≥2.0e6 CD34+-cells/kg with a target of 5.0e6 CD34+-cells/kg and <1.0e5 CD3+ t-cells/kg. up to 3 collections were allowed to achieve the CD34+-cell dose. eighty-six products were processed for 44 patients. three products were processed for 4 patients, two for 34 patients, and one product for 6 patients. all patients received the minimum CD34+ cell dose and 38 (86%) exceeded the target dose. Patients received a median of 8.1e6 CD34+-cells/kg (2.4e6-3.1e7) containing a median of 6.7e3 CD3+-cells/kg (1.1e3-8.5e4). a median of 7.0e10 (2.1e10-1.6e11) tnC were processed for each enrichment procedure. multivariate analysis showed that CD34+ cell recovery (65.6%±20.7%) and log tCD (4.8±0.5) were not associated with initial tnC and log tCD was not correlated with initial CD3+-cell content (1.8e10±1.0e10). However, there was an inverse correlation between CD34+-recovery and initial absolute CD34+-cells (p=0.005). Centers performed 2-34 CD34-enrichment procedures. the most common contaminants were monocytes (median 0.3%, 0-22%), b-cells (median 0.3%, 0-7.4%) and nK-cells (median: 0.03%, 0-4.7%). Data from 4 centers performing ≤4 procedures were pooled for comparison with centers performing 9-34 procedures for log tCD, CD34+-cell recovery, and CD34+-cell purity. Differences in log tCD were significant (p=0.007) but represented only a 0.6 log difference between the lowest (4.6) and the greatest (5.2) mean tCD. CD34+-recovery (53.6%-83.9%, p=<0.001) and purity (86.6%-98.3%, p=0.02) also differed between centers. in summary, although differences were seen products processed using the ClinimaCs CD34-enrichment were uniformly highly enriched for CD34+ cells, with good CD34+ cell recovery and very low t-cell content.

23IntRAMyOCARdIAL dELIVERy OF ACRX-100 EnhAnCES CARdIAC FunCtIOn And InCREASES VASCuLOGEnESIS In A PORCInE MOdEL OF ChROnIC hEARt FAILuREJ. Pastore1, T. J. Miller1, R. Aras1, M. S. Penn2; 1Juventas Therapeutics, Cleveland, OH, 2Cleveland Clinic, Cleveland, OH.

aCrX-100 comprises a non-viral Dna plasmid engineered to transiently express human stromal-cell Derived Factor 1 (sDF-1). sDF-1 is a strong chemoattractant of stem and progenitor cells to promote tissue preservation and blood vessel development in damaged tissue. a 57-pig safety and efficacy study evaluated cardiac functional response, toxicity and bio-distribution in a porcine model of heart failure after treatment with escalating doses of aCrX-100. all enrolled pigs had a left ventricular ejection fraction (lVeF) <40% and a left ventricular end systolic volume (lVesV) >56.7 ml 30 days post-infarct, when they received intra-myocardial injections of saline (control), or aCrX-100 at doses of 7.5 mg (low), 30 mg (mid) or 100 mg (high). at 60 days post-injection, aCrX-100 improved lVeF and lVesV at low and mid doses. aCrX-100 promoted vasculogenesis at all doses relative to controls, with significant increases in the low and mid doses 30 days post-injection. importantly, increased vessel density (>200 vessels/field) correlated with improved cardiac function. the difference in change in lVesV between vessel density groups (<200 vessels/field: 5.9±11.5 ml vs. >200 vessels/field: -8.6±13.4 ml-) was statistically significant (p<0.05) at 30 days post-injection, and the difference in change in lVeF (<200 vessels/field: -0.9±10.2% vs. >200 vessels/field: 7.5±5.5%) approached statistical significance (p=0.052). no aCrX-100 dose was associated with signs of toxicity, adverse effects on clinical pathology or histopathology. aCrX-100 distributed primarily to the heart and was essentially cleared from all organs within 90-days of treatment. these findings indicate that the no observable adverse effect level (noael) for aCrX-100 in the pig model of ischemic heart failure was 100 mg. based on these results, the FDa has allowed an inD to initiate an open label, 16 subject Phase 1 dose-escalation study to evaluate the safety of aCrX-100 for treating subjects with ischemic cardiomyopathy (nyHa Class iii with prior myocardial infarction).

22APOPtOSIS In CRyOPRESERVEd AutOLOGOuS PBSCS And dELAyEd EnGRAFtMEnt AFtER tRAnSPLAntAtIOn dESPItE SuFFICIEnt Cd34(+) CELLSL. Wu1, A. Al-Hejazi1, L. Filion1, R. Ben1, M. Halpenny2, A. Giulivi2, D. Allan1; 1University of Ottawa, Ottawa, ON, CANADA, 2Canadian Blood Services, Ottawa, ON, CANADA.

background: Delayed hematopoietic engraftment following autologous peripheral blood stem cell (PbsC) transplantation remains a risk despite adequate CD34(+) cell yields. loss of cell viability during cryopreservation and thawing contributes to reduced CD34(+) cells available for reinfusion although early induction of apoptosis in viable cells has not been fully explored as a potential factor.methods: 100 patients with lymphoma underwent autologous PbsC transplantation between 2001 - 2007. Fourteen patients (group a) had delayed neutrophil recovery (absolute neutrophils < 1.0x109/l) at day 30. an additional 28 age and sex-matched controls were selected (Controls). stored aliquots of cryopreserved PbsCs were analyzed by flow cytometry for total mononuclear and CD34(+) cell viability (7aaD exclusion) and induction of apoptosis (annexin-V positive). aldehyde-dehydrogenase expressing cells were also enumerated.results: group a and Controls were similar including gender, age, and CD34(+) cells in PbsC grafts (3.7 vs 4.3 x 106/kg, p=0.23) but group a patients had more prolonged days to neutrophil engraftment > 0.5x109/l (17.6 vs 12.0, p=0.0005) and platelet recovery > 20x109/l (30.1 vs 13.9, p<0.0001). the proportion of viable 7aaD(-) total mononuclear cells (mnCs) in group a was not different from Controls (90±1.0% vs 93±0.9%, p=0.06), however, patients in group a had more apoptotic total mnCs (7aaD-negannVpos) compared with Controls (56±4.2% vs. 37±4.8%, p=0.012). the viability of CD34(+) cells was not different between groups (84±3.1% vs. 85±3.0%, p=0.91) although a trend towards more apoptotic CD34(+) cells in group a was observed (22±3.7% vs. 14±1.8%, p=0.067). the proportion of CD34(+) cells that were alDHhi was not different between groups.Conclusion: early apoptosis in mnCs caused by cryopreservation and thawing may contribute to delayed hematopoietic recovery following autologous PbsC transplantation. our data suggest an important accessory role of viable non-apoptotic mnCs and underscore the need to develop cryoprotectants that reduce cellular injury associated with freezing and thawing.

24dECAdE-LOnG PERSIStEnCE OF AdOPtIVELy tRAnSFERREd AutOLOGOuS REdIRECtEd Cd4-ζ ChIMERIC RECEPtOR t CELLS In hIV+ Study SuBJECtSJ. Scholler1, N. Aronson2, G. Plesa1, R. G. Carroll1, G. Binder-Scholl1, K. Hege3, S. G. Deeks4, R. T. Mitsuyasu5, A. Vogel6, Z. Zheng1, J. Cotte6, W. B. Bernstein7, B. L. Levine6, C. H. June6; 1Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, 2Walter Reed Army Medical Center, Washington, DC, 3Cell Genesys, South San Francisco, CA, 4University of California San Francisco, San Francisco, CA, 5University of California-Los Angeles, Los Angeles, CA, 6Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, 7National Cancer Institute, Bethesda, MD.

the ability of adoptively transferred cells to persist long term and function in a homologous or enhanced fashion is a central goal of immunotherapy. to test the hypothesis that central memory t cells have long term persistence, we measured the presence of genetically modified t cells in patients with HiV in three trials evaluating adoptively transferred CD4/8 t cells transduced with the CD4-β chimeric receptor over time. We developed a quantitative PCr (qPCr) assay to measure the persistence of adoptively transferred CD4/8 t lymphocytes transduced with a chimeric receptor consisting of extracellular CD4 linked to intracellular CD3-β (“CD4-β” chimeric receptor). PbmC’s from a series of clinical trials in which HiV+ study subjects received autologous CD4-β gene modified t cells from up to 11 years post-infusion were available for this long term persistence analysis. the data from three clinical trials demonstrated that 37 of 39 subjects continue to have CD4-β modified cells detectable in PbmC, up to 11 years post infusion. While the majority of patients had an average CD4-β copy level in their PbmC populations of 0.01 to 0.1%, a considerable proportion had levels exceeding 0.1% and at relatively stable engraftment levels over time. rt-PCr analysis indicates that the CD4-β gene is transcriptionally active. the efficacy of the CD4-β modified t cells alone on HiV patient’s latent reservoirs was assessed by the level of coculture replication-competent HiV. at 8 weeks post-first infusion, the mean change in infectious virus declined from baseline by -0.34 log in one study and by -0.29 log in a second study. Functional studies of in vivo persisting CD4-β -modified cells may have positive implications for redirected t cell therapy. the data suggests that adoptively transferred CD4-β modified central memory cells can have active long term persistence of greater than 11 years.


may 23-26, 2010


Oral Abstracts

25PROLOnGEd COntROL OF VIREMIA AFtER tRAnSFER OF AutOLOGOuS Cd4 t CELLS GEnEtICALLy MOdIFIEd WIth A LEntIVIRAL VECtOR EXPRESSInG LOnG AntISEnSE tO hIV EnV (VRX496)P. Tebas1, D. Stein2, G. Binder-Scholl1, L. Zifchak1, A. Seda2, J. Cotte1, Z. Zheng1, A. Brennan1, A. Mackley1, D. Schullery1, J. Boyer1, T. Mikheeva1, F. Shaheen1, R. Mukherjee1, T. Brady1, F. Aberra1, M. Kalos1, F. Bushman1, R. Collman1, T. Rebello3, L. Humeau3, R. Cohen3, G. McGarrity3, B. Levine1, C. June1; 1University of Pennsylvania, Philadelphia, PA, 2Jacobi Medical Center, Bronx, NY, 3VIRxSYS Corporation, Gaithersburg, MD.

We are evaluating autologous CD4 lymphocytes genetically modified with a lentiviral vector (VrX496) expressing antisense targeting HiV env for the control of HiV infection and disease progression. in an ongoing phase i/ii trial, 17 HiV-1-infected subjects who were fully suppressed on Haart received, over a period up to 13 weeks, 3 or 6 infusions each of 1010 autologous CD4 t cells transduced with VrX496 and expanded ex vivo. six weeks after the last infusion, eligible subjects underwent a scheduled treatment interruption (sti) to evaluate effects on HiV rna viral load by viral load setpoint and CD4 cell count. subjects are monitored for persistence of vector modified cells, integration sites profiles between the cell product and over time in vivo, immunogenicity of the cell product, and effects on CD4 counts and viral load during treatment interruption. all subjects received infusions. CD4 counts were elevated during infusions, and remained stable around baseline levels after treatment interruption. integration site analysis to date has shown no clonal selection or preference for oncogenes. approximately 50% of subjects developed antibodies to the vector envelope protein VsV-g, which had no effect of persistence. 76% of subjects (13/17) underwent sti; 62% (8/13) of subjects who underwent sti had a historic viral load setpoint and were evaluable for the efficacy endpoint. 88% (7/8) of evaluable subjects had a decrease in setpoint (range log10 -0.26 to -0.98), which is significant compared to historical data on treatment interruption alone (p=0.035). in two subjects viral recrudescence was delayed 1 and 4 months over controls. this is the first report of prolonged control of HiV viremia during sti after the infusion of genetically modified t cells. We are evaluating genetic and immunologic factors as well as VrX496-driven sequence variations in HiV env, to explain the effects on viremia in these subjects.

27A nOVEL SCORInG SyStEM, thE CORd BLOOd APGAR, PREdICtS EnGRAFtMEnt AFtER CORd BLOOd tRAnSPLAntAtIOn: OPtIMIZAtIOn OF SELECtIOn OF CBuS FOR tRAnSPLAntAtIOnK. Page1, L. Zhang2, A. Mendizabal2, A. E. Balber3, T. Gentry3, J. Kurtzberg1; 1Duke University Medical Center, Durham, NC, 2The Emmes Corporation, Rockville, MD, 3Aldagen, Durham, NC.

background: using current graft selection criteria, primary graft failure and engraftment delays are major obstacles to the overall success of unrelated donor umbilical cord blood (Cb) transplantation (Cbt). up to 20% of patients receiving a Cbt experience primary graft failure resulting in part from inadequate potency of their donor cord blood unit (Cbu). in this single center retrospective study, we describe correlations of graft parameters with engraftment and present a novel scoring system, the Cord blood apgar (Cba) to optimize graft selection for Cbt.methods: the Cba was developed utilizing a database created from 435 consecutive single cord myeloablative transplants performed at Duke between 1/1/2000 and 12/31/2008. Correlations of pre-cryopreservation (pre-cryo) and post-thaw graft parameters (tnC, CD34, CFu, volume and mnC) with time to engraftment were analyzed. based on univariate analysis and hazard ratios, a weighted scoring system was developed and internally validated. transplanted Cbus were assigned two scores: a Pre-cryo score (PCs) using pre-cryo characteristics alone and a Composite score (Cs) based on combined pre-cryo and post-thaw graft characteristics.results: the overall cumulative incidences of neutrophil (day +42) and platelet (day +180) engraftment were 76.9% and 55%, respectively. Cba-PCs scores, which could be used for initial unit selection, were predictive of neutrophil (Cba-PCs≥7.75 vs. <7.75: Hr=3.5, p<0.0001) and platelet engraftment (Cba-PCs≥7.75 vs. <7.75: Hr=1.8, p=0.003). likewise, Cba-Cs scores, which could be determined using post-thaw parameters measured on an attached segment before unit release, were strongly predictive of neutrophil (Cba-Cs≥17.75 vs. <17.75: Hr=4.01, p<0.0001) and platelet engraftment (Cba-Cs≥17.75 vs. <17.75: Hr=1.74, p=0.006). the scoring was internally validated using test and training databases developed from the overall database.Conclusions: the Cba is strongly predictive of engraftment after Cbt and can be utilized for donor selection for search and release of Cbus. this should be validated in a prospective multi-institutional study.

26L-ASPARAGInASE LOAdEd InSIdE REd BLOOd CELLS AS A nEW CELL BASEd MEdICInAL PROduCtJ. BAILLY1, Y. BERTRAND2, Y. GODFRIN1; 1ERYtech Pharma, LYON, FRANCE, 2Institut d’Hématologie et Oncologie Pédiatrique and Université Claude Bernard, LYON, FRANCE.

l-asparaginase has been a mainstay of acute lymphoblastic leukemia (all) treatment since decades and its efficacy has been demonstrated in a broad range of patient’s profiles. However its use has been hampered by frequent and/or significant toxicities.l-asparaginase loaded red blood cells (grasPa®) extends the duration of action of the enzyme while reducing the side effects. thus, the red blood cell (rbC) acts as a microbioreactor, plasmatic asparagine diffuses through the rbC membrane to the intra cellular compartment where it is cleaved by the entrapped l-asparaginase. in addition encapsulation into rbC protects the enzyme of a binding by the anti-asparaginase antibodies.We developed an automatised, reproductible and safety process for manufacturing this red cell based medicinal product within 3 hours.First, the packed red cells are washed to remove the residual plasma and the preservative solution. then, l-asparaginase is mixed with the washed rbC and dialyzed against a hypotonic solution. at this step, the red cells swell, pores appears on the surface of their membrane and l-asparaginase enters inside the red cells. Dialysis parameters are adjusted in regard with erythrocytes osmotic fragility. after, a resealing solution is added “on line” to recover isotonicity and to definitively close the pores: l-asparaginase is entrapped.Cell medicinal product is controlled and released in accordance with biochemical and hematological specifications.the advantages of grasPa®, a red cell based medicinal product, were shown in a phase ii clinical trial. this is an effective therapy for depleting serum asparagines for 18 days with one single infusion of 150 iu/kg and with a much better safety profile.

28QuAntItAtIVE EX VIVO ChARACtERIZAtIOn OF huMAn REnAL CELL POPuLAtIOn dynAMICS VIA hIGh-COntEnt IMAGE-BASEd AnALySIS (hCA)S. M. Wallace, A. T. Bruce, S. Choudhury, P. Tatsumi-Ficht, B. R. Cox, B. J. Wagner, R. Kelley, S. C. Presnell; Tengion Inc, Winston-Salem, NC.

autologous cells can be harnessed and deployed as key components of products that functionally augment or replace degenerating organs and tissues. thorough ex vivo characterization of cellular component(s) is essential for establishing defined prototypes for in vivo evaluation. High-content image-based analysis (HCa) provides robust characterization of heterogeneous cell populations, yet quantitates phenotypic and functional data on a cell-by-cell basis, yielding insights into the relationships between cellular composition of selected prototypes and in vivo therapeutic outcomes.the present study used HCa and fluorescence-based assays to assess the albumin-transport functionality of specific tubular cell subpopulations in heterogeneous primary cultures of human renal cells established from 22 donors with (n=5) or without (n=17) chronic kidney disease (CKD) and compare the relative distribution of specific tubular cell phenotypes and functionality between CKD and non-CKD specimens.HCa analysis revealed that the cellular composition of renal cultures established from CKD and non-CKD donors was similar, representing tubular, glomerular, ductular, endocrine, and vascular compartments. assessment of protein transport function via receptor-mediated albumin uptake illustrated that cells possessing this function comprised a significant and similar proportion of CKD (40-80%) and non-CKD (40-80%) primary cultures. serial passage of these heterogeneous cultures under standard media and culture conditions resulted in a gradual loss of the protein-transport activity in both CKD and non-CKD cultures, but the transport-positive population was preferentially retained in CKD-derived cultures.in summary, HCa provided an accurate and meaningful comparative assessment of renal cell phenotype and function, yielding quantitative cellular-level data and revealing cell population dynamics that may be unable to be detected by other population-based assays (i.e., gene/protein expression). the synergy between advancements in image collection automation and evolution of more sophisticated analysis software packages has made application of HCa feasible in an industrial r&D environment, enabling detection and tracking of cell subpopulations throughout various processing steps.


Poster Abstracts

29dEMEthyLAtIOn OF thE FOXP3 LOCuS IS KEy tO PREdICtInG And MOduLAtInG SuPPRESSIVE FunCtIOn OF huMAn tREGST. Akimova1, T. Golovina1, T. Mikheeva2, L. Wang1, J. L. Riley2, W. W. Hancock3; 1CHOP, Philadelphia, PA, 2UPenn, Philadelphia, PA, 3CHOP/UPenn, Philadelphia, PA.

the ability of Foxp3+ t regulatory (treg) cells to suppress immune responses may provide a potential new therapy in autoimmunity and transplantation. However, their low frequency requires ex vivo expansion and there is a lack of reliable markers of human tregs. Demethylation of Cpg-rich islands in the first intron of FoXP3 appears unique to tregs versus other cells, but the association of FoXP3 demethylation in freshly isolated or expanded tregs with their suppressive function is unclear.using in vitro suppression assay, we studied three types of human tregs: magnetic beads isolated (4 donors, mean 77% FoXP3+), freshly FasC-sorted (4 donors, mean 94% FoXP3+) and FasC-sorted, then ex-vivo expanded tregs (4 donors, mean 89% FoXP3+), with autologous and allogeneic responder t-cells. FoXP3 was analyzed by methylation-sensitive PCr. We also tested the effects of three Dna methyltransferase inhibitors (Dnmti), Decitabine, Zebularine and rg108, in 22 suppression assays with magnetic beads isolated (9 donors) or expanded (4 donors) tregs.suppression by human tregs was directly correlated with % of unmethylated Cpg at the FoXP3 locus (r=0.593, p=0.033) and inversely correlated with % of methylated Cpg (r=-0.642, p=0.018). the methylation status of tregs did not correlate with FoXP3+ cell purity, but significantly correlated with FoXP3+ expression in tregs after suppression assays (r= -0.85, p=0.001), reflecting the direct relationship between the epigenetic status of tregs and the stability of FoXP3 expression. all tested Dnmti significantly enhanced treg suppression in vitro.We conclude that monitoring the extent of demethylation of Foxp3 within the first intron is the single best guide available to predict treg suppression, and is independent of the extent of Foxp3+ treg purity. moreover, treg function can be enhanced by Dnmti use, pointing to new ways to monitor and enhance human treg functions during clinical trials.

31EFFICIEnt GEnERAtIOn OF huMAn nK CELLS FROM CORd BLOOd Cd34+ hEMAtOPOIEtIC PROGEnItORSA. Bersenev1, B. Perussia1, K. S. Campbell2; 1Thomas Jefferson University, Philadelphia, PA, 2Fox Chase Cancer Center, Philadelphia, PA.

in this study we have established basic culture conditions to generate human natural killer (nK) cells from CD34+/CD45ra+/CD7+ hematopoietic progenitor cells (HPC) derived from umbilical cord blood. Differentiation to nK cells was efficiently driven within 1-2 weeks under stroma-free culture conditions, which included only Flt3l and il-2. the resulting cells expressed a diverse array of markers that are characteristic of immature and mature nK cells (CD56, CD161, CD94, nKg2D, Kir, and 2b4), but minimal expression of markers characteristic of dendritic cells (CD11c, CD1a) or t-cells (CD3). the nK cells were functionally mature, since they could be stimulated to generate iFn-gamma and demonstrated specific lysis of a target tumor cell line. therefore, our work has confirmed that cord blood CD34+/CD45ra+/CD7+ HPCs constitute a highly enriched nK progenitor population, and we have further established simple culture conditions to drive their differentiation to functionally mature nK cells.supported by niH r01 grant ai-055842.

30EnGInEEREd huMAn REGuLAtORy t CELLS EXPRESSInG LEntIVIRAL PdL1 undER CELL FAtE COntROL PREVEnt LEthAL XEnOGEnEIC GVhdS. Amarnath1, J. C. Wang2, J. L. Riley3, B. L. Levine3, C. H. June3, J. Medin2, D. H. Fowler1; 1National Cancer Institute, National Institute of Health, Bethesda, MD, 2IMS, University of Toronto, Toronto, ON, CANADA, 3UPENN, Philadelphia, PA.

Programmed death ligand-1 (PD-l1) on regulatory t cells (tregs) inhibits pathogenic effector t cells. We hypothesized that genetically-engineered human t cells forced to express PD-l1 would function as tregs to inhibit human-into-mouse xenogeneic gVHD. the approach we developed: (1) enforces stable PD-l1 expression; (2) utilizes t cell delivery vehicles that persist in vivo; and (3) incorporates an enhanced cDna for ‘cell fate control’. the recombinant lentiviral vector (lV) encodes cDna for a human CD19 and mutated tmPK fusion protein that activates aZt prodrug, and then encodes full-length human PD-l1. We manufactured human CD4+ t cells using co-stimulation, iFn-β, and rapamycin to generate anti-apoptotic th1 cells. after 6 days of culture and CD19-flow sorting, >90% of transduced t cells expressed PD-l1. the tmPK/aZt suicide axis was functional: after 72 h of in vitro aZt exposure, gene-transduced t cells had reduced viability (% viable: 17+2.5) relative to non-transduced cells (52+0.5). in the x-gVHD model, gene-modified t cell recipients had increased numbers of human t cells in the spleen that co-expressed CD19 and PD-l1 relative to non-transduced t cell recipients (p=0.02). a separate experiment involved cohorts that received: human th1 cells alone (C#1) or with PD-l1 lV-transduced t cells (C#2); purified human treg cells (C#3); or control lV-transduced t cells (C#4). on day 5, mice were challenged with lPs to induce x-gVHD. each cohort has similar human t cell engraftment. relative to C#1, both C#2 and C#3 had reduced numbers of iFn-g expressing human t cells (p<0.03) and reduced serum human tnF-a (p<0.04). recipients of tregs or PD-l1-transduced t cells had reduced lethality from x-gVHD (C#2< C#1, p=0.002; C#3< C#1, p=0.0001). thus, CD19/tmPK.PD-l1 lV yields stable and functional transgene expression, thereby permitting PD-l1 mediated immuno-gene therapy under cell fate control.

32dIFFEREntIAL CytOtOXIC RESPOnSE ELICItEd By uCB And PBMC AGAInSt OVARIAn CAnCER CELLSS. Blaydes Ingersoll, G. P. Stoltzfus, S. Ahmad, J. R. Edwards, N. J. Finkler, R. W. Holloway; Florida Hospital Cancer Institute, Orlando, FL.

ovarian cancer is the deadliest of the gynecologic neoplasms and while chemotherapy has impressive response rates initially, it has little impact on long-term survival. therefore, there is a compelling need to develop novel therapeutic regimes to treat this disease. We hypothesize that cellular therapy in combination with cytokines or chemotherapies may be a novel treatment option for refractory ovarian cancer. an in vitro model of immune cell-mediated cytotoxicity against ovarian cancer cells was established. red fluorescent protein (rFP) transfected ovarian cancer cells (sKoV3-rFP) were co-cultured in the presence of adult PbmC and il-2/iFnβ-2b either alone or in combination. PbmC in the presence of il-2/iFnβ-2b elicited up to 70% killing of sKoV3-rFP cells. because umbilical cord blood (uCb) cells are a well established source of immune cells for therapeutic interventions, we sought to investigate if mononuclear cells isolated from uCb could elicit a similar cytotoxic response. Cord blood mononuclear cells (Cbm) were isolated by density-gradient separation and washed for use in the assay. Cbm, in the presence of il-2 (25 ng/ml) and iFnβ-2b (500 u/ml) either alone or in combination, were co-cultured with sKoV3-rFP for 48 h. the Cbm cell doses tested were 20:1 and 10:1 for Cbm:sKoV3-rFP. the Cbm did not elicit a cytotoxic response against sKoV3-rFP cells (<10%; n=4) in any of the conditions tested. However, when adult PbmC are co-cultured with sKoV3-rFP cells under similar experimental conditions, the average cytotoxic response was 51% (n=6; p<0.01) for 20:1 and 35% (n=6; p<0.01) for 10:1 PbmC:sKoV3-rFP cells. these results show that the Cbm do not respond in the same manner as adult PbmC under these cytotoxicity assay conditions. studies are underway analyzing various cytokines and cell doses along with their concentrations and combinations to determine if Cbm are capable of eliciting a cytotoxic response against ovarian cancer cells.


may 23-26, 2010


Poster Abstracts

33ROBuSt And SuStAInEd EX VIVO EXPAnSIOn OF nK CELLS WIth ARtIFICIAL APCS BEARInG MEMBRAnE-BOund IL-21 IS ASSOCIAtEd WIth A dIStInCt PhEnOtyPE And tRAnSCRIPtIOnAL PROFILE.C. J. Denman, V. V. Senyukov, S. S. Somanchi, L. M. Kopp, M. Lima da Silva, H. Singh, L. Hurton, S. Olivares, S. Maiti, M. H. Huls, R. E. Champlin, L. J. Cooper, D. A. Lee; UT M. D. Anderson Cancer Center, Houston, TX.

introduction: nK cells have therapeutic potential for a wide variety of human malignancies. the major obstacle for adoptive nK cell immunotherapy is obtaining sufficient cell numbers, as these cells represent a small fraction of peripheral white blood cells, expand poorly ex vivo, and have limited life spans in vivo. to generate clinical-grade t cells for adoptive transfer, we developed K562-based aaPC expressing CD86, CD137l, CD64, CD19 membrane-bound il-21 (mil-21). Here we describe the expansion potential of nK cells from 18 donors including heavily-treated cancer patients, and describe transcriptional profiles associated with il-21 vs. il-15 signaling.methods: PbmC from 18 donors were expanded by weekly stimulation with K562 aaPC, and tested for receptor expression by flow cytometry, cytotoxicity using the calcein release assay, and 100 genes of interest by nCounter gene expression array.results: mil-21 promoted nK cells expansion of 20-fold each week for up to 7 weeks, with a peak expansion rate of 100-fold in the second week. mean expansion at day 21 was 110,719-fold. nK cells expanded with mil-21 had increased expression of Kir, nCr, CD16, and nKg2D, with cytotoxicity against leukemia, sarcoma, and carcinoma targets. the expanded nK cells retained donor Kir licensing, though with diminished inhibition, and demonstrated enhanced killing via aDCC. il-21-expanded nK cells showed increased expression of the activating receptor CD160 and decreased expression il-4, -5, -6, and -10, and FoXP3. expanded nK cells are susceptible to genetic modification with mrna or plasmid Dna via electroporation, and we have expressed chimeric receptors for CD33, gD2, and CD19 using this approach. this will be a fruitful system for further genetic modification of the aaPC to enable molding of the Kir repertoire during expansion and improved homing via trogocytosis, and for genetic modification of the nK cells for enhanced tumor targeting.

35IMMunOthERAPy uSInG REd BLOOd CELLS: AntIGEn And AdJuVAnt dELIVERy SyStEMA. BANZ, M. CREMEL, A. REMBERT, Y. GODFRIN; ERYtech Pharma, LYON, FRANCE.

Various strategies have been elaborate to deliver antigen to antigen-presenting cells (aPCs) in order to induce an efficient specific immune response. most of these strategies have encountered difficulties to target aPCs only and deliver both antigen and immune stimulator. We developed a system that allows delivering both antigen and adjuvant to aPCs by the same vehicle, the red blood cells (rbCs). this strategy is based on the physiological properties of rbC which are naturally removed from the circulation by aPCs. antigen and adjuvant can be entrapped into rbCs by a hypotonic lysis process leading to a homogeneous population with 100% of loaded-cells. ovalbumin (oVa) was chosen for the proof of concept. the number of molecules entrapped ranged from 9,800 to 71,000 depending on the oVa concentration used for the dialysis. C57bl/6 mice were injected intravenously either with oVa-loaded rbCs (rbCoVa) and free adjuvant, or with rbCs loaded with both oVa and adjuvant. results demonstrated the efficacy of rbCs in antigen delivery to aPCs and induction of oVa-specific b and Ctl responses. these responses were dependent on the dose-amount of antigen entrapped, and furthermore, the quantity of adjuvant necessary to induce a significant CD8+ t cell response can be dramatically reduced when it was encapsulated. in addition, we investigated the capability of this system to control the tumour growth in e.g7-oVa-bearing mice. after two injections of rbCoVa combined with adjuvant, the tumour growth was significantly delayed when compared with mice receiving a mix of free oVa and adjuvant. altogether, these results support the use of rbCs, as antigen and adjuvant carriers, for targeting DCs in the aim of inducing specific Ctl responses in the disease of cancer.

34REGuLAtORy tR1 CELL thERAPy OF ChROnIC InFLAMMAtORy dISEASESA. Foussat, V. Brun, H. Bastian; TxCell, SA, Valbonne, FRANCE.

il-10 producing regulatory cells, also called tr1 cells were discovered in 1997 based on their properties to prevent in vitro bystander t-cell proliferation and to inhibit in vivo chronic colitis in mice. in 2001, txCell biotech was founded on the idea that such regulatory cell populations could have a strong efficacy also in humans in severe chronic inflammatory conditions.in order to assess the potency of tr1 cell therapy in patients displaying severe Crohn’s disease, a reproducible manufacturing process for ovalbumin specific human tr1 cell was set-up in gmP conditions. Characterization of the human cell therapy product shows an in vitro suppressive activity on t-cell proliferation dependent on the production of both il-10 and tgF-beta. manufactured tr1 cells display a regulatory phenotype including Foxp3, gitr and Ctla4 surface expression and express a set of homing molecules crucial for the homing to inflammatory tissues. in vitro toxicity studies of human tr1 cell products (tumorogenicity, karyotyping, telomerase activity) showed a safety profile compatible with the use of these lymphocytes for cell therapy.based on these elements, a phase i/iia clinical trial was initiated in march 2008 in severe Crohn’s disease patients. in this trial, concomitant administration of the control antigen (ovalbumin) and of the autologous ovalbumin specific tr1 cells is performed in order to induce a massive release of suppressive cytokines in the inflamed gut tissues upon encounter of the cells with the antigen.

36A MEdICInAL MuShROOM EXtRACt BASEd On AGARICuS BLAZEI MuRILL InduCES EXPRESSIOn OF CELL SuRFACE MARKERS And PROduCtIOn OF CytOKInES In huMAn MOnOCytE-dERIVEd dEndRItIC CELLS (MddC) In VItROG. Hetland, D. T. Forland, A. M. Tryggestad, J. M. Tangen, T. Lyberg, E. Johnson; Oslo University Hospital, Oslo, NORWAY.

the edible basidiomycetes mushroom agaricus bm (abm) of brazilian rain forest origin is used in traditional medicine against cancer and several diseases. it has immunomodulating properties, probably due to high content of β-glucans and other biological response modifiers, and is shown to inhibit cancer and sepsis in mouse models. We examined stimulatory effects on mDDC cultures after 24h of the abm-based extract andosantm, also containing the mushrooms Hericium erinaceum (15%) and grifola frondosa (3%). We found down-regulated CD11c, de novo CD69 and enhanced CD86 expression on the cells, and dose-dependent increased levels of il-8, miP-1β, g-sCF, tnFβ, il-1β and il-6 in the cell culture supernatants. Whereas the synthesis of il-2, il-8 and iFnβ was similar for 10% of the extract and 0.5 µg/ml of e. coli lPs, andosantm induced a 2- to 10-fold higher production than did lPs of il-1β, tnFβ and g-sCF, respectively (Fig.). We conclude that stimulation of mDDC with an abm-based extract induced differential expression of cell surface markers for cell adhesion, activation and antigen presentation, and increased production of proinflammatory, chemotactic and some th1-type cytokines in vitro.


Poster Abstracts

37EXAMInInG thE EFFECtS OF GSK3ζ InhIBItIOn On t CELL ACtIVAtIOnG. Klamer1,2, K. Ko3, E. Song3, T. O’Brien3,4, A. Dolnikov1,3; 1Children’s Cancer Institute Australia, Lowy Cancer research Centre, Sydney, AUSTRALIA, 2University of New South Wales, School of Women’s and Children’s Health, Sydney, AUSTRALIA, 3Sydney Cord and Marrow Transplant Facility, Sydney Children’s Hospital, Sydney, AUSTRALIA, 4Centre for Children’s Cancer and Blood Disorders, Sydney, AUSTRALIA.

glycogen synthase kinase 3β (gsK3β) is a multifunctional serine threonine kinase which regulates over 80 substrates including inflammatory mediators nFβb, Creb, β-catenin and nFat. the anti-inflammatory effects of gsK3β inhibition have been well documented in murine models of inflammatory disease, human monocytes, dendritic cells and microglia, however, these effects have not been well described in t cells, the effector cell of many inflammatory diseases. one such disease is graft versus Host Disease, the main contributor of transplant related mortality in allogeneic hematopoietic stem cell transplantation. our aim is to examine the potential of gsK3β inhibitors to treat t cell mediated immune disorders by analysing their effects on cell activation and inflammatory mediator synthesis. Cell division tracking revealed that gsK3β inhibitors, 6-bromoindirubin-3-‘oxime (bio) and lithium, suppressed PHa induced cord blood derived t cell activation by inhibiting cell divisions and expression of effector t cell antigens CD25 and CD154. gsK3β inhibitor treated t cells retained proliferative potential after drug removal suggesting retainment of immuno-competency. using real time PCr we demonstrated that bio activated transcription of the anti-inflammatory il-10 cytokine in Jurkats and primary t cells. Furthermore, bio attenuated expression of pro-inflammatory cytokine genes tnFβ and iFnβ in activated t cells. additionally, expression of iβbβ mrna was suppressed. Comprehensive gene expression analysis is being conducted to identify genes and pathways involved in bio mediated suppression of t cell activation. Flow cytometric based intracellular cytokine staining is being conducted to examine il-10, tnFβ and iFnβ production. additionally, the effects of gsK3β inhibition on allogeneic t cell activation are being investigated in a mixed lymphocyte reaction where dendritic cells are being used to stimulate activation. our data proves that small molecule gsK3β inhibitors are a novel anti-inflammatory class of drugs, therefore providing rationale to examine their anti-inflammatory potential in in vivo settings of immune disease.

39ChARACtERIZAtIOn OF EX VIVO-EXPAndEd ALLOGEnEIC nAtuRAL KILLER CELLS FOR CLInICAL APPLICAtIOn In AntI-tuMOR IMMunOthERAPyO. Lim1, Y. Lee1, M. Jung2, H. Shin2, H. Jung1, J. Kang1, J. Her1, S. Kang1, B. Min1, Y. Hwang1,2; 1Mogam Biotechnology Research Institute, Yongin, KOREA, REPUBLIC OF, 2Green Cross Corp., Yongin, KOREA, REPUBLIC OF.

a novel method of expansion and activation of natural killer (nK) cells for clinical application under good manufacturing practice (gmP) compliance was established. Purified nK cells from 100 ml of peripheral blood were stimulated with interleukin-2 and anti-CD3 antibody in a closed culture bag system for 14 days. more than 1x10e9 of highly pure population of CD16+/CD56+ nK cells were obtained while contaminants such as t cells, b cells, or monocytes were analyzed by less than one percent.expanded nK cells showed potent efficacy on killing tumor cells. in response to target cells, effector cytokines such as iFn-β and tnF-β were efficiently secreted 10-fold more compared to resting nK cells. adhesion molecules, Dnam-1; activating receptors, nKg2D; and natural cytotoxicity receptors, nKp30, nKp44, and nKp46 were up-regulated by ex vivo-expansion resulting in the enhancement of cytolytic activity against tumor cells. this potent cytotoxicity of expanded nK cells revealed a more apparent result with perforin/granzyme-dependent necrosis over than trail or Fasl-mediated apoptosis of target cells. in addition, expanded nK cells effectively discriminated transformed cells from allogeneic normal peripheral blood lymphocytes and selectively killed target cells.For non-clinical testing for toxicity and kinetics, human nK cells were adoptively transferred into sCiD mice via tail vein. the administrated nK cells were mainly found in the lung, liver, and spleen for 48 hours and a few were circulated in the blood stream. one week after transfer, human nK cells were hardly detected by immunohistochemistry, flow cytometry or PCr. there were no reports concerning any significant adverse effects of nK cell transfusion.in conclusion, an innovative process of large-scale expansion of nK cells with up-regulated expression of cytotoxic effector molecules was developed. these ex vivo-expanded allogeneic nK cells were demonstrated to be safe and are expected to be effective for clinical application in anti-tumor immunotherapy.

38t CELL RESPOnSES In PAtIEntS VACCInAtEd WIth tELOMERASE (htERt)-MRnA tRAnSFECtEd FASt dEndRItIC CELLSE. I. Suso1, A. Rasmussen1, S. Aamdal2, S. Dueland3, G. Gaudernack1, G. Kvalheim4; 1Department of Immunology, Institute of Cancer Research, Oslo University Hospital, Oslo, NORWAY, 2Department of Clinical Research, Oslo University Hospital, Oslo, NORWAY, 3Department of Cancer Research, Oslo University Hospital, Oslo, NORWAY, 4Department of Cellular Therapy, Oslo University Hospital, Oslo, NORWAY.

two cancer patients were vaccinated with dendritic cells (DC) loaded with telomerase (htert) mrna to investigate the safety, tolerability and immunological response to vaccination prior to the start of a new phase i/ii clinical trial. one advanced lung cancer patient and one patient with a relapsed pancreatic adenocarcinoma, were treated with autologus fast DC (Jarnjak-Jankovic s. et al. 2007) transfected with mrna encoding htert. the patients first received four weekly injections followed by monthly booster injections. each vaccine aliquot contained 5-10 x 106 DC injected intradermally. Peripheral blood mononuclear cells (PbmC) were obtained prior to the four standard vaccinations, after 5 weeks, 12 weeks and monthly thereafter. thawed PbmC were stimulated in vitro with transfected DC. t-cell proliferation assays were performed with irradiated transfected DC and mock-transfected DC controls used as aPC. t-cell proliferation against a panel of 24 overlapping htert peptides was also assessed. in addition, htert-specific CD8+ t cells were monitored by pentamer staining. the treatment was well tolerated with minor side effects. immune responses against telomerase-transfected DC were detected in both patients and correlated with survival outcome. the pancreas patient still has stable disease since June 2007. the patients showed specific t-cell proliferation in response to the mrna-loaded DC in vitro after vaccination. We also detected stable populations of htert-specific CD8+ t cells in the both patients by pentamer staining in post-vaccination samples. samples from both patients were tested against a panel of 24 htert peptides and t-cell responses against several peptides were detected. these clinical experiences show that vaccination with htert-mrna transfected DC is safe and able to induce robust immune responses to several telomerase t-cell epitopes both in CD4+ and CD8+ t cells. based on these experiences all new DC vaccination protocols include the use of htert-mrna in combination with autologous tumour mrna

40COMMERCIALIZEd MAnuFACtuRInG PROCESS OF CVAC™, A dEndRItIC CELL VACCInE, FOR A PhASE II tRIAL In AdVAnCEd OVARIAn PAtIEntSM. Loudovaris1, J. Moloney1, E. Butler1, D. Ritchie2, M. Rogers3, D. Wall2; 1Cell Therapies Pty. Ltd., Melbourne East, AUSTRALIA, 2Peter MacCallum Cancer Centre, Melbourne East, AUSTRALIA, 3Prima BioMed, Armadale, AUSTRALIA.

ovarian cancer is one of the most common malignancies and cause of cancer deaths in women. to date no consolidation therapies have been identified in phase iii studies to improve survival. recent advances in immunology have shown that combining immunotherapy with other treatment may improve patient survival. immunotherapy, and especially dendritic cell (DC)-based vaccines, appears to be one of the most promising therapies. Prima biomed is developing a therapeutic vaccine, CVac™, composed of autologous DC loaded with mannan-muC1 Fusion Protein (m-FP) for investigational use. Previously, a Phase i/ii clinical trial in australia using CVac™ to treat ovarian cancer showed promising results. Prior to commencing a Phase iib trial process improvements were implemented. briefly, the process improvements include closed system elutriation, washing and culture and additional improved quality control testing. Here we present the data from 6 normal donor products manufactured to demonstrate the process improvement, with this data being used to support the successful inD submission for the Phase iib clinical trial. apheresis products containing a mean 1.1±0.1e+10 mononuclear cells (mnC) were shipped overnight to the manufacturing site. Products were enriched using the elutra system with a mean monocyte yield of 1.95±0.8e+09, purity of 77.4±9.6% and recovery of 75.5±12.6%. these monocytes were cultured ex vivo for 6 days in aim V media containing gm-CsF and il-4. on day 5 of the culture period the DC were pulsed with m-FP. on day 6 the culture were harvested and washed using the Cytomate™ system. the process generated 7.4±1.9e+08 viable DC, with a mean purity of 86.4 ± 5.8% and met all acceptance criteria. the process generated 13.5±3.5 doses of CVac™, with 9-12 doses required for the clinical treatment and quality control testing. the inD was approved in December 2009 and patient enrollment in the usa and australia is currently underway.


may 23-26, 2010


Poster Abstracts

41LEVELS OF CIRCuLAtInG PSA And hLA-dR- MOnOCytES In PROStAtE CAnCER PAtIEntS VACCInAtEd WIth ALLOGEnEIC PROStAtE CAnCER CELLS (APCC) WIth OR WIthOut AutOLOGOuS dEndRItIC CELLS (dCS)M. L. Maas, D. J. Padley, A. B. Dietz, M. Kohli, D. A. Gastineau, S. Vuk-Pavlovic; Mayo Clinic, Rochester, MN.

recently we found high levels of circulating CD14+Hla-Dr- monocytes in late-stage prostate cancer patients (Vuk-Pavlovic et al., Prostate, 2010, in press). laboratory evidence indicates that these cells can suppress immunity and cannot mature into functional DCs. to determine if PCa-specific vaccination affects these cells, we monitored their levels in patients injected with aPCC (designated onyCap-23 and P4e6) without or with autologous mature DCs. each dose consisted of 8×106 of each aPCC with, when applicable, 10×106 DCs. DCs were matured from isolated CD14+ cells; the preparations contained more than 99 percent CD83+ cells. DCs were combined with aPCCs and stored in liquid nitrogen vapor. the cells were thawed and injected intradermally into axillary and inguinal regions. the first two doses were combined with bCg and administered two weeks apart. the next dose was injected two weeks later and the subsequent doses at one month intervals. Circulating Psa was measured prior to each vaccination and the percent of CD14+Hla-Dr- cells was measured at every other vaccination. Patient 1 received four doses of aPCCs. after skipping a dose, subsequently he received two doses of aPCC+DCs. He withdrew after six months due to disease progression. Patient 2 received 11 of the scheduled 14 aPCC+DC treatments. in both patients, pretreatment Psa velocity was positive; treatment changed it into negative, but after two and five months, respectively, Psa levels started rising again. level of circulating CD14+Hla-Dr- cells in Patient 1 was normal (3.6±0.8%)and stable over the six weeks it was measured; in Patient 2, it was 58.0 percent before treatment and 11.6 percent at four months; then it started increasing again. the observed correlation between vaccination and levels of Psa and CD14+Hla-Dr- cell levels requires further studies.

43CGMP PROduCtIOn OF uMBILICAL CORd BLOOd-dERIVEd t-REGuLAtORy CELLS In SuPPORt OF A FIRSt-In-huMAn CLInICAL tRIALD. H. McKenna1, D. J. Sumstad1, C. G. Brunstein2, D. M. Kadidlo1, K. L. Hippen2, J. Curtsinger2, C. H. June3, B. L. Levine3, J. S. Miller2, B. R. Blazar2, J. E. Wagner2; 1Molecular & Cellular Therapeutics, University of Minnesota, Saint Paul, MN, 2Blood & Marrow Transplant Program, University of Minnesota, Minneapolis, MN, 3Pathology & Laboratory Medicine, University of Pennsylvania, Philadelphia, PA.

introduction: t regulatory cells (tr) are a subset of CD4+ t cells that express both CD25 (il-2rβ) and FoxP3. tr are critical in auto-, allo-, and tumor immunity. several groups have shown that tr can successfully inhibit gVHD in animal models. We report our experience in support of the first clinical trial using umbilical cord blood (uCb)-derived tr with double uCb transplant. methods: uCb was enriched for CD4+/CD25+ t regulatory cells using the ClinimaCs® device. selected cells were re-suspended at 1 x 106 cells/ml in X-ViVo 15 with 10% ab serum, and anti-CD3/antiCD28-coated beads were added (cell:bead = 1:3). the culture was supplemented with 300 iu/ml il-2 starting on the 3rd day of culture. a viable cell count was determined every 2-3 days, and media and il-2 were added to maintain the cell concentration at 0.5 x 106 cells/ml and il-2 concentration at 300 units/ml. beginning on day 14, viable cell counts were obtained every 2-3 days to adjust cell concentration per algorithm. Cells were cultured in flask(s)/cell factories for 18 +/-1 days. the final product was released for infusion after successful lot release testing. results: see table. median nucleated cell fold-expansion was 208 (13-1796). in total, 23 patients were treated. no severe infusional adverse events were observed. When compared to historical outcomes, there was no adverse impact on sustained donor chimerism, gVHD, infection, relapse and PFs. tr were detectable up to 14 days post-infusion. Conclusion: tr were successfully expanded under cgmP. overall clinical results are promising.lot release testing summary

test specification result (mean)% CD4+/CD25+ >/= 60% 84%% CD4-/CD8+ </= 10% 1%Viability (7-aaD) >/= 70% 96%anti-CD3/CD28 bead Count < 100 beads/3 million cells 4 beads/3 million cellsendotoxin < 5eu/kg 0.97 eu/kggram stain no organisms no organisms

42AnALyZInG IntEGRAtIOnS OF tRAnSPOSOnS tO GEnERAtE CLInICAL-GRAdE tuMOR-SPECIFIC t CELLSS. Maiti1, J. Zhang1, B. Moriarity2, S. Liang1, M. Dawson1, M. Figliola1, L. Zhang1, H. Huls1, P. Kebriaei1, S. Kelly1, R. Champlin1, D. Largaespada2, P. Hackett2, L. Cooper1; 1M.D. Anderson Cancer Center, Houston, TX, 2University of Minnesota, Minneapolis, MN.

the development of cost-effective and effective therapy requires (i) integration of desired transgene, (ii) long-term transgene expression, and (iii) minimal risks of insertional mutagenesis. We have adapted non-viral sleeping beauty (sb) system for clinical use to electro-transfer Dna plasmids expressing a chimeric antigen receptor (Car) as a transposon (tn) and hyperactive sb11 transposase. after gene transfer, t cells rendered specific for the b-lineage-specific antigen CD19 are selectively propagated to clinically-sufficient numbers on clinical-grade artificial antigen presenting cells (aaPC) in a Car-dependent manner. FisH and Q-PCr demonstrate that these t cells contain a single genomic insertion of tn per cell. Pyrosequencing of tn integration sites from 14 genetically modified t-cell preparations were characterized for their genomic distribution and the relationship between “expected” (integrating at any ta dinucleotide repeat) and “observed”. tn insertion frequencies follow the pattern of chromosome length and not the number of refseq genes. although no significant preferential integration occurred around the transcription start sites (tss) of oncogenes, mirnas, and Cpg islands, there was a slight enrichment observed around general tss. as recombinant lentiviral plasmids are being used in clinical trials, we compared these sb integration data to transgene insertions occurring after viral transduction. Compared to the sb system, the lentivirus’ insertions demonstrated a much increased propensity to insert around any tss. For all genes, tn are less likely to be found near a tss than lentivirus (e.g., 8.4% versus 12.9% p-value 3X10-9). thus, the sb system appears to insert a Car tn in a more random distribution compared with lentiviral vectors. these data are the first to comprehensively evaluate the distribution of Car tn integration sites in t cells genetically modified and propagated using an approach adapted for human trials and provide additional rational and reassurance for the clinical testing of sb non-viral gene transfer systems.

44OPtIMIZEd MAtuRAtIOn COCtAIL FOR CLInICAL GRAdE dEndRItIC CELL VACCInAtIOnJ. Michalek1,2, K. Skalova1, K. Mollova1; 1Babak Research Institute, Masaryk University, Brno, CZECH REPUBLIC, 2Dept. of Pediatrics, University Hospital Brno, Brno, CZECH REPUBLIC.

Dendritic cells (DCs) are professional antigen presenting cells able to activate both helper (th) and cytotoxic (tc) lymphocytes. Here we tested 5 variants of DC maturation coctails for clinical trials application. immature DCs were prepared with plastic adherence of mononulear cells obtained by leukapheresis from 12 healthy volunteers and grown in complete media with gm-CsF and il-4 for 5 days. tumor lysate was added overnight and maturation was induced with maturation coctail (mC)-1 containing: tnF alpha alone; mC-2: tnF alpha + il-1 beta + iFn gamma + prostaglandin e2 + tlr 7/8 ligand r848; mC-3: tnF alpha + il-1 beta + il-6 + prostaglandin e2; mC-4: iFn gamma + r848; mC-5: iFn gamma + lipopolysacharide (lPs). Phenotypic characterization of immature and mature DCs was performed by flow cytometry. Cytokine expression of il-12p70 and il-10 and their ratio was quantified by cytometric bead array and allogeneic t cell activation with matured DCs was studied in mixed lymphocyte culture (mlC). the results clearly demonstrated similar phenotypic characteristic of DCs matured with different mCs while significant differences were noted with cytokine il-12 production as well as il-12/il-10 ratio between different maturation coctails. il-12 production was the highest with the mC-5, intermediate with mC-4 and low with other mCs. similar findings were noticed in mlC - high il-12 production by DCs corresponded with higher activation of t cells. in conclusion, broad differences in functional capacity of DCs matured under different conditions were noticed. these finding led us to selection of mC-5 for the clinical trials with cancer immunotherapy using dendritic cells.this work was supported with grants msmt nPVii 2b08066 and msmt nPVii 2b06058.


Poster Abstracts

45t-CELL thERAPy FOR Out BREd CAnInES WIth SPOntAnEOuS B CELL nOn hOdGKIn LyMPhOMAC. M. O’Connor1, S. Sheppard2, H. Huls1, M. Johnson2, C. June3, S. Craig1, D. A. Lee1, R. E. Champlin1, H. M. Wilson2, L. J. Cooper1; 1The University of Texas M.D. Anderson Cancer Center, Houston, TX, 2Texas A&M University College of Veterinary Medicine, College Station, TX, 3University of Pennsylvania Abramson Family Cancer Research Institute, Philadelphia, PA.

Canine cancer provides a therapy model for human malignancies because of the large size, intra-species genetic diversity, human genetic similarity, and spontaneously occurring tumors that develop despite an intact immune system. Canine b-lineage lymphoma is genetically, molecularly, and physiologically similar to aggressive nHl. We have begun to investigate whether infusion of autologous t-cells can improve the outcome of canine lymphoma. lymphocyte recovery after lymphodepleting chemotherapy is a predictor for improved survival in human clinical trials and chemotherapy can act as an immune modulator to improve the anti-tumor effect of t-cells. We have adapted the canine nHl model to assess whether restoring depleted canine CD3+ t-cell pools can potentiate chemotherapy. We report a new platform to ex vivo propagate clinically-sufficient numbers of t-cells derived from dogs receiving chemotherapy using K562-derived artificial antigen presenting cells (aaPC) genetically modified to express desired t-cell co-stimulatory ligands and which can be loaded with clinical-grade oKt3 to cross-link canine CD3. PbmC were isolated from dogs with nHl and numerically expanded on β-irradiated oKt3-aaPC with exogenous il-2 and il-21. autologous t-cells were infused into canine nHl patients receiving chemotherapy using an intra-patient dose escalation scheme. there were no toxicities over grade 2 and infused fluorescently-labeled t-cells were detected for at least 35 days after adoptive transfer. the infused products were primarily CD3+CD8+ cells, which were able to correct the iatrogenic loss of CD8+ t-cells after chemotherapy. these data demonstrate that oKt3-aaPC can be used to generate clinical-grade canine t-cells for the safe reconstitution of chemotherapy-induced lymphopenia. in aggregate, since these aaPC have been produced by PaCt as a master cell bank for human clinical trials, canine nHl serves as a platform to assess add-back of t-cell therapy in humans and paves the way for infusing genetically modified canine t-cells that have redirected specificity for nHl.

47LARGE SCALE BIOREACtOR EXPAnSIOn OF tuMOR SPECIFIC t LyMPhOCytES FOR AdOPtIVE CELL thERAPyA. M. Sadeghi; Division of Clinical Immunology, Uppsala, SWEDEN.

backgroundthe aim of this study was to create a closed cell culture system with the WaVe bioreactor system 2/10 with perfusion and the techniques for tube-welding in order to create an improved milieu for expansion of tumor infiltrating t lymphocytes (tils). our hypothesis was that the bioreactor allows for optimized provision of nutrients and removal of spent media while minimizing culture volumes. this might lead to a better quality of expanded cells with lower amounts of exhausted cells compared to static expansions in culture bags.Procedurestumor infiltrating lymphocytes from 4 melanoma patients were expanded and compared in parallel using the WaVe bioreactor and traditional static culture methods. Parameters like viability, final cell numbers, phenotype and effector function were measured.resultsour results show that the bioreactor is suitable for large-scale expansion of tumor specific lymphocytes. the WaVe bioreactor in this setup was equipped with perfusion allowing optimized provision of nutrients and removal of spent media while minimizing culture volumes. the bioreactor allowed for improved cell growth and higher final cell yields when compared with that obtained using static culture conditions. Phenotypic characteristics of tils were compared prior and post expansion and showed no consistent difference between the two expansion methods. tils harvested had the phenotype and function corresponding to intermediate to late effector cells.Discussionthe WaVe bioreactor is suitable for large-scale expansion of tils. Perfusion of fresh media allows for much higher cell densities and improved cell growth in constant and minimal culture volumes. in conclusion expansion of tils in the bioreactor is a resource-effective method of reaching large numbers of t cells for cell therapy in the clinic.

46EX VIVO EXPAndEd Cd4+Cd127LO/-Cd25+ POLyCLOnAL tREGS: PhASE I CLInICAL tRIAL FOR thE tREAtMEnt OF RECEnt OnSEt t1dA. Putnam, M. Lee, W. Liu, H. Escosa, T. Brusko, J. Bluestone; UCSF Diabetes Center, San Francisco, CA.

over the past decade regulatory t cells (tregs) have been shown to be essential for maintaining immune homeostasis and controlling self-reactivity. animal and human pre-clinical studies support the use of regulatory t cells (tregs) as a cellular therapy for the re-establishment of immune homeostasis in patients with ongoing autoimmune diseases. studies in several experimental systems have shown that the adoptive transfer of polyclonal tregs can suppress ongoing autoimmunity, delay tissue allograft rejection and block graft versus host disease. in an effort to generate therapeutically relevant numbers of sufficiently pure polyclonal tregs for the treatment of autoimmune diabetes, CD4+CD127lo/-CD25+ t cells from both new onset type 1 diabetic (t1D) patients and controls were isolated on an aria ii cell sorter using cgmP/cglP-grade monoclonal antibodies and expanded ex vivo in a cgmP clean room facility. using a combination of anti-CD3/anti-CD28-coated beads plus il-2, the highly purified tregs (greater than 95%, fully demethylated, FoXP3+) could be expanded on average ~ 1000 fold in 14 days (range 598-1145, n = 7). the expanded cells maintained FoXP3 expression (mean 94.9%, range 91-97.4%) as well as potent suppressor cell activity in vitro. We are currently preparing to initiate a dose escalating, phase i clinical trial in t1D, transferring up to 2.9 x 109 cells. overall, this study illustrates the potential of generating a pure population of tregs on an approved large scale basis for the therapeutic treatment of autoimmunity and sets the stage for using these cells, as well as second generation antigen-specific tregs to treat immunologic diseases.

48PRIMEd EX-VIVO CO-StIMuLAtEd AutOLOGOuS t-CELLS And POSt-ASCt InFLuEnZA VACCInAtIOn FOR MuLtIPLE MyELOMAE. A. Stadtmauer, D. Vogl, B. Levine, N. Aqui, K. Macdonald, C. June, K. Sullivan; university of pennsylvania, Philadelphia, PA.

introduction: Patients with myeloma (mm) are vulnerable to flu infection from altered humoral and cellular immunity from the disease and therapy. methods: We performed a randomized open-label single center study of two influenza vaccine schedules for patients with high-risk mm responding after conventional induction therapy. of 21 patients enrolled, 11 were randomly assigned to group X and received pre- and d+14 post-transplant flu vaccine with the commercially available influenza vaccine (Fluzone). the 10 patients in group y received only post-transplant vaccination. all patients received t-cell harvest, high dose melphalan and asCt and ex-vivo (anti-CD3/anti-CD28) co-stimulated autologous t cells (at-cells) infusion. immunological assessment was conducted d+60, 100 and 180. the primary study endpoint was immune response to influenza as measured by the serotype-specific antibody response on day 100 using a standard hemagglutination inhibition assay (Hai). results: the median age was 55 (range 48-68), 13 m, 8 F, 18 Caucasian, 2 african american, 1 Hispanic, igg 11, iga 7, light chain 3. With a median follow-up of 1 year, 83% (18/21) patients are alive with 48% (17/21) alive in remission. Hai titers, however were higher for group X when compared to group y both for H3n2 (p= 0.007) and for H1n1 (p=0.009). Hai titers for group y remained near baseline throughout all time points while group X peaked day 60 and remained elevated day 180. 51% of group X had 4 fold or greater increase in titer vs 13% in group y. group X t-cells had higher % CFse stained than group y. in a b-cell elisPot group X had a greater number of influenza responsive b-cells than group y (p=0.01). Conclusion: this randomized trial of flu vaccine schedule for mm patients undergoing asCt suggests superior flu immune reconstitution when vaccine is administered to prime autologous t-cells prior asCt and at-cell infusion.


may 23-26, 2010


Poster Abstracts

49COnStItutIVELy ACtIVE AKt In t CELLS IMPARtS RESIStAnCE tO nEuROBLAStOMA MEdIAtEd SuPPRESSIOnJ. Sun1, D. M. Spencer1, C. M. Rooney1,2; 1Baylor College of Medicine, Houston, TX, 2Center for Cell and Gene Therapy, Houston, TX.

neuroblastoma (nb) is a common extra-cranial childhood malignancy with poor response to conventional therapy in patients with high-risk disease. our group has shown that adoptive transfer of nb-specific t-cells produce tumor responses including complete remissions with minimal toxicity. However, tumor specific t-cells are susceptible to multiple inhibitory stimuli from the nb environment, including lack of costimulation, secretion of inhibitory molecules and recruitment of t regulatory cells (t-reg). the akt/protein kinase b (PKb) plays a central role in t-cell proliferation, effector functions and survival and in t-reg mediated suppression. We hypothesized that expression of constitutively active akt (cakt) in t-cells should sustain t-cell survival and function in the complex inhibitory microenvironment of nb.the expansion of t-cells expressing cakt from a retrovirus vector was significantly greater than of vector-transduced t-cells and was explained by decreased apoptosis resulting from upregulated anti-apoptotic molecules including mcl-1, bcl-xl and bcl-2. cakt-cells also secreted higher levels of il-2 and iFn-β likely a result of their sustained expression of nF-βb. cakt-cells were relatively resistant to suppression by and conversion into t-regs as measured by functional analyses and FoxP3 expression. to determine if constitutive akt expression provides a survival advantage to t cells in a nb model, we redirected the antigen-specificity of CD3/28 blasts to the lan-1 nb cell line, which highly expresses the disialoganglioside (gD2). t-cells expressing cakt and a chimeric antigen receptor (Car) specific for gD2 proliferated in response to lan-1 and eliminated the tumor more efficiently in cocultures than t-cells expressing gD2 Car alone. cakt expressing t cells were resistant to apoptosis induced by lan-1 as well as by anti-Fas and tgF-β relative to control t cells. in conclusion, cakt transduced t cells showed resistance to multiple evasion strategies employed by nb tumors and may therefore enhance anti-tumor activity of adoptively transferred t-lymphocytes.

51FLOW CytOMEtRIC MEthOd FOR dEtECtIOn OF In VIVO REd CELL BOund IGGW. Beres, L. McGuire, S. J. Nance, G. Meny; American Red Cross, Philadelphia, PA.

background: the immunohematology research laboratory is developing a flow cytometry based method to assist in the diagnosis of patients with autoimmune hemolytic anemia (aiHa) by detecting red cell-bound immunoglobulins. method: aliquots from patient blood samples submitted for Dat evaluation were utilized in assay development with the approval of the institutional review board. the blood samples were stored at 2-6°C until the day of testing. red blood cells from each sample were washed three times using 0.85% buffered saline solution and prepared at a 1% suspension of rbC in 0.85% buffered saline solution. a FitC-labeled anti-human igg (β) goat F(ab’)2 antibody (invitrogen, Camarillo, Ca) was used to detect the presence of cell-bound igg. the antibody was used at a 1:100 dilution in 0.85% buffered saline solution containing 1% bovine serum albumin (bsa) and added at a 1:1 ratio to a cell button of 1% rbC suspension. the sensitized sample was incubated for 30 to 60 minutes in the dark at room temperature, washed and resuspended in 0.85% buffered saline / 1% bsa. a control sample was also prepared following the same procedure with the exception of the sensitization of the antibodies. the samples were stored in the dark until they were tested. a flow cytometer is an expensive and sensitive piece of equipment that the immunohematology research laboratory did not have. the research laboratory obtained use of the facility’s Clinical services Cellular therapy laboratory’s © becton Dickinson FaCsCaliburtm (Franklin lakes, nJ) flow cytometer via collaborative scheduling. a Cellular therapy technologist performed the acquisition and analysis of 50,000 cells from each sample at optimal settings. Conclusion: a flow cytometry method to detect red-cell bound immunoglobulin was developed using resources shared between a clinical Cellular therapy laboratory and an immunohematology research laboratory.

50A REtROSPECtIVE COhORt Study OF EtAnERCEPt VERSuS CORtICOStEROIdS FOR thE tREAtMEnt OF IdIOPAthIC PnEuMOnIA SyndROME AFtER ALLOGEnEIC hEMAtOPOIEtIC StEM CELL tRAnSPLAntAtIOn.R. Tizon1,2, N. Frey2, D. F. Heitjan2, K. Tan2, S. C. Goldstein2, E. Hexner2, M. Vozniak2, J. Davis2, B. D. Fuchs2, A. Loren2, S. M. Luger2, A. Perl2, E. A. Stadtmauer2, D. Tsai2, D. Porter2; 1Memorial Sloan Kettering Cancer Center, New York, NY, 2Hospital of the University of Pennsylvania, Philadelphia, PA.

introduction: idiopathic Pulmonary syndrome (iPs) occurs in 5-25% of all hematopoietic stem cells transplant (HsCt) recipients within 100 days of transplant. iPs is characterized by progressive respiratory failure as a result of lung injury due to cytotoxic chemotherapy, cell-mediated immune injury and inflammatory cytokines. standard treatment approaches to iPs have involved supportive care, broad-spectrum antibiotics and high dose corticosteroids, with high mortality rates between 50-80%. mouse models suggest a central role for donor t cell derived tumor necrosis Factor-β (tnF-β) in iPs pathogenesis. based on positive reported series of corticosteroids in combination with etanercept (enbrel; amgen, thousand oaks, Ca), a soluble tnF-β protein, we report our institutional outcomes of consecutive iPs patients treated with corticosteroids with and without etanercept. to date, there have been no reports directly comparing high-dose corticosteroid therapy with anti-tnF alpha treatment for iPs.methods: this is a retrospective cohort study in adult HsCt pts with confirmed or suspected iPs (n=39). twenty-two patients were treated with high-dose corticosteroids for iPs (group 1) according to our standard practice, while 17 patients received corticosteroids in combination with etanercept (group 2). endpoints such as differences in survival at Day +28 after iPs treatment, days on mechanical ventilation, and six month and 2 year survival were assessed between the two groups.results/Discussion: survival at day +28 of iPs onset was significantly better for recipients of etanercept plus steroids (group 2) compared to recipients of steroids alone (group 1) at 88.2% (95% Ci 64-100%) vs 36.4% (95% Ci 17-59%) (p=0.001). this benefit did not translate into long-term survival, as no difference was noted in survival at day +180 or at 2 years after iPs onset. newer therapies and the pending results from the ongoing bmt-Ctn phase iii trial comparing corticosteroids with and without etanercept may provide a positive impact on patient outcomes.

52ASSESSMEnt OF tIL FOR RESIduAL MELAnOMA uSInG FLOW CytOMEtRy.N. Garlie, A. Choithani, R. Siebenlist, J. Richards; Aurora St. Luke’s Medical Center, Milwaukee, WI.

adoptive therapy using tumor infiltrating lymphocytes (til) has been used in experimental treatments in conjunction with interleukin-2 (il-2) over the past two to three decades. one current method of generating til for adoptive transfer is to culture tumor enzyme digests in the presence of high dose il-2 for a 10-20 day period followed by a two week rapid expansion protocol. given that til are expanded from a surgically resected tumor an underlying issue is the level of tumor that remains after til expansion. to address this issue a multiparametric flow cytometry assay was developed to evaluate the presence of tumors within a til population. the assay was designed to identify viable tumor cells within a til population. the live/Dead green viability dye was used to identify viable cells, which could then be fixed and stained for intracellular antigens. to identify tumors, antibodies to the intracellular tumor antigens gp100, mart-1, tyrosinase and s100 where combined with an antibody against mCsP a surface antigen to detect tumor cells while anti-CD3 was used to identify til. identification of melanoma cells lines spiked into a t cell culture was achieved in 100% of samples tested. more importantly, the assay was able to detect tumor in melanoma enzyme digests from patient samples and clearance of tumors during til expansion with il-2. this work demonstrates that flow cytometry maybe used to evaluate expanded til cultures for the presence of tumor.this work was supported by the northwestern mutual Foundation.


Poster Abstracts

53BIOBuRdEn REduCtIOn In AutOLOGOuS ISLEt tRAnSPLAntAtIOnS. Irwin; Medical University of SC, Charleston, SC.

autologous islet transplants frequently require that potentially infected tissue be processed for immediate transplant, ethically justified by the urgent medical need of the procedure and lack of equally effective substitute therapies.Current processing methods cannot ensure sterility of the final product without functional or structural damage, and the critical need to maximize islet yields means that the infusion of the final product must occur before effective sterility testing can be completed.in order to resolve this quandary, autologous islets were prepared for transplant in a gmP capable laboratory, and endogenous bioburden was measured at various points during the procedure. our data has shown that a three log bioburden reduction can be reliably achieved using a mechanized shaking device during digestion of the extraneous tissue that is superior to a manual shaking method. most importantly, however, preliminary data supports data published elsewhere that the increased bioburden has not affected graft function or quality of life.

55uSE OF thE EndOSAFE PtS FOR MEASuRInG EndOtOXIn LEVELS In PLAtELEt LySAtED. J. Padley, G. W. Butler, A. L. Mohr, C. Liwski, J. L. Gilgenbach, D. A. Gastineau, A. B. Dietz; Mayo Clinic, Rochester, MN.

We manufacture platelet lysate (Pl) as a culture supplement for clinical cellular therapy products. Pl supports and enhances growth of a variety of cell types. endosafe Pts (Pts) uses FDa licensed lal cartridges to measure endotoxin. Pts tests samples in duplicate with two internal endotoxin controls. the spiked wells test for inhibition or enhancement (i/e) by the test material, reported as spike recovery, (sr) which must be between 50 and 200% of the expected value. Duplicate wells are compared and the coefficient of variation (CV) between them must be <25%. Prior to using Pts each sample type undergoes i/e testing to determine the optimal dilution. Pts cartridge sensitivity and product endotoxin release limit (erl) determine the maximum valid dilution, mVD. Prior to release each lot of Pl is tested for endotoxin with Pts. our erl for Pl is < 0.5 eu/ml. initially we used a cartridge of 0.01 eu/ml sensitivity, the lowest available, with mVD of 1:50. We have manufactured and endotoxin tested 62 lots of Pl. testing 62 lots required 112 cartridges (mean 1.8 per lot). Fifty five had endotoxin <0.5eu/ml. Four were above the erl, (0.77 + 0.19 eu/ml;mean + sD). endotoxin was inconclusive for 3 lots. of the initial tests for each lot 47 had acceptable sr and CV. of 15 failed assays 8 had high CV, 6 low sr, and 1 both. During this study a 0.005 eu/ml sensitivity cartridge was introduced by the manufacturer, changing our mVD to 1:100. 32 of 46 initial tests with the 0.01 cartridge and 15 of 16 with the 0.005 cartridge were valid. We were aware our initial mVD was not optimal, but we were limited by the erl. our experience demonstrates the importance of choosing both an appropriate erl and sufficient cartridge sensitivity to minimize repeat testing.

54CGMP VALIdAtIOn OF A FLOW CytOMEtRy IdEntIty ASSAy FOR GMP-PROduCEd StEM CELLSC. J. Larson; WuXi AppTec, Inc., Philadelphia, PA.

objective: Flow Cytometry is a valuable and well-established tool in the evaluation of cultured stem cells for identity. as more stem cell products reach commercialization, the need for validated flow cytometry assays to satisfy regulatory filings becomes apparent. We describe here a completed gmP validation of an identity assay where stem cells were known to be positive for two markers and negative for a third.methods: the assay was conducted in three runs and measured intra and inter-assay precision using triplicate values from three different points along antibody titration curves for markers on reference cells. From the titration curves, the limit of detection was also evaluated. accuracy and specificity were evaluated using a control cell that had an antigen profile opposite the reference cells and were spiked in with reference or run separately. Finally, robustness was evaluated as a function of incubation period for the combined antibodies on the reference cells at five different periods.results: the maximum value for either marker was 6.0% for intra and 12.0% for inter-assay precision. the loD demonstrated at least a 40-fold separation of the negative from the antibody concentrations used in the final assay. accuracy targets from spiking studies demonstrated a maximum of 17.7% from the expected target. specificity demonstrated a difference in mFi between cell populations of at least 10-fold. Finally, robustness confirmed that all incubation periods produced substantially equivalent mFi values resulting in the middle time-point being selected for the final assay.Conclusion: the validation effort described here met all internal acceptance criteria and fell within previously established ranges. a gmP-level flow cytometry identity assay was created following this validation which has been used for release and stability studies of gmP produced lots designated for clinical use.

56REGuLAtORy COnSIdERAtIOnS FOR BIOLOGICS InCORPORAtEd IntO COMBInAtIOn PROduCtSM. Henderson; Regulatory & Clinical Research Institute (RCRI), Minneapolis, MN.

Combination products, whether drug/device; drug/biologic; biologic/device; cross-labeled product; or kitted product, require modified regulatory strategies from design to post-market compliance. FDa has established the office of Combination products to help manufacturers with combination product approvals and assigned various types of combination products to specific divisions of FDa. in the eu no parallel organizational structure exists. the manufacturer of a combination product is responsible for determining which directive applies to the product under development. a brief summary of the regulatory considerations unique to combination product development, along with an overview of the two rules recently proposed by FDa, covering current good manufacturing practices (cgmP) and adverse event reporting, will be presented.


may 23-26, 2010


Poster Abstracts

57SAFEty BEGInS At thE SOuRCE- thE IMPORtAnCE OF RAW MAtERIAL SELECtIOn And COntROLA. Imam, T. Fletcher; BD Biosciences Advanced Bioprocessing, Miami, FL.

both safety and efficacy are required for a therapy to succeed. However, the treatment inD mechanism established in 1987 allows investigational drugs to be provided outside controlled clinical trials to treat patients with serious or life threatening diseases and for decades has biased the focus onto the effectiveness of treatments compared to their safety. With the advancements in developing therapeutic products that successfully meet performance criteria, it is now time for the industry and its regulators to shift the emphasis towards producing safe products.so far, comparatively little effort is made to look at components and ingredients that directly or indirectly become part of the finished product and may pose a risk to the end user. this type of risk can be minimized by increasing control of raw materials during their selection as well as during the conception and design of new therapeutics.as safety is paramount to the success of a therapeutic product, it needs to be addressed using a two-pronged approach. one is the design of a comprehensive quality strategy for the selection, qualification and approval of raw materials and their suppliers. the other equally significant element is to identify appropriate raw materials as a key input during the design process.this parallel strategy must be treated as an interdependent approach for providing resolution in addressing potential risks associated with the use of animal origin materials in the form of viruses and organism such as bse agents.a simple solution would be to source only animal-free materials which have been approved by consensus both within the industry and by the regulatory agencies. until those standards have been established, it is imperative that product design efforts incorporate a risk-based approach that would effectively minimize the potential for inappropriate raw materials to introduce risks into new products.

59GEnE thERAPy And thE BRAIn: IS AFRICA EthICALLy, LEGALLy And SOCIALLy REAdy?G. M. Wanderi; School of Medicine, University of Nairobi, Nairobi, KENYA.

untreated mental illness is a significant policy problem with high fiscal and social costs, particularly among the urban poor in african cities. HiV-associated dementia (HaD), brain cancer and HiV-associated opportunistic brain infections have increased in incidence and mortality over the past decade.thus the remarkable research advances in neuroscience in the west, along with progress in genomics, informatics and molecular medicine opens new vista in the development of innovative therapeutic interventions to many such brain disorders like depression, addiction, neuro-degeneration and dementia in africa.implementation of gene-based vaccinations or treatments offer an attractive strategy for the prevention and treatment of these brain diseases but currently poses medico-legal, ethical, financial-economic and logistic problems. However, since these treatments would have a beneficial effect over generations, they necessitate the necessary investment towards making such approaches efficient and safe.the implications of such new research in neuroscience for individuals, society, and culture in developed nations has been substantial with great emphasis placed on setting policy guidelines to manage and respond to these innovations. However, african nations remain largely in the dark due to a slower pace of innovation, adaptation and technological infrastructure in neuroscience research. already clinical trial treatments based on these approaches are recruiting volunteers from african nations.We postulate through literature review that, with their greater diversity of culture, language and ethnicity as well as the increasing scientific capacity and growing communication infrastructure, african nations will need to most urgently tackle the questions that the blend of genetics and neuroscience research is raising.

58OVERVIEW OF REGuLAtIOnS And StAndARdS FOR StEM CELL-BASEd thERAPIES: An IntERnAtIOnAL PERSPECtIVET. Nguyen1, B. von Tigerstrom2; 1Centre of Genomics and Policy, McGill University, Faculty of Medicine, Montreal, QC, CANADA, 2College of Law and School of Public Health, University of Saskatchewan, Saskatoon, SK, CANADA.

in 2009, the united states Food and Drug administration (FDa) approved the first clinical trial using human embryonic stem (hes) cells. this is considered an important milestone for stem cell research as it opens the door for other stem cell-based therapies. However, as stem cell research continues to move towards more clinical applications, it is important to question how these stem cell-based products will be regulated. this is an important concern yet is often given little attention compared to other legal or ethical issues in ongoing stem cell debates. stem cell-based products are novel and have complex characteristics due to their source, potency or use, and therefore these products may be defined and classified in different ways under certain regulations. even then, stem cell-based products may not fit perfectly within existing categories leaving these products potentially unmonitored or unregulated. as a result, this exposes challenges with respect to ensuring safety and quality in the processing, distribution and international circulation of human cell and tissue products. this study seeks to illuminate particular barriers to harmonizing international laws and policies related to clinical translation of stem cell and to investigate potential methods for overcoming these challenges. this study will provide a comparative analysis of the regulatory frameworks surrounding the clinical translation of stem cells in various jurisdictions, such as Canada, united states, France, australia, belgium, China and the united Kingdom. this examination will also be informed by standards established by the issCr (guidelines for the Clinical translation of stem Cells) and directives established by the european Commission. it is concluded that while the challenges to harmonization are diverse and important, so too are the benefits of establishing uniformity in the regulation of stem cell-based therapies worldwide.

60PhARMACOLOGICAL EVALuAtIOn OF nOVEL IndOLE-2-CARBOXAMIdES AS POtEnt LIPId-LOWERInG AGEntS In tRItOn-WR1339-InduCEd hyPERLIPIdEMIC RAtST. Al-Qirim; Al-Zaytoonah University, Amman, JORDAN.

the lipid-lowering effects of two novel anti- hyperlipidemic agents, bmi2C [n-(4-benzoylphenyl)-5-methoxy-1H-indole-2-carboxamide] and DDmi2C [n-(9,10-dihydro-9,10-dioxoanthracen-2-yl)-5-methoxy-1H-indole-2-carboxamide], were studied using hyperlipidemic rats as an experimental model; hyperlipidemia was developed by intraperitoneal injection of triton Wr-1339 (200 mg/kg body weight). at a dose of 15 mg/kg body weight, bmi2C and DDmi2C significantly reduced elevated plasma triglyceride levels after 7 and 24 h. Furthermore, bmi2C and DDmi2C significantly reduced elevated plasma total cholesterol levels after 24 h. interestingly, high-density lipoprotein-cholesterol levels were significantly increased in all treated groups, this effect may be due to the activation of lipoprotein lipase. these findings indicate that the two studied novel compounds have a promising potential in the treatment of hyperlipidemia and atherosclerosis.


Poster Abstracts

61IntRACEREBRAL tRAnSPLAntAtIOn OF BM-MSCS REduCES AMyLOId-BEtA dEPOSItIOn And RESCuES MEMORy dEFICItS In ALZhEIMER’S dISEASE MICE By MOduLAtIOn OF IMMunE RESPOnSESJ. Bae1, W. Min1, I. Jeon1, H. Jin2; 1Kyungpook National University School of Medicine, Daegu, KOREA, REPUBLIC OF, 2Kyungpook National University, Daegu, KOREA, REPUBLIC OF.

alzheimer’s disease (aD) is characterized by the deposition of amyloid-β peptide (aβ) and the formation of neurofibrillary tangles. transplantation of bone marrow-derived mesenchymal stem cells (bm-msCs) has been suggested as a potential therapeutic approach to prevent various neurodegenerative disorders, including aD. However, the actual therapeutic impact of bm-msCs and their mechanism of action in aD have not yet been ascertained. the aim of the present study was therefore to evaluate the therapeutic effect of bm-msC transplantation on the neuropathology and memory deficits in amyloid precursor protein (aPP) and presenilin 1 (Ps1) double-transgenic mice. Here we show that intra-cerebral transplantation of bm-msCs into aPP/Ps1 mice significantly reduced aβ deposition. interestingly, these effects were associated with restoration of defective microglial function, as evidenced by increased aβ-degrading factors, decreased inflammatory responses, and elevation of alternatively activated microglial markers. Furthermore, aPP/Ps1 mice treated with bm-msCs had decreased tau hyperphosphorylation and improved cognitive function. in conclusion, bm-msCs can modulate immune/inflammatory responses in aD mice, ameliorate their pathophysiology, and improve the cognitive decline associated with aβ deposits. these results demonstrate that bm-msCs are a potential new therapeutic agent for aD.

63EXPAnSIOn OF huMAn MESEnChyMAL StEM CELLS In A nOVEL SERuM-FREE And XEnO-FREE CuLtuRE SyStEML. Chase1, S. Boucher2, Y. Zheng3, A. Campbell4, J. Bradford5, U. Lakshmipathy6, M. Vemuri2; 1Life Technologies, Madison, WI, 2Life Technologies, Frederick, MD, 3National University Singapore, Singapore, SINGAPORE, 4Life Technologies, Grand Island, NY, 5Life Technologies, Eugene, OR, 6Life Technologies, Carlsbad, CA.

as human mesenchymal stem cells move towards clinical application as a cell-based therapy, the need for clinical-grade culture reagents has become an absolute necessity. in response to this need, we have developed a novel xenogenic (xeno)-free and serum-free culture system (stemPro® msC sFm XenoFree), designed specifically for the expansion of human mesenchymal stem cells (msCs). When grown under optimized conditions, human bone marrow-derived msCs (bm-msCs) expanded using a completely xeno-free and serum-free workflow (including stemPro® msC sFm XenoFree complete medium, Cellstart™-coated flasks and cell harvest with the recombinant enzyme tryple™) revealed an expansion rate similar to msCs expanded in 10% msC Qualified Fbs-containing medium. Cells expanded under both conditions revealed a similar phenotype when analyzed by multiplex flow cytometry, gene array analysis, mesoderm differentiation assays and karyotype analysis. in addition, primary msCs can be isolated using xeno-free/serum-free medium from ficoll-purified bone marrow mononuclear cells. to further enhance the xeno-free/serum-free culture system, we developed a protocol using a chemically defined, animal-origin free substrate that, when added directly to stemPro msC sFm XenoFree medium, can support the expansion of msCs without the need for pre-coated flasks. Cells expanded using this improved workflow displayed an expected phenotype, including a standard cell surface antigen profile and retained multi-lineage mesoderm differentiation potential. Finally, application of the xeno-free/serum-free culture system on multipotent adipose-derived msCs revealed successful expansion and retained differentiation potential.

62StEMPRO® LIPOMAX™, A dEFInEd XEnOFREE LIPId-RICh SuPPLEMEnt FOR PRIMARy And StEM CELL RESEARCh And thERAPy APPLICAtIOnSS. Boucher1, L. Chase2, M. Vemuri1; 1Life Technologies, Frederick, MD, 2Life Technologies, Madison, WI.

Cellular processes in primary and stem cells require a diverse panel of biochemical components to maintain membrane equilibrium, preserve somatic homeostasis, and retain self-replicating capability. one critical component for efficient cellular activity is the presence of lipid molecules. as primary and stem cell culture systems evolve to more defined media systems without serum, it becomes necessary to add back lipid components to compensate for the loss of lipid-rich materials in serum. to address this gap, we developed a defined xenofree cell culture lipid supplement - stemPro® lipomaX™. lipomaX is a proprietary formulation prepared from pooled human plasma. since earlier observations indicated that adipose-derived stem cells (aDsC) expand poorly under serum-free condition, we conducted studies to determine if this xenofree lipid supplement could enhance expansion of these cells. using a serum-based media as a control, we performed a titration curve study to identify an optimal lipomaX dose for enhanced proliferation of aDsC in stemPro msC sFm, a serum-free media system designed for multipotent mesenchymal stromal cells (msC). We observed enhanced expansion when lipomaX was applied at a dilution of 1:50 to 1:200 in serum-free media as compared to serum-containing control. in multi-passage studies, application of lipomaX to serum-free media was found to yield higher total net number of aDsC as compared to unsupplemented serum-free media. in collaboration with a translational research lab, when lipomaX was supplemented to xenofree (XF) media prototype, they observed enhanced aDsC expansion as well. in addition, when aDsC were expanded in XF media prototype supplemented with lipomaX and then induced to undergo adipogenesis or chondrogenesis, there was increased number of lipid-vesicle forming adipocytes and an increase in the size of chondrogenic pellets. results from our studies and others suggest that primary and stem cell function may benefit from addition of lipid-rich supplement in serum-free media systems.

64huMAn uMBILICAL CORd PERIVASCuLAR CELLS: ROLE OF SPECIFIC GEnES In thEIR dIFFEREntIAtIOn tO OStEOBLAStS S. CIAVARELLA, M. DE MATTEO, F. DAMMACCO, F. SILVESTRIS; UNIVERSITY OF BARI MEDICAL SCHOOL, BARI, ITALY.

mesenchymal progenitor cells (mPCs) include a fibroblast-like cell population with both high proliferation rate and multilineage differentiation capacity. recently, human umbilical cord perivascular cells (HuCPVCs) have been proposed for bone regeneration though a better understanding of the mechanisms driving their differentiation is necessary to improve the osteogenic potential. Here, we investigated the regulation of major genes, such as PParβ, runx2 and their co-regulator taZ, that are reciprocally involved in the in vitro adipocyte or osteoblast differentiation of HuCPVCs. adherent fibroblastic-like cells from the umbilical cord stroma, were analyzed for the expression of specific mesenchymal markers and cultured in appropriate media to induce either adipocyte or osteoblast differentiation. During the 35 day period of culture, the cells were periodically evaluated by fluorescence microscopy to verify their cytoskeletal features and by real time rt-PCr to measure the mrna levels of runx2, PParβ, as well as of both osteocalcin and adiponectin, namely the downstream genes. in addition, both transcriptional and protein levels of taZ, and its cellular localization were evaluated. undifferentiated HuCPVCs co-expressed high levels of mesenchymal antigens showing their typical F-actin stress fibers pattern. after the induction, osteogenic cells showed higher content of condensed fibers of F-actin as compared to those undergoing adipocyte differentiation. also, they became positive to the alizarin red staining and expressed higher levels of runx2 with respect to PParβ. in osteogenic differentiated HuCPVCs, taZ was expressed at much higher magnitude than in adipogenic-induced cells and this accumulation in nuclei was related to its primary role in activating runx2. thus, the HuCPVC differentiation is regulated by specific transcriptional programs that include exclusive genes in their final osteogenic fate.


may 23-26, 2010


Poster Abstracts

65tRAnSCRIPtOME PROFILInG AFtER tRAnSPLAntAtIOn OF huMAn AdhEREnt AduLt PROGEnItOR CELLS IntO RAt MOdELS OF ISChEMIC CnS InJuRyJ. Hamilton1, R. Mays1, A. Ting1, S. Savitz2, J. Carroll3, R. Deans1; 1Athersys, inc, Cleveland, OH, 2University of Texas, Houston, Houston, TX, 3Medical College of Georgia, Augusta, GA.

stem cell transplantation is emerging as a therapeutic treatment following ischemic insult to the Cns. little is known regarding the mechanistic interaction between the injured tissue environment and transplanted cells. We have previously demonstrated functional improvement in animal models of Cns ischemia after iV infusion of multistem®, an adherent pluripotent adult stem cell product. We have examined transcriptional changes in the injured tissue environment using microarray and qPCr. Human multistem® was infused intravenously into two Cns animal models: adult rats subjected to mCa occlusion (vehicle or 4 million cells, 24 hours after surgery); or neonatal rats subjected to hypoxic-ischemic injury (vehicle, 100,000 or 1 million cells, administered 7 days after injury). animals were sacrificed 3 days after infusion. rna was isolated from brain, lung, and spleen, and was subjected to qPCr and microarray analyses to identify gene expression changes. qPCr analysis revealed detectable human mrna sequences within brain, lung, and spleen, but not at sufficient levels to examine by microarray. rat-specific microarray analyses of brain mrna identified broad effects of multistem® treatment: in both injury models, approximately ½ of the genes that were significantly induced by injury were attenuated by multistem®; conversely, approximately 1/3 of the genes that were significantly downregulated by injury were induced by multistem®. Further examination of these changes identified multiple gene ontology classifications and pathways that are significantly impacted in multistem®-infused compared to vehicle-infused animals, including immune response, cell proliferation, and cell death / apoptosis. of particular note was the down regulation of receptors on inflammatory cells and activated endothelium, which correlates with in vitro studies demonstrating that multistem® reverses selectin and adhesion receptor induction mediated by multiple inflammatory pathways. the observed gene expression changes suggest multiple mechanisms through which functional benefit from stem cell treatment of ischemic injuries of the Cns may be achieved.

67thE POtEntIAL uSE OF MESEnChyMAL PROGEnItOR CELLS FROM AdIPOSE tISSuE FOR CLInICAL APPLICAtIOnSR. Eshel, E. Zigman-Hoffman, A. Faragy, G. Senior, E. Gur, B. Meilik, E. Naparstek;

tel-aviv sourasky medical Center, tel-aviv, israel.mesenchymal Progenitor Cells (mPCs) are non-hematopoietic stromal cells that can be found in many organs and are capable of differentiating into mesenchymal tissues such as bone, cartilage, skeletal muscle and adipocytes. Due to their multilineage differentiation, easy access and lack of immune response, mPCs have various potential therapeutic applications such as tissue regeneration.the aim of our study was to develop a safe and clinically applicable method for the isolation, preservation and thawing of adipocyte derived mPCs for reconstructive applications.For that purpose, mPCs were isolated from fat tissue after liposuction procedure using collagenase digestion. the isolated cells were used to establish long-term culture either immediately following the harvest, or after cryopreservation and thawing.the in vitro cultures were established in Dmem containing 10%FCs for three weeks. the adherent cells were trypsinized and analyzed by flow-cytometry.both freshly isolated and cryopreserved and thawed cultured cells showed a typical membrane phenotype characteristic for mPCs with median expression of CD90+(94%), CD73 +(93%), CD105+(97%), CD45- and CD34-.We conducted a clinical trial in order to asses the safety and the potential use of fresh or thawed adipose tissue derived autologous mPCs for minor aesthetic applications such as wrinkle filling. twenty patients (19 F, 1m, median age 47±17) were recruited for study, 10 received single injection of fresh mPCs and 10 received a second injection of thawed cells. no side effects were reported and after a follow-up of 10 months stable improvement was evident in most of the treated patients.our results confirm previous observations and demonstrate that adult mPCs can be easily isolated from fat tissue and potentially used as a safe source of autologous cells for soft tissue filling.moreover, our data clearly show that these cells can be cryo-preserved for further use while maintaining their potential implementation in reconstructive surgery.

66A COMPARAtIVE Study OF EFFECt OF thE OXyGEn MICROEnVIROnMEnt On In VItRO thE AnGIOGEnESIS OF huMAn AdIPOSE tISSuE- OR uMBILICAL CORd-dERIVEd StROMAL PROGEnItOR CELLSB. Kim1,2, J. Kim1, S. Han1, H. Park1, Y. Yoon2, B. Do1; 1HurimBioCell Inc., Seoul, KOREA, REPUBLIC OF, 2Dept of Life Science, Hanyang University, Seoul, KOREA, REPUBLIC OF.

mesenchymal stem cells (msC) have an excellent therapeutic potential because of their multipotency and self-renewal properties in many reports. several approaches have been reported the neovascularization ability of msC in different condition. to regeneration of the angiogenic defect, numerical amount or stably differentiated progenitor endothelial cells is necessary. the present study was designed to determine the angiogenic ability of human adipose tissue (hat-sPCs) or umbilical cord derived stromal progenitor cells (huC-sPCs) on oxygen condition in vitro assay. Commercially obtained human umbilical vein endothelial cells (HuVeC) were used as a positive control group for in vitro angiogenesis assay. isolated hat-sPCs and huC-sPCs were grown to confluence and seeded into matrigel-coated wells and exposed to normoxia or hypoxia followed by angiogenic media exchange. after 6 hours, cord-like structures were recorded and lengths of induced structure were measured. recovered cells by dispase were analyzed by polymerase chain reaction (PCr) for gene expression of hypoxia inducible factor-1 (HiF-1) or vascular endothelial growth factor (VegF). and endothelial cell marker, CD31 and vWF, expression were examined by immunocytochemistry. a msC profile was confirmed by flow cytometry with the representative markers CD31, CD34, CD45, and CD90 was determined in the both cells. the cultured hat-sPCs showed positive expression for CD34, CD90 and negative expression for CD31, CD45. meanwhile, the cultured huC-sPCs showed positive for CD73, CD90 and negative for CD31, CD34 and CD45. mesh-like structure formation appeared in the positive control group (HuVeC) and hat-sPCs group under normoxia. although huC-msCs group did not showed any cord-like structure under normoxia, differentiated into endothelial-like cells under hypoxia. besides, endothelial marker expressions and hypoxia-induced gene expressions were accompanied by morphological changes. these results showed that hat-sPCs and huC-sPCs have a different ability in different microenvironments for regeneration of angiogenic structure.

68StEM CELL LInES FROM CORd BLOOd, BOnE MARROW And PLACEntA ShOW dIFFEREnCES In GEnE EXPRESSIOn And FunCtIOnG. Brooke1, F. Zaibak2, T. Rossetti1, R. Pelekanos1, A. Chalk3, C. Wells3, N. Mattigan3, C. Filby2, R. Williamson2, K. Atkinson1; 1Mater Medical Research Institute, Brisbane, AUSTRALIA, 2Murdoch Childrens Research Institute, Parkville, AUSTRALIA, 3University of Queensland, Brisbane, AUSTRALIA.

rational: multipotent stem cells from placenta and cord blood share many properties with bone marrow mesenchymal stem cells (msC), and have been suggested as a useful alternative for cell therapy. the exact relationship between the cells is unknown.methods: the differentiation potential, immunosuppressive properties and gene expression profiles of four independently derived human cord blood, placental and bone marrow stem cell lines were compared to fibroblast lines.results: We show the stem cells lines from each tissue source have unique differentiation and immunosuppressive potentials. at the global gene expression level, the stem cell lines were similar, sharing 93% of genes expressed. Principal component analysis confirmed that cells from each of the three tissues are unique, and that all differ from fibroblasts. interestingly, one of the four cord blood lines clustered with bone marrow, suggesting multiple stem cell populations may exist in cord blood. Plurinet analysis suggests that cord blood lines have a signature containing more pluripotency-associated genes. using DaViD analysis, a number of differentially expressed markers that may distinguish the different cell lines were identified, including genes that could explain differences observed in the capacity of the lines to mediate immunosuppression and adipogenesis.Conclusion: although bone marrow, cord blood and placental stem cell lines share many characteristics, each expresses a unique subset of genes associated with differences in cell function.


Poster Abstracts

69FORMAtIOn OF EndOdERM And LunG EPIthELIAL CELLS FROM CORd BLOOd-dERIVEd StEM CELLSC. Filby1, N. Elwood1, P. Van Kooy2, J. Kozlovski1, R. Williamson1, F. Zaibak1; 1Murdoch Childrens Research Institute, Parkville, AUSTRALIA, 2University of Melbourne, Parkville, AUSTRALIA.

rationale: unrestricted somatic stem cells (ussCs), derived from umbilical cord blood collected at birth are able to expand for >50 population doublings while retaining the potential to differentiate into cells of all three germline layers. However, differentiation of ussCs into endodermal or lung cell progenitors, has never been demonstrated. this study aimed to determine if ussCs can form endodermal or lung epithelial cells.methods: ussC lines were cultured in: (1) small airway growth medium (sagm) for 7, 14 or 21 days; or (2) serum-free media containing 100 ng/ml activin a for 3 days. Controls were cultured in either serum-free media or ussC media. expression of endoderm/lung development markers were determined by PCr (ttF1, sox17, Foxa2, gata6, CFtr & sP-C). induced ussCs were assessed for their ability to form cells representative of each of the three germline layers.results: undifferentiated ussC lines do not express CFtr, sox17, or sPC, but do express gata6. ussCs cultured in sagm express sP-C, but not CFtr, sox17 or ttF1. activin a induction does not increase the expression of sox-17, Foxa2, ttF-1, sP-C or CFtr in ussCs. activin a-induced ussCs retain the capacity to form all three germline layers following directed differentiation. Preliminary analysis indicates that ussCs express activin a receptors, but also express many activin a inhibitory ligands.Conclusions: although sagm-treated ussCs express sPC, they do not express CFtr. Furthermore, unlike human embryonic stem cells, activin a treated-ussCs do not form sox17+ endoderm. together, these results indicate that ussCs are inherently different to hesCs.

71MESEnChyMAL StEM CELLS thERAPy FOR REFRACtORy EXtEnSIVE CGVhdC. Fu, D. Wu, B. Gu; Jiangsu Institute of Hematology, Suzhou, CHINA.

obJeCtiVe: to assess whether treatment with mesenchymal stem cells (msCs) is an effective adjunct therapy for refractory extensive chronic graft-versus-host disease (cgVHD) resistant to conventional therapy.metHoDs: 3 patients with steroid-resistant extensive cgVHD were treated with msCs. one patient received one dose, two received three doses. the msCs were obtained from third-party Hla-mismatched cord blood .the first dose of msCs was 4 x 107/kg, the second dose was 5 x 107/kg, and the third dose was 5x 107/kg. Patients were observed for 4 weeks before next infusion. meanwhile the proportion of CD3+, CD4+, CD8+, CD19+, CD4+, CD25+, FoXP3+, FoXP3+CD4+ and FoXP3+CD25+ was examined with double fluorescent-labeled antibodies and flow cytometry before and 4 weeks after the msCs infusion.results: no patients had side-effects during or immediately after the infusions of msCs,except that once one patient got fever during the infusion and was completely controlled. one patient with three doses of treatment had complete response, and the other got partial response, the third one is being observed. Complete resolution was seen in the involvement of skin,liver,gastro intestine. Partial response was seen in the improvement of skin,joints,oral cavity and eye. response rate was not related to donor Hla-match. the ratio of CD4/CD8 and the proportion of regulatory t cells were significantly higher than that before msCs treatment.ConClusion: this finding has practical implications and suggests that third-party cells can be prepared and stored frozen to be used for steroid-resistant extensive cgVHD therapy. it is concluded that msCs may prevent the lethal cgVHD after allogeneic hematopoietic stem cell transplantation and raise the survival rate by increasing the ratio of CD4/CD8 and proportion of regulatory t cells in vivo.

70A SInGLE InJECtIOn OF ALLOGEnEIC MESEnChyMAL StROMAL CELLS IS SuFFICIEnt tO RESCuE LEthALLy IRRAdIAtEd MICE By PROMOtInG REGEnERAtIOn OF thE GAStROIntEStInAL EPIthELIuM And REStORInG EndOGEnOuS hAEMAtOPOIESIS.M. François, E. Birman, J. Galipeau; McGill University - Lady Davis Institute, Montreal, QC, CANADA.

mesenchymal stromal cells (msC), which are pluripotent stem cells located in the stroma of various tissues, possess regenerative and haematopoietic properties currently investigated in many clinical applications. in the present study, we investigated the ability of bone marrow-derived msC to protect from and/or repair radiation-induced injuries in a mouse model of lethal gamma radiation exposure. a single injection of 6x106 C57bl/6 msC given 24 hours following 9gy total body irradiation protected all treated-mice while 50% mortality was recorded in the Pbs control group. Histological analysis of the gastrointestinal (gi) epithelium performed at day 5 post msC/Pbs injection revealed that villis of the Pbs group were half the size the villis of the msC group. Ki-67 immunostaining also indicated an elevated amount of proliferating gi stem cells at day 5 in the msC group compared to the control group. Hematological analyses also demonstrated that msC protected irradiated mice from developing anemia in addition to accelerating granulocytes recovery. Histological analyses performed on bone marrow of the femur revealed zones of regeneration by day 17 post-treatment in the msC group while bone marrow of the control group remained hypocellular. Furthermore, mice treated with msC presented enlarged spleen containing a total number of lin-/C-kit+/sca-1+ hematopoietic stem cells significantly higher than the Pbs group and comparable to the non irradiated mice, which clearly indicated the presence of extramedullar haematopoiesis. amongst the possible factors secreted by msC implicated in the regeneration of the bone marrow and the gut, stromal-derived factor 1 (sDF-1/Cxcl12), highly express in msC, has been reported to interact via its receptor CXCr4 with haematopoietic and epithelial stem cells. in conclusion, these results clearly demonstrated the potential of msC as a complementary treatment for radiotherapy or for the treatment of accidental radiation exposure.

72thE OPtIMAL MESEnChyMAL CELL PROduCt: IS BOnE MARROW thE AnSWER?F. Grynspan1, M. Meiron1, N. Drori-Carmi1, H. Leibowitz1, E. M. Horwitz2; 1Pluristem Therapeutics Inc, Haifa, ISRAEL, 2The Children Hospital of Philadelphia and the University of Pennsylvania School of Medicine, Philadelphia, PA.

the importance of mesenchymal and mesenchymal-like stromal cells (msCs) in future cell therapeutics is irrefutable. the role of these cells in tissue repair, immune modulation, and angiogenesis is well established in pre-clinical and clinical studies. However, the optimal cell source and the characteristics required of the expanded cells to constitute a viable cell product remain unclear. the preparation of bone marrow-derived msCs requires marrow collection which is an invasive procedure for a volunteer donor. in contrast, placenta is a rich source of msC-like cells which utilizes an ongoing supply of readily available tissue, the placenta, without burdening a donor. moreover, placenta, as a cell source; allows for the ex vivo generation of a very large number of cells with limited population doublings allowing for easily standardized product characteristics with minimizing the risks associated with substantial ex vivo expansion protocols. Placenta adherent stromal Cells (PasCs) (n>50 samples) were prepared from placenta obtained from full-term deliveries and characterized for the expansion profile, the immunophentype (including adherent, progenitor and immunogenic markers), the immunomodulatory effect on lymphocyte subpopulations (CD4, CD8, CD4+CD25+FoxP3+), secretion levels of inflammatory cytokines (iFnβ, ββFβ, il-10, il-5, il-4, il-2) and angiogenic growth factors. the most salient results included a PasCs-associated reduction of t lymphocyte proliferation of 10-50% from controls upon alloreactive and mitogenic stimulation. the decrease in mnC proliferation was accompanied by a 70-fold reduction of tnFβ secretion. VegF secretion, a prototypic angiogenic factor, increased from 0.023-1.5 ng/106 cells under normoxic conditions to 1.4 to 5.0 ng/106 cells in hypoxic culture. Complete results from the analysis of this extensive database will be presented. animal models as well as initial results of early phase clinical trials suggest that PasCs comprise easily manufactured, highly potent cell therapy for tissue repair.


may 23-26, 2010


Poster Abstracts

73IMMunOMOduLAtIOn PROPERtIES OF PLACEntA dERIVEd AdhEREnt StROMAL CELLS.F. Grynspan1, N. Netzer1, M. Meiron1, P. Reinke2, H. Volk2; 1Pluristem Therapeutics Inc, Haifa, ISRAEL, 2Charité - Universitaetsmedizin Berlin and The Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Berlin, GERMANY.

mesenchymal stromal cells (msCs) role in tissue repair in both pre-clinical and clinical studies has made these cells natural candidates for cellular therapeutic product development, albeit their mechanism of action remains largely unknown. msCs self-renewal capacity and multi-lineage differentiation have been extensively studied but an emerging body of data suggests that paracrine and immunomodulatory activities play a major role in their therapeutic properties. the immunomodulatory effect of Placenta derived adherent stromal cells (PasCs) on mnCs was studied following polyclonal allogeneic t cell and mitogen stimulation (PHa, Con a and anti CD3/ CD28). the reduction in CD4+ and CD8+ t cell proliferation was accompanied by a reduction in tnFβ and iFnβ. il-10 secretion was unaffected in these systems although a high increase in the secretion of il-10 alongside a significant reduction in tnFβ was measured following lipopolysaccharide (lPs) stimulation. t cell response to recall antigens in the presence of PasC was tested with mnCs cell derived from four normal donors. PasC had no effect on the memory CD4+ cell response to recall antigens and no significant changes were observed in the secretion levels of six th1/th2 cytokines including iFnβ, ββFβ, il-10, il-5, il-4, and il-2 . mnCs stimulated with a CmV peptide in the presence of PasCs showed a 70% reduction in CD8+ t cells degranulation, suggesting that PasCs affect the antigen-specific cytotoxic response. nK cell mediated cytolysis activity was not affected by PasCs, as measured by the calcein-labeled nK sensitive target cells at different mnCs/target ratios. Furthermore, allo and mitogenic stimulations enhanced the CD4+CD25+FoxP3+ cell population suggesting an induction of regulatory t cells. taken together, it is likely that PasCs mechanism of action involve selective immunosuppression properties which helps modulate immune responses.

75CO-CuLtuRE OF huMAn uMBILICAL CORd BLOOd dERIVEd MESEnChyMAL StEM CELLS REduCES EndOtOXIn-InduCEd APOPtOSIS OF ALVEOLAR EPIthELIAL CELLS And InFLAMMAtORy REACtIOn OF ALVEOLAR MACROPhAGES In VItRO J. Ha, S. Choi, W. Oh, Y. Yang, H. Jeon; Medipost Biomedical Institute, Seoul, KOREA, REPUBLIC OF.

Despite extensive research into the pathogenesis of acute lung injury and the acute respiratory distress syndrome (ali/arDs), efficient and innovative therapies have not been developed. Current treatment is primarily supportive, with lung protective ventilation and a fluid conservative strategy. Pharmacologic therapies that reduce the severity of lung injury in experimental study have not yet been translated into effective clinical treatment options. in this study, we firstly investigated the anti-apoptotic effect of human umbilical cord blood-derived mesenchymal stem cells (huCb-msCs) on endotoxin treated alveolar epithelial cells (a549). as a result, huCb-msCs down-regulated apoptosis of a549 caused by high dose of lipopolysaccharide (lPs) treatment (250 µg/ml) compared with that of a549 single culture group. in addition, in order to examine anti-inflammatory function of huCb-msCs, another co-culture study with huCb-msCs and lPs treated alveolar macrophages cell line (nr8383) was performed, which resulted in reduction of CinC-1 and tnF-β level via direct interaction manner. However, these effects of huCb-msCs were not replicated by the other fibroblast as negative control. in conclusion, huCb-msCs markedly protected endotoxin-induced apoptosis of alveolar epithelial cells and reduced pro-inflammatory cytokine level from stimulated alveolar macrophage by endotoxin, which suggested that huCb-msCs can have therapeutic potentials and be applicable treatment for ali/arDs.

74BOnE MARROW MESEnChyMAL StEM CELL thERAPy FOR uRInARy InCOntInEnCE: In VItRO StudIESM. Gunetti, I. Ferrero, D. Rustichelli, K. Mareschi, E. Errichiello, F. Fagioli; Stem Cell Transplantation and Cellular Therapy Unit; Pediatric Onco-Hematology Division, Regina Margherita Children’s Hospital, Turin, ITALY.

background. urinary incontinence (ui) is an involuntary leakage of urine. some studies have pointed out the possibility of treating ui with stem cells from muscle biopsies.the particular characteristics and high plasticity of bone marrow (bm) msCs make them ideal candidates in cell therapy strategies to treat a number of degenerative and post-traumatic diseases caused by damage or cell loss, such as ui.aims. aims of the study were to:- evaluate the expression of myogenic markers in bm msCs.- evaluate the in vitro capacity of msCs to differentiate into muscle cells.methods. msCs isolated from healthy donors bm were in vitro cultured using some myogenic differentiation mediums. these data were compared to msCs cultured in stemness maintaining medium through morpghological analysis, immunohistiochemical staining and molecular assays.results. msCs isolated and cultured in myogenic differentiation mediums, were positive for myogenic markes: myogenin, myosin, Desmin, sarcomeric alpha actin, myosin Heavy Chain, β-sma. msCs cultured in stemness maintaining medium showed the same expression profile.immunofluorescent data were confirmed by early and late myogenic gene expression profile (β-sma, miogenin, myoD, myf 5, myf6, m-caderin) by real time rt-PCr and early and late myogenic protein expression (myogenin, myosin, Desmin, sarcomeric alpha actin, myosin Heavy Chain, β-sma) by Westwern blot analysis.msCs cultured in stemness maintaining medium were also positive, as resulted by PCr analysis, for the presence of Ca2+ l channels.Conclusions. taking together all these data demonstrated that basal msCs, cultured in stemness maintaining medium, express early myogenic gene and markers. this could be a very important starting point to use bm msCs in cell therapy for myogenic repair. our future aims will be to optimize ex-vivo myogenic differentiation cultures using specific feeder-layer or using co-cultures of msCs and muscle cells.

76FunCtIOnAL ChARACtERIStICS OF huMAn AduLt BOnE MARROW-dERIVEd SOMAtIC CELLS: A unIQuE POPuLAtIOn OF MESEnChyMAL SuPPORt CELLS B. Heimbach, G. Kopen, V. Ragaglia, W. Righter; Garnet BioTherapeutics, Malvern, PA.

We have developed a novel, proprietary process for the isolation and expansion of non-hematopoietic allogeneic human adult bone marrow-derived somatic cells (habm-sC) in commercial quantities. Human abm-sC are stable mesenchymal support cells that do not form unwanted tissues when administered in vivo. We describe here some functional aspects of these cells.abm-sC secrete a wide variety of factors, including known promoters of angiogenesis; angiogenin, il-8 and VegF. We assessed the ability of abm-sC to alter angiogenesis, specifically the ability to induce migration of endothelial cells. Cells from different levels of expansion and multiple product level lots were compared. Human abm-sC induced migration of endothelial cells, supporting the hypothesis that they may play a role in angiogenesis. this effect was evident across multiple levels of expansion and product lots demonstrating consistency in the culture process and supporting homogeneity of the cell banks. the physical presence of the cells was not required for the effect to be observed suggesting that angiogenic effects of abm-sC in-vivo may persist long after the cells are gone.Contraction of three-dimensional collagen gels in-vitro is a biological process that mimics events observed in dermal wound healing. in the present study we assessed whether or not habm-sC can contract collagen gels when formulated immediately following removal from cryostorage. habm-sC-embedded gels contracted in a concentration- and time-dependant manner. gels embedded with heat-inactivated cells did not contract over time, demonstrating that the process of gel contraction requires functional engraftment.these data support the application of habm-sC in biological situations were endothelial migration or enhanced wound healing are therapeutically beneficial.


Poster Abstracts

77COMPARAtIVE Study OF MESEnChyMAL StEM CELLS dERIVEd FROM ARtERIAL, And VEnOuS, And WhARtOn’S JELLy EXPLAntS OF huMAn uMBILICAL CORdI. ISHIGE1, T. Nagamura-Inoue1, M. J. Honda2, A. Tojo1; 1Institute of Medical Science, University of Tokyo, Tokyo, JAPAN, 2Nihon University, Tokyo, JAPAN.

baCKgrounD: mesenchymal stem cells (msCs) can be isolated from several tissues types such as bone marrow (bm), umbilical cord blood (uCb), umbilical cord (uC), and so on. of these, uCb and uCs have advantages because of their easy availability, non-invasiveness. Despite some of the practical advantages of the uC, however, the best components of the uCs from which to isolate msCs for clinical applications remain controversial. to find the best part in uC for clinical use, we compared the characteristics of the migrating cells from Wharton’s jelly (uCWJ), vein (uCV), and arteries (uCa).metHoDs: uCs were dissected, diced into small fragments, and aligned in explants cultures from which migrating cells were isolated. isolated cells from each three components were determined their surface markers, their relative capacities for proliferation, and multi-lineage differentiation (osteogenesis, adipogenesis, and chondrogenesis). mixed lymphocyte reaction (mlr) was also performed.results: the fibroblast-like, spindle-shaped cells were cultured from each components, which negative for CD31, CD34, CD45, CD271, and Hla-class ii, but positive for CD13, CD29, CD44, CD73, CD90, CD105, and Hla-class i. uCV cells exhibited a significantly higher frequency of CFu-Fs than did uCWJ and uCa cells. individual msCs could be selectively differentiated into osteoblasts, adipocytes, and chondrocytes. When compared for osteogenic potential, uCWJ cells were the least effective precursors, whereas uCa-derived cells developed alkaline phosphatase activity with or without an osteogenic stimulus. the uCWJ cells inhibited the lymphocyte proliferation for responder CD4+ and CD8+ cells upon the stimulation in mlr.ConClusions: Cells derived from three different components exhibited msC-like phenotypes and capacity for multi-lineage differentiation with varying precursor potentials. uC, especially uCV and uCa, could provide a better source of msCs than bm for experimental and clinical applications. Further investigation is necessary to determine the potentials for the regeneration of organs which are important for clinical applications.

79IL-8 MEdIAtEd huCB-MSCS MIGRAtIOn tOWARd u87-MG, huMAn GLIOMA CELL.D. Kim, J. Kim, Y. Yang, W. Oh, J. Chang; MEDIPOST, Seoul, KOREA, REPUBLIC OF.

Human umbilical cord blood mesenchymal stem cells (huCb-msCs) are being widely used as therapeutic cell source in clinical trial. recently, we found that huCb-msCs can migrate toward several glioma cell lines by interleukin-8 (il-8). recombinant il-8 enhanced migration of huCb-msCs toward u87mg however pretreatment of il-8 receptor antibody reduced its migration in a transwell chamber. in addition, when expression of il-8 in u87mg cells was down regulated by sirna, il-8 expression and tropism of huCb-msCs toward u87mg were decreased. since tropism of huCb-msCs was reduced by il-8 sirna, il-8 receptor, CXC chemokine receptor 1 (CXCr1) was overexpressed in huCb-msCs to evaluate migration capacity toward u87mg compared to control huCb-msCs. huCb-msCs expressing CXCr1 showed enhanced migration capacity toward to u87mg and il-8. Collectively, we suggested that glioma tropism of huCb-msCs is mediated by il-8 and CXCr1.this study was supported by a grant from the national r&D Program for Cancer Control, ministry for Health, Welfare and Family affairs, republic of Korea (0820040)

78BM-MSCS PREVEnt thE LOSS OF nIEMAnn-PICK tyPE C MOuSE PuRKInJE nEuROnS By CORRECtInG ABnORMAL SPhInGOLIPId MEtABOLISM And InCREASInG SPhInGOSInE-1-PhOSPhAtEH. Jin, W. Min, I. Jeon, J. Bae; Kyungpook National University, Daegu, KOREA, REPUBLIC OF.

niemann-Pick type C (nP-C) disease exhibit neuronal sphingolipid storage and cerebellar Purkinje neuron (Pn) loss. although it is clear that Pns are compromised in this disorder, it remains to be defined how neuronal lipid storage causes the Pn loss. our previous studies have shown that bone marrow-derived mesenchymal stem cells (bm-msCs) transplantation prevent Pn loss in nP-C mice. the aim of the present study was therefore to examine the neuroprotective mechanism of bm-msCs on Pns. We found that nP-C Pns exhibit abnormal sphingolipid metabolism and defective lysosomal calcium store compared to Wt Pns. bm-msCs promote the survival of nP-C Pns by correction of the altered calcium homeostasis, restoration of the sphingolipid imbalance, as evidenced by increased sphingosine-1-phosphate (s1P) levels and decreased sphingosine, and, ultimately, inhibition of apoptosis pathways. these effects suggest that bm-msCs modulate sphingolipid metabolism of endogenous nP-C Pns resulting in their survival and improved clinical outcome in mouse.

80StEM CELL SuRFACE GLyCOMICS tO IMPROVE thE QuALIty And EFFICACy OF CELLuLAR thERAPEutICSA. Kotovuori1, S. Natunen1, A. Heiskanen2, J. Nystedt1, M. Mikkola1, T. Hirvonen1, H. Anderson1, V. Pitkänen1, T. Otonkoski3, P. Lehenkari4, S. Laitinen1, T. Satomaa2, L. Valmu1; 1Finnish Red Cross Blood Service, Helsinki, FINLAND, 2Glykos Finland, Helsinki, FINLAND, 3University of Helsinki, Helsinki, FINLAND, 4University of Oulu, Oulu, FINLAND.

Detailed characterization of cells is essential in achieving cellular therapy products with constant quality, safety and efficacy. While the karyotype and expression of surface markers are routinely examined, and there is plenty of knowledge on the gene expression profiles and proteomes of different types of stem cells, glycosylation of stem cells has received less attention. yet glycosylation largely defines the structure of the cell surface, and thus biological properties of the cell. glycosylation is cell type specific, changes during development and differentiation, and plays important roles in cell adhesion and migration. therefore we expect glycosylation to both reflect the state of a stem cell and to be involved in important biological processes affecting the outcome of stem cell therapy.to get an overview of stem cell glycobiology, we have studied both pluripotent embryonic stem cells and multipotent adult stem cells (mesenchymal and hematopoietic). a panel of complementary methods including mass spectrometry, various immunochemical methods and transcript profiling was used to characterize the glycomes of stem cells.stem cell glycomics has revealed xenocontamination by non-human glycan structures derived from culture media, as well as changes in the glycome upon differentiation. therefore glycosylation reflects key aspects of the quality of stem cell products, and analysis of glycosylation should be included in quality control routines. in addition, we have utilized certain aspects of stem cell glycobiology to achieve improved cellular therapeutics. as glycosylation affects the migratory and adhesive properties of cells, manipulating the stem cell surfaces by altering their glycosylation will result in optimized targeting of stem cells into desired tissues. We have also developed a defined animal product free matrix based on glycomic information, for the culture of embryonic stem cells and induced pluripotent stem (iPs) cells. understanding the glycosylation of stem cells will help to develop more effective and safe therapies.


may 23-26, 2010


Poster Abstracts

81hyPOXIC COndItIOn EnhAnCES PROLIFERAtIOn And MAIntEnAnCE OF uMBILICAL CORd BLOOd MESEnChyMAL StEM CELLS In XEnOFREE MEdIuMM. Lampinen1, A. Laitinen1, H. Anderson1, L. Chase2, S. Boucher2, M. Vemuri2, S. Laitinen1; 1Finnish Red Cross Blood Service, Research and Development, Helsinki, FINLAND, 2Life Technologies, Primary and Stem Cell Systems, Frederick, MD.

background: one challenge for improving the therapeutic utility of adult stem cells is in vitro expansion under animal component-free condition without diminishing their self-renewal and tissue-specific lineage differentiation capacity. umbilical cord blood mesenchymal stem cells (uCb-msC) have high expansion capacity but after several cell divisions, they reach senescence and their phenotype is changed, which likely reflects altered cellular function.aims: to meet the challenge of expanding cells in serum-free medium, we lowered the oxygen pressure in cell culture down to 3%. this mimics the physiological hypoxia of tissues and stem cell ‘niches’, since several in vitro and in vivo studies have demonstrated that hypoxia has an important role in regulating stem cell proliferation.results: using a novel xeno-free medium, stemPro®msC sFm XenoFree medium (XF) from invitrogen, we demonstrated expansion of different uCb-msC donor cell lines under normoxic and hypoxic condition. low oxygen enhanced the growth of all cell lines tested and enhanced their replicative life span compared to their normoxic counterparts. one donor line demonstrated growth throughout the multi-passage study. there was some variability in expansion rates among the three donor cell lines. the benefit of hypoxia on cell growth was even more pronounced with one donor line grown in XF than observed with in earlier experiment using standard serum-containing medium.Flow cytometry analysis of commonly used msC markers showed unaltered phenotype for all donor cell lines, in both oxygen conditions, throughout the culturing steps. moreover, osteogenic and adipogenic differentiation capability were well retained under normoxic and hypoxic culture conditions up to 5 passages in XF.Conclusions: stemPro®msC sFm XenoFree medium is well-suited for expansion of uCb-msC. all donor cell lines demonstrated expansion up to seven passages and maintained their stem cell phenotype. We observed that hypoxia-enhanced cell proliferation was more pronounced in XF than in standard serum-containing medium.

83MuLtIPOtEnt MESEnChyMAL StEM CELLS FROM AMnIOtIC FLuId ORIGInAtE nEuRAL PRECuRSORS WIth FunCtIOnAL VOLtAGE-GAtEd SOdIuM ChAnnELS And ShOW hIGh IMMunOMOduLAtIOn PROPRIEtIESK. Mareschi1,2, M. Muraro1, D. Rustichelli1, C. Cravero1, V. Comunanza3, E. Carbone3, R. De Fazio4, S. Sdei4, C. Benedetto4, F. Fagioli1; 1Pediatric Onco-Hematology Department, Regina Margherita Children’s Hospital, Torino, ITALY, 2Department of Pediatrics, University of Turin, Torino, ITALY, 3Department of Neuroscience, NIS Centre, University of Turin, Torino, ITALY, 4Department of Gynecology and Obstetrics, University of Turin, Torino, ITALY.

objective. We investigated whether human amniotic fluid (aF) contains mesenchymal stem cells (msCs) and evaluated their phenotypic characteristics, differentiation potential and immunomodulation proprieties in vitro comparated with msCs isolated from bone marrow (bm).methods. aF was harvested during routine prenatal amniocentesis. We evaluated cellular growth, immunophenotype, stemness markers and differentiative potential during in vitro expansion. regulatory CD4+ CD25+ t cell activity and maturation of Dendritic Cells (DCs) collected from PbmCs of healthy donors were evaluated in co-colture with msCs isolated from human aF and bm to study the immunomodulating effects.results. aF msCs showed a high proliferative potential, were positive for CD90, CD105, CD29, CD44, CD73, CD166; showed oct-4 and nanog molecular and protein expression and differentiated into osteoblasts, adypocytes and chondrocytes. in neural Progenitor mantainnce medium aF-msCs expressed neural markers and increased na+ channel density. inactivation of the ttX-sensitive channels accelerated and became more similar to native neuronal voltage-gated na+ channels. CD4+CD25+ regulatory t cells significantly increased in presence of msCs; in fact CD4+CD25+ t cells in the control group were 5.5%, 23.4% in co-culture with aF-derived msCs and 16% in co-culture with bm-derived msCs.msC co-cultures strongly inhibit the differentiation on monocytes to DCs. there was a significant reduction in the expression of CD83 mature DCs treated with msCs, suggesting their skew to immature status. a decreased expression of presentation molecules (Hal-Dr) and costimulatory molecules (CD80 and CD86) were also observed.Conclusion. these data suggest that aF is an important multipotent stem cell source with a high proliferative potential able to originate potential precursors of functional neurons. moreover, these cells showed a stronger immunosuppressive effect compared to the bm-derived msCs. this effect was mediated by inducing the generation of CD4+CD25+ regulatory cells and by suppressing monocyte differentiation into DCs, thus indicating the important role of aF-msCs on immunoregulation.

82AnALytICAL MEthOd VALIdAtIOn OF FLOW CytOMEtRy-BASEd IdEntIFICAtIOn And PuRIty tESt FOR huCB-MSCSI. Lee, M. Jung, W. Oh; Biomedical Research Institute, MEDIPOST Co., Ltd., Seoul, KOREA, REPUBLIC OF.

the flow cytometry assay is widely being used to examine the cell identification and purity test for cell therapy. However, the analytical method validation of flow cytometry assay is limited because of the absence of suitable guidance and reference materials. Due to the nature of the cell types measured by flow cytometry, it is critical to select a fit-for-purpose reference cells suitable to identify the intended target cells. Having successfully completed validation steps for human umbilical cord blood-derived mesenchymal stem cells(huCb-msCs), we selected C2C12(mouse myoblast cell line) as placebo cells expected structurally similar to or closely related to the huCb-msCs but a positive response is not obtained. also, we selected human peripheral blood mononuclear cells(hPbmCs) as impurities for huCb-msC. C2C12 were similar in cell size and complexity of cytoplasm with huCb-msCs. For accuracy and specificity, C2C12 was spiked into huCb-msCs. there were no detectable huCb-msCs signal present in all C2C12 cells analysed and the recovery values were in 104.8% ~ 128.8%, which met the acceptance criterion. Precision was also determined by % rsD values and the values were shown less than 10% CV. the r2 value for all runs performed in this validation study were > 0.960 showing the flow cytometry methods for identification of huCb-msC are suitable quantitative analysis. also, the analytical procedure was not influenced by antibody reaction time, reaction cell number and antibody titer within the tested range (≤ 1.0 %CV). For purity validation, hPbmCs was spiked into huCb-msCs. there were no detectable hPbmCs signal present in all huCb-msCs analysed and the recovery values of hPbmCs were in 86.3% ~ 121.8%, which met the acceptance criterion. in this results, we confirmed that C2C12 and hPbmCs are suitable for use as reference materials of huCb-msC and all experimental results met the acceptance criterion.

84COMPARISOn OF PROPERtIES OF MESEnChyMAL StEM CELLS dERIVEd FROM huMAn BOnE MARROW And CORd BLOOdL. A. Marquez-Curtis1, Y. Qiu1,2, A. Janowska-Wieczorek1,2; 1Canadian Blood Services, Edmonton, AB, CANADA, 2University of Alberta, Edmonton, AB, CANADA.

mesenchymal stem cells or multipotent stromal cells (msC) established from bone marrow (bm) and cord blood (Cb) hold tremendous potential for tissue regeneration and cellular therapy. aside from immunomodulatory properties the clinical efficacy of msC depends on their ability to differentiate and to migrate to damaged/injured tissues. in this study, we compared msC derived from bm and Cb in terms of their expansion rates, morphological characteristics, tri-lineage differentiation potential and expression of early stem-cell related transcription factors. also, expression of the complement C5a receptor, which is involved in the migration of msC to injury sites where C5a is generated, was examined. adherent monolayers of msC established from bm and Cb were maintained in media supplemented with fetal calf serum for up to 12 passages, and assessed for their ability i) to differentiate (to adipocytes, chondrocytes and osteocytes), ii) to express transcripts for nanog, oct-4, sox-2, rex-1 and C5ar, and iii) to migrate towards C5a. We found that although confluency of primary cultures is achieved faster for bm-derived msC, in subsequent passages Cb-derived msC displayed more than 2-fold increase in proliferation rate. Cb-derived msC showed more primitive thin spindle-shaped morphology compared to the wider, flattened morphology of bm-derived msC characteristic of a later progenitor phenotype. bm-derived msC differentiated into adipocytes, chondrocytes and osteocytes more readily than Cb-derived msC. msC from both sources expressed transcripts for nanog, oct-4, sox-2 and rex-1, which were sustained up to 12 passages. C5ar mrna was also expressed by both bm- and Cb-derived msC, but Cb-derived msC were more strongly chemoattracted to C5a across reconstituted basement membrane. in conclusion, this study suggests that differences in properties of bm- and Cb-derived msC may reflect the distinct functional activities of the niches they come from, and these should be considered when designing clinical protocols.


Poster Abstracts

85CFu-F LEVELS In BOnE MARROW ASPIRAtES OF CARdIAC PAtIEntS tREAtEd WIth MESEnChyMAL StEM CELLSI. McNiece, S. Landa, F. Hussein, L. Ylisastigui, J. Hare; University of Miami, Miami, FL.

Cellular therapy for ischemic cardiomyopathy offers the potential for repair of damaged myocardium. our group has several clinical trials ongoing to evaluate the role of bm msCs in patients with myocardial infarction. as part of these studies we have performed colony forming assays (CFu-F) on bm mnCs as a potential potency measure for generation of msCs. the CFu-F assay has been used in previous studies of the role of msCs in stromal cell formation in vitro.bm cells were obtained from cardiac patients and from young normal donors and the mnCs isolated and plated for CFu-F formation. the number of CFu-F varied between patients with a median of 17 CFu-F (range o to 115) per 106 bm mnCs. the levels of CFu-F were higher in normal donors (p<0.05) with a median of 80 CFu-F (range 15 to 125) per 106 bm mnCs. the median age of normal donors was 25 years (range 18 to 38) compared to patients who were 56 years (range 39 to 79). these data suggest that the level s of CFu-F are decreased in older patients with cardiac disease compared to young healthy individuals. We also compared the levels of CFu-F in bm aspirates to the number of msCs generated for patients enrolled in clinical trials to receive msCs. based on a target dose of 200 million msC, 2 of 10 patients failed to achieve this dose. one of these patients had zero CFu-F and the second had 11.5 CFu-F per 106 bm mnCs. these preliminary data suggest that the CFu-F levels in bm aspirates may be predictive of the potential for msC expansion in culture. these studies continue to accrue patients and further CFu-F assays will be performed as a potential potency test for msC production.

87PLACEntA dERIVEd MESEnChyMAL-LIKE StROMAL CELLS- nOt ALL CELLS WERE CREAtEd EQuALR. Ofir1, N. Drori-Carmi1, M. Meiron1, O. Yaniv1, L. Lior1, M. Jack1, L. Pinzur1, E. Zehavi-Goldstein1, F. Grynspan1, E. M. Horwitz2; 1Pluristem Therapeutics Inc, Haifa, ISRAEL, 2The Children’s Hospital of Philadelphia and The University of Pennsylvania School of Medicine, Philadelphia, PA.

Human placenta is a rich source of mesenchymal-like stromal cells that are ideally suited for clinical applications. However, the placenta is a complex organ which includes tissue from both maternal and fetal origin raising the possibility of different mesenchymal cell populations. in this study, we isolated and characterized cells derived from different portions of full term human placentas: Decidua basalis (maternal origin), the Chorionic Villi (fetal origin), and the amniotic membrane (fetal origin).Comparison of characteristics between the isolated, ex-vivo expanded cell populations, obtained from fourteen different placentas, revealed typical mesenchymal marker expression (CD105+, CD73+, CD90+), but Decidua basalis-derived msCs showed higher levels of expression of the adhesion molecules CD106, CD166, CD44 and CD54 compared to cells derived from the Chorionic Villi and amniotic membrane.the average diameter of cells derived from amniotic membrane (19.55+0.25µm) was higher than that of cells derived from Chorionic Villi or Decidua basalis (17.82+0.33µm 16.4+0.43µm, respectively, P<0.001 for each) and the proliferation rate, measured as the Population Doubling per Day of cells derived from the Decidua basalis was significantly higher (0.425+0.03) than that of cells derived from the Chorionic Villi or amniotic membrane (0.325+0.055 and 0.25+0.065, respectively, P<0.001 for each). all three cell populations suppressed PHa-stimulated mononuclear cell proliferation in vitro, indicating immunomodulatory capability, with amniotic membrane derived-cells showing the greatest reduction (73.6+5.2%, P<0.001 compared to each other group), Chorionic Villi-derived cells, intermediate, and Decidua basalis-derived cells the least (53.4+3.0, P<0.001 compared to each other group). surprisingly, a correlation was not found between immunosuppressive capability and the therapeutic effect of the cells following in vivo administration in a rat model for stroke.these data indicate substantial phenotypic and functional diversity among mesenchymal-like stromal cell populations obtained from different anatomic regions of the Human Placenta which may suggest a differential therapeutic potential of these cells.

86COMPARAtIVE AnALySIS OF MESEnChyMAL StROMAL CELLS FROM AduLt And uMBILICAL CORd BLOOd.J. J. Montesinos1, G. Fajardo-Orduña1, E. Flores-Figueroa1, E. Hernández-Estevez1, P. Flores-Guzmán1, M. Castro-Manrreza1, S. Castillo-Medina1, S. Orozco2, H. Mayani1; 1Oncology Hospital, Mexico City, MEXICO, 2Especialties Hospital, Mexico City, MEXICO.

bone marrow (bm) has been recognized as the main source of mesenchymal stem cells (msC). in recent years, some reports have shown the presence of msC in umbilical cord blood (uCb) and adult mobilized peripheral blood (amPb). in the present study we have obtained msC from these three sources and characterized them in a comparative manner. msC were detected in 9/9, 11/104 and 1/108 samples from bm, uCb and amPb, respectively. the frequency of such cells was significantly lower in uCb and amPb sources (1 msC/200 x 106 and 1 msC/119 x 106 mononuclear cells, respectively) as compared to bm (1/31,000 mononuclear cells). similar to their marrow counterpart, uCb- and amPb-msC populations comprise several morphologically-distinct cell types, including neural-like cells. msC from the three sources showed similar patterns of cell surface antigen expression, i.e., they were positive for “mesenchymal” antigens and several adhesion molecules, and were negative for hematopoietic and endothelial markers. their capacities to differentiate into adipocytes, osteocytes and chondrocytes were also similar; however, the type of cartilage produced by bm-derived msC was different from the one produced by uCb- and amPb-msC cells. Finally, not only we observed msC with neural morphology, even without any neural differentiation stimuli, but we detected the expression of neural proteins. a possible explanation for this observation would be the presence of several msC subpopulations, each one committed to a particular cell lineage, including the neural lineage. our results indicate that amPb is not a rich source of msC. it is noteworthy, however, that the only amPb-derived msCs obtained, possess similar biological properties to those of uCb- and bm-msCs. in conclusion, uCb-msC instead of amPb may prove useful in the development of cellular therapy protocols.

88CLInICAL-SCALE ISOLAtIOn OF MESEnChyMAL StROMAL CELLS (MSC) FROM BOnE MARROW WIth thE CLInIMACS® PLuS InStRuMEnt And Cd271 MICROBEAdS.K. Pütsch, J. Schmitz, J. Schmitz; Miltenyi Biotec GmbH, Bergisch Glacbach, GERMANY.

mesenchymal stromal cells (msCs) have shown their potential for cell therapy in clinical trials, for example to treat acute graft-versus-host disease and for the regeneration of tissue. several groups have indicated that isolated CD271+ bone marrow mononuclear cells (bm-mnCs) show a 100-fold greater clonogenic potential and a one to three log greater expansion rate of msCs in culture compared to cells isolated by plastic adherence. expanded CD271+ msCs express msC markers, such as CD73, CD90 and CD105 and preserve their multilineage differentiation potential into adipocytes, osteoblasts and chondrocytes.in this study we show isolation of CD271+ cells using the ClinimaCs® Plus instrument, an automated cell separation system based on maCs® technology which enables the operator to perform clinical-scale magnetic enrichment of target cells or depletion of unwanted cells in a closed and sterile system.method: Human bm was harvested from sternum. CD271 bright cells were purified from bm using CD271 microbeads and the ClinimaCs® Plus instrument. the clonogenic potential of the CD271-positive fraction was compared to CD271-negative cells and unseparated bm. Furthermore, we analyzed the expansion potential of all fractions. Cultured cells were also analyzed regarding their differentiation potential and expression of cell surface markers.results: starting from 80 ml human bm, CD271bright cells were isolated with a purity of about 85%, yielding about 8×104 CD271+ cells. Comparing the different fractions, the best clonogenic and expansion potential was achieved with CD271bright cells. all msCs obtained showed a similar differentiation potential and cell surface marker expression.Conclusion: We successfully developed a protocol for the isolation of CD271bright cells in a closed and sterile system with high purity and recovery. With their increased expansion potential CD271-positive cells represent an optimal homogenous starting population for a time effective clinical-scale msC expansion.


may 23-26, 2010


Poster Abstracts

89StEM CELL duROtAXIS - tRAFFICKInG tO RIGId FIBROtIC SCARSM. Raab; University of Pennsylvania, Philadelphia, PA.

scarring in the heart after a myocardial infarct, scarring in the skin after wounding, or scarring of the liver in cirrhosis - all lead to rigidification of tissue through extensive collagen crosslinking and can also lead to accumulation or ‘homing’ of adherent cells, including mesenchymal stem cells (msCs). it is unknown how msCs localize and whether tissue rigidity plays a role. ‘Durotaxis’ is a term recently coined to describe the tendency in 2D culture for sparsely plated fibroblasts to crawl towards a stiff, collagen-coated gel made adjacent to a soft gel. stiffness-induced motility would perhaps suggest cell homing to a stiff matrix, but clear evidence for accumulation of any cell type has been lacking. We cultured human msCs on simple collagen-i coated gels with gradients in stiffness similar in magnitude to scars such as in the border zone of an infarcted heart. We first show that msCs are indeed migratory with crawling speeds that depend on stiffness. on stiffness gradients, we find that msCs migrate toward the stiffer side of a substrate with definitive accumulation taking several days. Proliferation appears unaffected by stiffness in these systems, but to eliminate the potential for differential expansion, cell division was pharmacologically inhibited and durotaxis was once again documented. Consistent with past reports for cell spreading on homogeneous gels, the spread area of msCs also increased along the stiffness gradient, which indicates that msCs are not only responding to substrate stiffness but also tending to adhere more on stiff gels. the orientation of the centrosome in migrating msCs also appears to be affected the stiffness of these gels. in addition to gels, we decellularize heart tissue with detergents to obtain a tissue-derived matrix, on which msCs adhere and migrate. there is potentially a stiffness dependence for msC migration with this substrate as well.

91huMAn EMBRyOnIC StEM CELL thERAPy In CEREBRAL PALSy: A 15 PAtIEnt CASE SERIESG. Shroff; Nutech Mediworld, New Delhi, INDIA.

Human embryonic stem Cells (hesC) grown in an animal and feeder free media and formulated in commercially available compositions were administered to 15 patients diagnosed with cerebral palsy with moderate to severe cognitive impairment with baseline DQ scores ranging from 9 to 35 on the Vineland behaviour adaptive test seeking treatment between august 2008 and september 2009. Patients ranged from 15 months to 5years of age . eleven were male and Four were female. each patient received hesC administered in a structured regimen using intramuscular and intravenous routes. the patients received an intramuscular injections of hesc on a daily basis. one intravenous injection was given once a week .the first phase of treatment was for two months followed by a break of 3 to 6 months. the next lot of treatment was for one month and then followed by a break of 3 to 6 months and so on.the patients underwent medical exams wherein their progress and any adverse effects were monitored. the treatment and follow-up duration ranged from 2 to 12 months. all patients were followed up using the DQ and also a specially devised parameters which could monitor any small recovery. all patients in this series showed improvement in the DQ score and in the sPeCt scans in the first 2 months of therapy. Further improvements were observed in those patients who continued to receive treatment and were followed up showed continual improvement.observed improvements in score were sustained, with no patient showing a relapse to lower DQ scores during the period of observation and follow-up . this case series suggests significant benefits and the potential for sustained reversal of disability and no further deterioration in patients receiving hesC therapy for moderate to severe progressive Cerebral Palsy.

90ChARACtERIStICS OF A nOn-hEMAtOPOIEtIC ALLOGEnEIC BOnE-MARROW dERIVEd CELL POPuLAtIOn thAt CAn BE MAnuFACtuREd tO PhARMACEutICAL StAndARdS And QuAntItIES.B. Heimbach, G. Kopen, V. Ragaglia, W. Righter; Garnet BioTherapeutics, Malvern, PA.

We have developed a novel, proprietary process for the isolation and expansion of non-hematopoietic allogeneic human adult bone marrow-derived somatic cells (habm-sC) in commercial quantities. Potentially billions of clinical doses can be generated from a single donor bone marrow. We describe here the kinetics, surface characteristics, and secretion profile of these cells.habm-sC can achieve approximately 40 population doublings with a consistent doubling time of less than 30 hours. under defined conditions, these cells retain the ability to form self-renewing colonies for multiple generations and maintain their typical morphology without evidence of senescence. the cells retain limited and measurable replicative capacity and have a normal karyotype. Human abm-sC are stable mesenchymal support cells that are co-positive for CD13, CD18bP, CD44, CD49c, CD90, and Hla Class i, but lack expression of CD10, 11b, CD31, CD34, CD45, CD62l, CD80, CD86, CD106 and Hla Class ii.their phenotypic expression profile was analyzed at each level of expansion and across multiple lots from the same donor. these cells secrete factors involved in modulating inflammation (il-6, tgFb1, stnFri and ii, il-1ra), augmenting regeneration (bDnF, sDF) and altering wound repair (activin a, mmP1). habm-sC respond to appropriate stimulation (e.g., tumor necrosis factor alpha [tnF-β]) with increased secretion of growth factors and cytokines. il-6 can be induced up to 20 fold over basal conditions. While other factors, such as mmP-13 or gm-CsF, which are not detected under standard culture conditions, are highly expressed after the cells are exposed to tnFa.the kinetics, surface profile and secretion pattern of habm-sC are distinct and reproducible. in addition, these cells can be processed under gmP standards for clinical application.

92ChARACtERIZAtIOn OF huMAn MESEnChyMAL StEM CELLS (hMSC) FROM BOnE MARROW In SERuM FREE COndItIOnSL. J. Solmesky, M. Weil; Tel Aviv University, Tel Aviv, ISRAEL.

hmsC are characterized by their self renewal capacity and by their multipotency. hmsC are easily isolated from bone marrow samples by using their property to adhere onto a plastic surface. another particular feature of hmsC is their capacity to survive without serum. this culture condition is an advantage for the study and characterization of the effects of specific molecules (endogenous or exogenous) on these cells without serum interference. thus, we could determine their role in cell survival as well as in cell adhesion, migration, differentiation and proliferation.in serum free culture conditions, we found that hmsC maintain their typical cell surface markers expression, cell proliferation and differentiation potential, even after prolonged periods in culture.using this serum free approach we confirmed that a common survival pathway between bmP and Wnt exists, controlling psmad1 levels downstream. in addition, we found that ra does not affect the hmsC viability but rescues the effect of noggin or sFPr2, elevating the proportion of psmad1+ nuclei. However, at longer periods, ra causes a decrease in cell survival.microarray analysis showed that the transcripts whose expression is regulated by ra are mainly related to proliferation, differentiation, migration and adhesion, being most of them down-regulated by ra treatment. several experiments confirmed that ra significantly reduces the erk1 derived pathways and this directly affects the different hmsC biological characteristics mentioned above. moreover, in contrast with these ra effects, we found that in serum free conditions egF+bFgF treatment promotes erk pathway activation which drives hmsC proliferation, differentiation and migration.altogether, being hmsC capable to survive under serum free conditions we were able to exploit this rare cellular property and characterized the specific signaling factors that drive or block their stem cell biological properties.reference:solmesky, l.J., abekasis, m., bulvik, s., and Weil, m. (2009). stem Cells Dev 18:1283-92.


Poster Abstracts

93EFFECt OF WOund MICROEnVIROnMEnt On MESEnChyMAL StEM CELL PROLIFERAtIOn And dIFFEREntIAtIOn.W. H. Stepp, T. S. Brown, N. J. Crane, E. Elster, D. K. Tadaki; Naval Medical Research Center, Silver Spring, MD.

Heterotopic ossification (Ho)--the formation of osseous bone in soft tissue--has been described in patients with brain and/or spinal cord injury, total hip arthroplasty, and extremity injury. We have observed a significantly higher incidence rate in combat wounded patients compared to similar civilian injuries. it has been suggested that mesenchymal stem cells (msC) play a role in this process and we sought to characterize this role as it relates to our patient population.We initiated a study of the effect of local wound environment on msC proliferation and differentiation. Wound fluids (WF) from patients were added to msC cultures at a concentration of 0.4% of total culture volume. We demonstrate that WF from Ho patients enhanced proliferation of msCs cultured in basal media as compared to the basal media alone and non-Ho patient WF. We then examined the effect of WF on msC differentiation. the results of those studies showed no differentiation of msC cultured in basal media with either Ho or non-Ho WF. interestingly, we observed greatly enhanced osteogenesis of msCs cultured in osteogenic differentiation media with Ho WF compared to osteogenic media alone or with non-Ho WF. in the Ho WF cultures, we see large amounts of mineral deposition (ranging from 791-1469 um of alizarin red) at 10 days post-induction with osteogenic growth factors. this is a stark contrast to those cultures where wound fluid is derived from non-Ho forming wounds (0-30 um of alizarin red stain) after 10 days.these findings have a significant impact on the use of msC in regenerative medicine application for wound healing and soft tissue regeneration following traumatic injury. We believe that further characterization of the microenvironment is necessary to prevent undesirable differentiation of the msC. We are currently examining the effect of the microenvironment on other differentiation pathways of msCs.

95EnhAnCEd ChOndROGEnIC POtEntIAL And IMMunOSuPPRESSIVE ACtIVIty OF huMAn MESEnChyMAL PROGEnItOR CELLS CuLtuREd In A nOVEL XEnO-FREE MEdIuMB. J. Short1,2, R. Wagey1, A. Kokaji1, B. Hoac1, T. Thomas1, A. Eaves1, B. Wognum1; 1StemCell Technologies Inc, Vancouver, BC, CANADA, 2University of British Columbia, Vancouver, BC, CANADA.

introduction: mesenchymal progenitor cells (mPCs) are an important cellular source for cell therapy. We have developed a humanized, serum-free medium (mesenCult®-XF) for the expansion of human mPCs and studied the proliferation, differentiation and immunosuppressive potential of the mPCs generated in this medium.materials and methods: Clonogenicity was analyzed by low density plating of bone marrow mononuclear cells. Proliferation was examined by serial passage and chondrogenesis was assessed by micromass culture in mesenCult®-XF chondrogenic differentiation medium. For testing of immunosuppression, mPCs were co-cultured with activated t-cells labelled using carboxy-fluorescein-diacetate (CFse). t-cell division was analyzed by flow cytometry.results: mPCs cultured in mesenCult®-XF or serum-containing medium revealed similar CFu-F frequencies (119 ±33 and 109±16 per 106 cells plated; ± sD, n=3), but mesenCult®-XF derived colonies were significantly larger. mPCs cultured for 9 passages in mesenCult®-XF showed an average fold expansion of 8.5 ± 1.4; ± sD (n=3), at each subculture, which was ~3-fold higher than that observed in serum-containing medium. Compared to serum-cultured mPCs, chondrogenic differentiation potential was greatly enhanced in cells cultured for 2, 4 or 6 passages in mesenCult®-XF, with extensive production of type ii collagen and alcian blue positive extracellular matrix components. mesenCult®-XF mPCs inhibited t-cell proliferation completely after 3 days and partially (20% proliferating t cells,) after 7 days of co-culture, whereas serum-mPCs showed much weaker inhibition (50% and 80% proliferating t cells after 3 and 7 days, respectively).Discussion and ConclusionsmPCs expanded faster and more extensively in mesenCult®-XF, exhibited enhanced chondrogenic differentiation and showed more robust immunosuppressive activity in vitro than mPCs cultured in serum-containing medium. the ability to expand functional human mPCs in a xeno-free medium will enable further research into the use of these cells for clinical applications such as tissue replacement and immunosuppression.

94GROWth ChARACtERIStICS OF EndOMEtRIAL REGEnERAtIVE CELLS (ERCS) CuLtIVAtEd In dIFFEREnt MEdIAS. Thirumala1, T. Ichim2, N. Riordan2, F. Ramos2, M. Murphy3, E. J. Woods1; 1General Biotechnology LLC, Indianapolis, IN, 2Medistem Inc, San Diego, CA, 3Indiana University School of Medicine, Indianapolis, IN.

the aim of the present study was to compare the basic growth properties of endometrial regenerative Cells (erCs) cultivated with different culture media in order to establish the optimal culture conditions thus providing a basis for further studies on designing standardized culture conditions for clinical scale production of erCs. For the culture of erCs, the following media were tested i) Dmem/F12 (Fisher scientific, usa) with 10% Fbs (gibco, usa) ii) mesenCult® basal medium (stem cell technologies, Canada) with 10% Fbs (atlas biologicals, usa) iii) mesencult® aCF media (stem cell technologies, Canada) iv) mesenCult® Proliferation Kit (stem cell technologies, Canada) and v) stemPro serum Free media (invitrogen usa). 1% Pen-strep-amphotericin (Fisher scientific usa) was added wherever required. Cells were seeded at 1000, 4000 and 8000cells/sq cm of culture area. For all conditions, the time taken by the cells to reach approximately 80% confluence was estimated. the data suggest that initial plating density has significant impact on the time required to reach confluence with more time taken by the cells at lower seeding densities. among all the media tested, the optimal media seems to be mesenCult® Proliferation Kit with no significant difference from mesencult® basal media with 10% Fbs from atlas biologicals or Dmem/F12 with 10%Fbs from gibco.For subsequent studies, mesencult® basal media with 10% Fbs from atlas biologicals was selected for calculating the doubling time and cumulative population doublings using the established formulae. the data indicated that the cells grew at a doubling time of approximately one doubling every 21 hours based on quantification of cell number using microscope counting. Further, erCs were passaged in culture for 140-150 population doublings before the cells appeared senescent. the regenerative ability along with higher rate of proliferation and prolonged culture in vitro supports the feasibility of using erCs for therapeutics development.

96huMAn MAPC LABELEd WIth A FLuORInE MRI tRACER REtAIn MuLtI-LInEAGE dIFFEREntIAtIOn POtEntIALB. Helfer1, A. Balducci1, E. Ahrens1,2, A. K. Wesa1; 1Celsense, Inc., Pittsburgh, PA, 2Carnegie Mellon Institute, Pittsburgh, PA.

Human mesenchymal multipotent adult progenitor cells (maPC) have great clinical promise to treat a wide variety of diseases, from stroke to cardiac ischemia. a question remains as to the trafficking and persistence of maPC following therapeutic administration, and few clinical tools are readily available. a vital prerequisite of a successful cellular tracer is one which permits the non-invasive detection and quantification of cells without impacting the therapeutic index or functional characteristics of the cellular product.Cell sense is a fluorocarbon-based tracer for fluorine magnetic resonance imaging (mri) providing a quantitative method to localize transplanted cells in vivo. We tested the ability of this tracer to label maPC and evaluated the cells functionally. maPC were labeled with up to 7 x 1012 fluorine molecules per cell as detected by nuclear magnetic resonance (nmr). the viability of labeled maPC was equivalent to unlabeled cells (> 90%). Following labeling, maPC were cultured under conditions to induce their proliferation and differentiation into distinct lineages: adipocytes, chondrocytes and osteoblasts. Progeny were confirmed with histochemical staining identifying lipids in mature adipocytes (oil red o), mineralization of osteoblasts (alizarin red s) and production of glycoproteins by chondrocytes (alcian blue). maPC cultures differentiated into mature adipocytes, osteoblasts and chondrocytes, with no evident differences in growth of cultures, staining or morphology between labeled and unlabeled cultures.Differentiation is comprised of synchronized processes (including sequential gene expression, cellular division and terminal maturation) necessary for progenitors to develop into discrete tissues with diverse functions. the lack of interference in these highly complex biological processes suggests that the therapeutic index of maPC may be preserved in labeled cells. Demonstrating the homing and persistence of transplanted cells is likely necessary for clinical approval; this non-toxic, biologically inert cellular tracer is uniquely suited for visualizing maPC-based therapies by mri in translational and clinical studies.


may 23-26, 2010


Poster Abstracts

97ABStRACt WIthdRAWn.

99EXPAnSIOn OF huMAn MESEnChyMAL StEM CELLS uSInG A MICROCARRIERS-BASEd CuLtIVAtIOn SyStEMM. Bracke, D. Schop, E. L. Borgart, J. D. de Bruijn; Xpand Biotechnology, Bilthoven, NETHERLANDS.

background: mesenchymal stem cells (msC) have great potential as tissue regenerative source as they have the capacity to form various types of tissue such as bone, cartilage, endothelium and cardiac muscle. an important source for adult stem cells is bone marrow. However, the yield of multipotent stem cells in bone marrow is very low, and cells need to be isolated and expanded up to a million-fold for effective therapeutic use.aim: in order to expand stem cells in a controlled, reproducible and cost-effective way, a bioreactor system based on 3D culturing conditions is being developed. this study describes the technology to isolate and expand bone marrow hmsCs in a microcarrier-based cultivation system.methods: Human msC, isolated from bone marrow of multiple donors, were seeded on Cytodex 1 microcarriers (4.000 cells/cm2, 20 cm2 carrier/ml) in a stirred vessel bioreactor (325-800ml). Cells were expanded for up to 15 days under different cultivation conditions varying in feeding regimen (e.g. gradual addition of fresh medium and/or glucose solution), pH and/or Do control, refreshing and stirring rate. During cultivation, cell growth was followed using alamarblue and light microscopy. Cell metabolism was monitored in terms of nutrient consumption (glucose, glutamine) and metabolite production (lactate and ammonia). expanded cells were analysed by FaCs flowcytometry and multipotency was characterized by adipo-, osteo- and chondrogenic differentiation.results & Conclusion: Continuous proliferation of hmsCs on microcarriers was observed, and depending on the culturing regimen, an average population doubling of 2.8 to 4.1 occurred. Furthermore, 3D expanded cells exhibit similar multipotency as 2D cultured msCs, and were able to form chondro-, osteo- and adipocytes.using the described technology for the development of a generic adult stem cell expander will allow use of expanded stem cells for many different clinical applications, including myocardial and vascular regeneration.

98PERFuSIOn CuLtuRE OF t LyMPhOCytES In thE WAVE BIOREACtOR™ SyStEM 2/10C. Annerén, L. Pauler, C. Sund-Lundström; GE Healthcare Bio-Sciences AB, Uppsala, SWEDEN.

Developing rapid, large scale t cell manufacturing processes as well as reducing the cost for disposables, media and labor is becoming increasingly important. We here present a robust and simple process for producing large numbers of peripheral blood t cells at high cell densities using the WaVe bioreactor system 2/10 with perfusion. t cells were activated and induced to proliferate upon binding to anti-CD3/CD28 beads and transferred to the disposable, functionally closed presterilized Cellbag™ bioreactor at a density of 3-5 x 10e5 cells/ml on day 3-4. in response to activation, the cells underwent the expected physical, biologic and phenotypic changes including increase in cells size, upregulation of CD25 surface antigen expression and a massive increase in proliferation. after the maximum volume of the Cellbag bioreactor (1l or 5l, depending on bag size) had been reached the perfusion was started. the cell density gradually increased, whereas pH and levels of metabolites and nutrients were kept constant by increasing the perfusion rate. the cells expanded 200 to 300-fold in the bioreactor and 2-6 x 10e10 cells at a density of 1-2 x 10e7 cells/ml were harvested after 11 to 13 days of culture. the expanded t cells remained biologically functional and could be re-activated to produce high levels of cytokines. the cost savings on consumables and labor by using the WaVe bioreactor system, compared to static bags, are significant due to the handling of one single Cellbag bioreactor and the automated exchange of media. Handling only one bag and the high cell densities achieved also reduces the risk of cross-contamination and the time and effort needed to concentrate and harvest cells at the end of the culture.

100CREAtInG A MuLtI-uSER CGMP CELLuLAR PROCESSInG FACILIty tO EnABLE tRAnSLAtIOnAL RESEARCh In thE ACAdEMIC EnVIROnMEntR. Haley; Duke University Medical School, Durham, NC.

introDuCtion: a Clinical translational science award was used to facilitate clinical translation by creating a cgmP shared facility. this facility enables translational research applications in cellular, tissue, immunologic and molecular therapy. the laboratory is set up to accommodate a single cell collection product as a batch as well as manufacturing of enzymatically produced oligonucleotide products and aseptic fill for immunotherapeutics.aPProaCH: building separate laboratories for each small, phase i translational project is too expensive for many candidate project investigators. this is a frequent barrier to translation. our collaborative effort bridged traditional boundaries between departments to pull together funding along with the necessary scientific, operational, and administrative expertise, to provide a multi-purpose space.metHoDs: a preparatory laboratory and two iso 7, (Class 10,000) clean rooms were outfitted. requirements from the Departments of surgery, Pediatrics, medicine, and allergy and immunology were incorporated. Procedures for environmental monitoring, cleaning, and shared space utilization were enforced by documented training for users. regulations are followed to ensure that products intended for human use are made in an organized, safe manner. resources that facilitate production of clinical grade biological material are currently advancing novel approaches to cancer, inborn errors of metabolism, and immunologic diseases in adult and pediatric populations.results: the cgmP shared resource facility is currently enabling the following preclinical and clinical protocols: dendritic cells modified by mrna for treatment of melanoma; aptamer dimer production for a clinical trial treating prostate cancer; t cell expansion of allogeneic cord blood grafts for immune reconstitution; and clean aliquoting and storage of food allergen for a clinical trial.ConClusions: by establishing a cgmP facility and leveraging specialized technical and regulatory expertise, Duke translational research institute has consolidated a pan-institutional resource that can be accessed cost-effectively to move promising therapy from the bench to the bedside in the academic environment.


Poster Abstracts

101dEVELOPMEnt OF A XEnO-FREE MICROCARRIER-BASEd huMAn MESEnChyMAL StEM CELL EXPAnSIOn SyStEMC. Johnston, J. Morris, M. P. Henshaw, L. L. Kelley; University of Utah, Salt Lake City, UT.

Widespread clinical use of human mesenchymal stem cells (hmsC) is limited by the challenges of scale-up from standard monolayer tissue culture techniques and the requirement for fetal bovine serum (Fbs). We developed a xeno-free spinner flask culture system using microcarrier beads and human platele lysate (Pl) instead of Fbs. eight types of microcarriers were evaluated using the manufacturer’s recommended concentrations and cultured in 125 ml spinner flasks in β-mem and 5% Pl. after 24 hours the suspensions were stained with the liVe/DeaD® Viability/Cytotoxicity assay (molecular Probes) to assess cell attachment to the microcarriers. in most cultuers a variety of unsatisfactory outcomes were observed including no cell attachment, presence of dead cells, microcarrier clumping or the formation of a fibrin web in the media. However, two microcarriers, microHex (nunC) and Plastic Plus 102-l (Hyclone) demonstrated excellent cell attachment and maintenance of cell viability. subsequent experiments were performed with microHex microcarriers. optimal hmsC seeding density was determined by incubation of the microcarriers with cell concentrations ranging from 5,000-40,000 cells/cm2. attachment efficiency increased proportionally with increasing cell numbers up to 20,000 cells/cm2 after which significant clumping was observed. the hmsC doubling time was determined and found to be shortest when using 7,500 cells/cm2 cultured in β-mem with 10% Pl under reduced o2 tension (15 vs 20%). Dissociation of hmsC from microcarriers was accomplished with tryple™ (invitrogen), a recombinant animal-free trypin substitute, yielding 90±7% viable cells (n=11) allowing further phenotypic and functional characterization. When compared to standard monolayer techniques using tissue culture flasks, the microcarrier-based system yielded ample numbers of expanded hmsC at a greatly reduced cost in labor, culture media, culture supplement additives, Pl and disposables. transition to a xeno-free microcarrier-based culture expansion system will greatly facilitate the regulatory process, reduce cost and expedite the use of hmsC in clinical trials.

103LARGE-SCALE CELL COnCEntRAtIOn And WAShInG uSInG A MOdIFIEd IntRAOPERAtIVE BLOOd RECOVERy dEVICE hAEMOnEtICS CELL SAVER 5 SyStEM.D. J. Powell Jr.1, J. Cotte1, Z. Zheng1, A. L. Brennan1, L. Chueng1, L. Truong2, G. Lee2, M. Holmes2, B. L. Levine1; 1University of Pennsylvania, Philadelphia, PA, 2Sangamo BioSciences, Richmond, CA.

background aims: large-scale washing and harvesting of cell therapy products is routinely performed using automated devices employing single-use sterile disposable sets with the capacity to concentrate cells at a rate of 100-400mls/minute. However, commonly used equipment, including the baxter Fenwal Harvester and baxter Cytomate systems, have been discontinued and identification of a suitable replacement for washing and harvesting 5-20l cell therapy cultures is essential. We describe validation of the Haemonetics Cell saver 5, programmed with user-defined settings, as a reproducible, efficient replacement system for clinical-grade cell washing and harvesting. methods: large-scale human t cell cultures were washed and harvested using the Cell saver 5, and when possible, compared to the baxter Fenwal Harvester. Cell recovery and viability was assessed post-harvest. recognizing that various cell culture supplements or gene delivery vectors may have different cell adhesion and/or stability profiles, we conducted spiking experiments using bovine transferrin and/or recombinant adenovirus to determine the capacity of the instrument and settings to reduce these soluble residuals. results: optimized instrument settings (250ml bowl; 3850rpm centrifugation; 250ml/min flow rate) were established in preliminary evaluation runs. Cell saver 5-based harvesting performed on fresh t cell cultures (n=7; mean 8.7x109 cells; range 2-8.4x109 cells) yielded excellent cell recovery (97.0±6.8%) and viability (96.4±1.3%). in five parallel runs, the Cell saver 5 provided statistically similar cell recovery (92.0±8.8% vs. 83.7±5.7%) and viability (95.5±1.7% vs. 96.7±0.6%) compared to the baxter Fenwal Harvester. substantial reductions in soluble transferrin (n=3; mean 10,394-fold) and adenoviral particles (n=5; mean 80.5-fold) were observed. Conclusions: large-scale cell harvesting was feasible using the Cell saver 5 with settings adapted for t cells, and resulted in high cellular recovery and viability, as well as substantial reductions in supplemented soluble residuals. the Cell saver 5 system and settings described here are thus a suitable replacement for now-obsolete cell harvesting devices.

102dEVELOPMEnt And IMPLEMEntAtIOn OF A StRAIGhtFORWARd REAGEnt And VEndOR QuALIFICAtIOn PROGRAM BASEd On IMPACt SCOREC. Johnston, N. Balliette, A. Havens, L. L. Kelley; University of Utah, Salt Lake City, UT.

reagent and vendor qualification is an important aspect for facilities that manufacture cell and tissue-based products for patient use. the responsibility for qualification resides with the manufacturer. to ensure consistency in our approach and to minimize the extent of resources required, we developed an in-house reagent and vendor qualification program based on determination of an impact score. the impact score is calculated by ratings given to each of three categories including quality, availability and safety. the quality rating is determined by the step in the process in which the supply/reagent is used, the length of contact time with the product, whether re-validation is required if the supply/reagent is changed and the concentration in the final product. the availability rating is determined by whether the supply/reagent is available from single versus multiple suppliers. the safety rating is derived from usP <1043> guidelines and is determined by whether the supply/reagent is a licensed biologic, approved for human use, sterile, endotoxin levels, guidelines for manufacture (cgmP or iso9000), grade (usP or nF) and presence or absence of animal proteins. enumeration of the various ratings lead to a total impact score that correlates with low, medium, high and critical impact. the total impact score and the phase of the clinical trial (pre-inD to bla) dictate the requirements for vendor qualification and range from no action to questionnaire to audit. importantly, we only apply this qualification process to supplies/reagents that come into direct contact with the product under manufacture. our experience to date indicates that approximately 8 vendors per inD protocol require qualification via questionnaire. our facility has performed one on-site audit for a critical single-source supplier located out-of-state. in summary, use of a system based on impact scoring results in a consistent approach without being arduous, resulting in assurance that supplies/reagents are appropriately qualified.

104APPLICAtIOn OF A hIGh COntEnt IMAGInG SyStEM FOR ChARACtERIZAtIOn And QuALIty ASSuRAnCE OF StEM CELL dERIVEd PROduCtS.E. P. Roquemore, E. Price, C. Hather, R. Ismail; GE Healthcare, Cardiff, UNITED KINGDOM.

stem-cell derived materials offer promise for predictive toxicology testing, therapeutic drug development and regenerative medicine. there is a basic requirement for better characterization and quality assurance in preparation of differentiated stem cell products. We demonstrate the utility of a rapid automated high content imaging system for verification, validation and quality assurance testing of human stem cell-derived cardiomyocytes. Cell populations are characterized using immunofluorescence detection of cardiomyocyte markers. expression is correlated with flow cytometry analysis, and utility of the cells is demonstrated in functional assays. the in Cell analyzer 2000 high content imaging system improves the quality assurance workflow, efficiency and data quality.


may 23-26, 2010


Poster Abstracts

105EStABLIShMEnt OF A CLInICALLy APPLICABLE MEthOd FOR LyMPhOCytE EXPAnSIOn uSInG RECOMBInAnt huMAn FIBROnECtIn FRAGMEnt (Ch-296; REtROnECtIn)Y. Yamaki1, T. Wakeda1, M. Kaida1, Y. Hoshi1, N. Takahashi1, S. Yamagata1, M. Shimada1, A. Kondo2, Y. Ikarashi2, K. Aoki2, Y. Takaue1, Y. Heike1; 1National Cancer Center Hospital, Tokyo, JAPAN, 2National Cancer Center Research Institute, Tokyo, JAPAN.

[introduction] retronectin (rn) is widely used in retroviral gene therapy to enhance gene transfer efficiency. together with anti-CD3 antibody, rn is known to enhance t-cell proliferation while conserving the naive phenotype. it has been demonstrated that in comparison to more differentiated phenotypes, naive t-cells are more active in mediating tumor regression. thus, t-cells expanded with rn (rn-laK cells) are expected to have greater potential to suppress tumor growth than t-cells expanded by conventional methods, which show differentiated phenotypes in vivo. in this study, we developed and evaluated a clinically applicable method for expansion of rn-laK cells using a closed-bag system.[method] From 13 media, we selected Kbm561, which showed superior proliferation efficiency in the number of cells and proportion of naive t-cells. mononuclear cells (mnCs), prepared from 60 ml of peripheral blood collected from healthy volunteer donors, were cultured in an rn and anti-CD3 antibody-coated culture bag. Cells were cultured for 14 days and were evaluated in accordance with the notification from the Japanese ministry of Health, labour and Welfare, ‘regarding securing Quality and safety for Drug or medical Devices being processed with Human (autologous) Cells and tissues’ (PFsb notification no.0208003).[results] 1.5×107 mnCs could be expanded to approximately 1.2-2.5×1010 rn-laK cells. the number of naive cells was larger among rn-laK cells when compared to laK cells expanded by a conventional method using anti-CD3 antibody alone. Furthermore, rn-laK cells were compatible with the PFsb notification no.0208003.[Conclusion] We were able to sufficiently expand rn-laK cells for clinical use from only 60 ml of peripheral blood. these cells may be useful in donor lymphocyte infusion (Dli) for post-transplantation relapse patients whose donors are not fit for apheresis in order to collect cells for Dli.

107EnGInEERInG OF hMSC WIth LEntIVIRuS EXPRESSInG huMAn FIX FOR thE tREAtMEnt OF hEMOPhILIA BM. Dodd1, A. Markar1, L. Marquez-Curtis2, J. Wen1, A. Janowska-Wieczorek2, G. Hortelano1; 1McMaster University, Hamilton, ON, CANADA, 2University of Alberta, Edmonton, AB, CANADA.

baCKgrounD aims: Hemophilia b is an inherited X-linked bleeding disorder caused by a defective coagulation factor iX (FiX), which is found in 1 out of 30,000 male births each year. Current treatment involves regular infusions of either plasma derived or recombinant FiX, at very high cost. the aim of our study was to investigate a cheaper alternative treatment using cell therapy. as human mesenchymal stem cells (hmsC) have increasingly been considered in cellular therapies due to their immunomodulatory properties and differentiation potential, here we have engineered hmsC to secrete FiX.metHoDs: mononuclear cells were obtained from cord blood (Cb) or human bone marrow (bm) and msC cultured for up to 10 passages and assessed for their differentiation potential. at passage 4-6 msC were subjected to electroporation, to allow the entry of Dna. Further, a lentivirus vector containing a CmV promoter and the human FiX cDna with a mini human intron i was also constructed and tested.results: electroporation using both square waveforms and exponential waveforms was able to introduce a plasmid Dna containing the marker gene luciferase into hmsC. optimal voltage using a square waveform was found to be 130V, the optimal pulse length was 30msec and the optimal number of pulses is 1. Human Cb msC were transduced with a single round of lentivirus-FiX particles. FiX expression was approximately 1 µg/106 cells/day, which was higher than the expression achieved in transduced murine g8 myoblasts. in contrast, msC electroporated with the lentivirus vector secreted <100ng FiX/106 cells/day. experiments using noD-sCiD mice transplanted with hmsC transduced with lentivirus-FiX and experiments using FiX-deficient mice are currently being carried out in our lab.ConClusions: hmsC transduced with lentivirus secrete high levels of hFiX and thus may be suitable to assess the feasibility of non-hematopoietic msC therapy as a treatment for hemophilia b.

106tEMOZOLAMIdE RESIStAnt ζζ t CELL-BASEd IMMunOthERAPy FOR GLIOBLAStOMA MuLtIFORMEJ. E. Bowersock1, A. Dasgupta2, G. Gillespie1, H. Arnouk1, H. Spencer2, L. S. Lamb1; 1University of Alabama at Birmingham, Birmingham, AL, 2Emory University, Atlanta, GA.

background: Cellular immunity is severely depressed following standard therapies for glioblastoma multiforme (gbm). We have previously shown that expanded/activated ββ t cells eliminate residual gbm and improve survival in a mouse intracranial xenograft model. the potential for this approach in the setting of chemotherapy-induced injury to the tumor could enhance its effectiveness in eliminating residual/resistant gbm. in this report, we investigate the cytotoxic function of mgmt gene-modified innate effector cells in the presence of temozolamide (tmZ).methods: the nK-92 cell line was used for preliminary experiments to test gene modification and cytotoxicity against a tmZ-resistant K562 cell line. nK-92 and Zoledronate/il2 (Zol/il-2) expanded/activated ββ t cells were transduced with a P140Kmgmt siV construct. nKg2D ligand expression and t cell phenotype were assessed by flow cytometry of surface antigens and intracellular cytokines. Flow-based cytotoxicity assays were used to determine potency of the cell product.results: P140Kmgmt-modified nK-92 cells were resistant to increasing concentrations of tmZ. non-modified or gene-modified nK92 cells were cytotoxic to either non-modified or modified targets in the presence of tmZ. genetic modification of nK92 cells did not alter their ability to kill target cells. Zol/il-2 expanded ββ t cells were successfully transduced with the P140mgmt siV construct. the cells expressed th1 cytokines tnF-β and iFn-β and were highly cytotoxic against u251mg, u87mg and D54mg cell lines and recognized multiple stress-induced nKg2D ligands on gbm cell lines. tmZ treatment resulted in enrichment of CD133+ gbm progenitor cells which were also significantly reduced in a four hour cytotoxicity assay.Conclusions: Zol/il-2 expanded/activated ββ t cells can be successfully transduced with the P140Kmgmt construct, conferring resistance to tmZ without reduction in potency.this study was supported by niH nCi grant # P50 Ca 097247, ninDs grant # r21 ns 57341 and the brain tumor society samuel gerson leadership Chair.

108Cd19-SPECIFIC t CELLS SELECtIVELy EXPAndEd On ARtIFICIAL AntIGEn PRESEntInG CELLS ARE RESIStAnt tO IMMunOSuPPRESSIVE EFFECtS OF tACROLIMuSM. Figliola, M. Dawson, M. Lima Da Silva, H. Singh, H. Huls, D. A. Lee, P. Kebriaei, R. E. Champlin, L. J. Cooper; UT MD Anderson Cancer Center, Houston, TX.

Donor-derived t cells infused after allogeneic hematopoietic stem-cell transplantation (HsCt) are used to prevent and treat malignant relapse. However, the ability to enhance the graft-versus-tumor (gVt)-effect may be compromised by the presence of immunosuppressive medications, such as tacrolimus, taken by the recipient (to achieve serum concentration of ~10 ng/ml) to prevent and treat graft-versus-host-disease (gVHD). to enhance the gVt-effect for CD19+ malignancies we have genetically modified peripheral blood-derived t cells to redirect specificity for CD19, through the electro-transfer of a Dna plasmid coding for a chimeric antigen receptor (Car). our approach uses the sleeping beauty (sb) transposon/transposase and CD19+ artificial antigen presenting cells (aaPC) to selectively propagate clinically-sufficient numbers of Car+ t cells for human trials. the current study was designed to observe the effect of tacrolimus on our ability to generate Car+ t cells. twenty-one days after electroporation and weekly additions of β-irradiated aaPC, there was an expected outgrowth of CD19-specific t cells expressing 86% Car. these genetically modified t cells were subsequently co-cultured in the absence or presence of tacrolimus (10 ng/ml or 100 ng/ml) on aaPCs. after 14 days the t cells were again measured for Car expression and assayed for their Car-dependent killing of CD19+ tumor cells. somewhat surprisingly, we found that t cells exposed to tacrolimus continued to numerically expand while maintaining Car expression and Car-mediated killing (Figure). these data support the adoptive transfer of allogeneic CD19-specific Car+ t cells into patients receiving tacrolimus after HsCt.


Poster Abstracts

109AMnIOtIC FLuId-dERIVEd StEM CELLS GEnEtICALLy EnGInEEREd FOR CELL-BASEd thERAPyJ. L. Haller, J. A. Frank; The National Institutes of Health Clinical Center, Bethesda, MD.

the current therapy for glioblastoma multiforme is poor and there is a need for more effective selective cell-based therapies. in this study human amniotic fluid-derived stem cells (aFsC) (mta Wake Forest) were genetically engineered as targeted cellular delivery vehicles. aFsCs were engineered with the Herpes simplex virus - thymidine kinase (HsV-tk) and the fluorescent reporter tdtomato (tdtom) to facilitate interrogation in vitro and enable tracking of the cells in vivo. this study reports on the stem cell properties and the in vitro cytotoxic effects of the tdtom/tk aFsC by direct co-culture of C-6 glioma cells and treatment with prodrug ganciclovir (gCV) as evaluated by mts cellular proliferation assay. Flow cytometry results indicated that tdtom/tk-aFsC retained their “stem-like” properties expressing embryonic antigen (ssea)-4 and oct4 as compared to primary cell line. mts analysis was performed following co-culture of 0.5 103 tdtom/tk-aFsC with an equal ratio of C-6 glioma cells for 7 days. treatment with 10µm gCV achieved at least 75% reduction in cell number and was not exceeded at higher drug dosages. our current results indicate that at the minimal ratio of 1:1and lowest dosage tested (10µm gCV) there was significant cell kill by the tdtom/tk-aFsC of C6 glioma and the same co-culture conditions with wild type aFsC resulted in values equal to untreated control cells. the bystander effect noted may serve as indication of formation of gap junctions as cytotoxic gCVtriphosphate is unable to passively diffuse into neighboring cells. Future studies will directly investigate the ability of tdtom/tk-aFsC to target the implanted primary tumor mass as well as infiltrate invasive disseminated tumor cells in a rat glioma model. tdtom/tk -aFsC will permit monitoring the success of cellular therapy in real time in vivo employing fluorescent as well nuclear imaging modalities.

111EFFICIEnt GEnE dELIVERy PLAtFORM FOR nEuRAL StEM And PROGEnItOR CELLS U. Lakshmipathy1, R. Quintanilla1, C. MacArthur1, Y. Liu2, F. Boyce3, M. Rao1; 1Life Technologies, Carlsbad, CA, 2The Scripps Research Institute, La Jolla, CA, 3Massachusetts General Hospital, Cambridge, MA.

modified stem cells are valuable tools for dissecting basic cell and developmental biology and enable their use in drug discovery and cell therapy. this required identification of methods that deliver gene of interest at high frequency into stem cells with minimal toxicity. We have developed baculovirus-based platform that utilizes non-integrating vectors for the rapid creation of transient or stably engineered embryonic and adult stem cells.baculoviruses adapted for mammalian expression, termed bacmam, has been reported to achieve high levels of transduction in hard-to-transfect mammalian cells. using a vector system with VsV-g and WPre, we show that bacmam can consistently transduce neural stem cells at over 80% efficiency. the expression of gFP persists in diving cells for 5-8 days and with random differentiation into neuron, astrocytes and oligodendrocytes. glial precursor cells and differentiated glial cells can also be transiently transduced to high efficiency using this system. the utility of this platform was further extended for the creation of stable cells by inclusion of the ebna1 and oriP ebV-based elements for episomal maintenance. this novel integrated platform provides a fast and efficient method to create labeled stem cells for downstream therapeutic and screening applications.

110IRRAdIAtIOn EnhAnCES thERAPEutIC POtEntIAL OF MESEnChyMAL StEM CELL-BASEd tRAIL GEnE dELIVERy In GLIOMAS. Kim1, W. Oh2, S. Jeun1; 1The catholic university of Korea, Seoul, KOREA, REPUBLIC OF, 2MEDIPOST Co.,Ltd., Seoul, KOREA, REPUBLIC OF.

irradiation is a standard therapy for glioma as well as many other cancers. Previously we defined the tumor targeting properties and antitumor effects of human umbilical cord blood-derived mesenchymal stem cells (uCb-msCs) as tumor necrosis factor-related apoptosis-inducing ligand (trail) gene delivery vehicles for glioma therapy. in the present study, we investigated the effect of tumor irradiation on the tropism of uCb-msCs toward glioma cells and that trail delivered by uCb-msCs synergistically enhanced trail-induced apoptosis in irradiated tumors. the migration assays showed increase of msCs migration towards conditioned media from irradiated glioma cells and irradiated tumor site in glioma-bearing mice compared with unirradiated cells. irradiated glioma cells had enhanced expression of interleukin-8, which was confirmed by elisa and immunohistochemistry in vitro and in vivo. We assessed the effect of combined treatment with irradiation followed by trail-secreting uCb-msCs on apoptosis of glioma cells. the sequential treatment of cells significantly enhanced more apoptosis than single agent alone treatment in either trail-sensitive or -resistant cells through upregulation of death receptor Dr5 and induction of caspase activation. moreover, in vitro coculture, experiments on transwell plates, and in vivo survival experiments in xenografted mice showed that msC-based trail gene delivery to irradiated tumors had more therapeutic efficacy than single treatment. these results suggest that tumor irradiation enhances the localization of uCb-msCs and the therapeutic potential of combined treatment with irradiation and trail can be useful for msC-mediated trail gene therapy.

112OVARIAn CAnCER CELLS uBIQuItOuSLy EXPRESSEd hER-2 And CAn BE dIStInGuIShEd FROM nORMAL OVARy By GEnEtICALLy REdIRECtEd t CELLSE. Lanitis1,2, D. Song1, L. Zhang1, R. G. Carroll3, R. Sandaltzopoulos2, G. Coukos1, D. J. Powell1; 1Ovarian Cancer Research Center, Philadelphia, PA, 2Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis, GREECE, 3Abramson Family Cancer Research Institute, Philadelphia, PA.

background: Her-2-specific t cells can be induced by vaccination or generated de novo by genetic engineering, however it remains uncertain to what extent t cell-based ‘’Her-2-directed’’ immunotherapy can be utilized for the treatment of advanced ovarian cancer.objective: to investigate the role of Her-2 as the resolute tumor antigen for t cell-based adoptive immunotherapy of ovarian cancer.methods: to determine the relative expression of Her-2 in ovarian cancer and normal ovarian surface epithelial cells (ose) real-time PCr, flow cytometry and western blot was performed. Human t cells were genetically engineered to express the C6.5 Her2-specific chimeric immune receptor (Cir). Her-2 Cir-transduced t cells were tested for their capacity to recognize and kill Her-2 expressing tumors and ose cells.results: Her-2 overexpression was observed in all established ovarian cancer cell lines (13/13) and short-term cultured cell lines (7/7). Consistent with these results, all tumor cells derived from primary ascites (22/22) and solid tumor (12/12) expressed Her-2, albeit at variable levels. Compared to tumor, normal ose expressed lower but detectable levels of Her-2. genetically redirected t cells recognized and reacted against all ovarian cancer cell lines (16/16), primary ascites (5/5) and solid tumor (5/5) tested, however little or no reactivity was observed against normal ose (1/4).Conclusions: our results show that Her-2 is expressed by all ovarian cancer cells but is limited in normal epithelium. this contrasts previous reports suggesting Her-2 overexpression in only 20-30% primary ovarian tumors via immunohistochemistry (iHC). importantly, all Her-2 expressing tumors but not normal epithelium are recognized by genetically engineered Her-2 specific t cells. these results suggest that the C6.5 Cir can discriminate between cancer and normal cells and therefore minimize the potential for “off target” reactivity. these findings provide the rationale for the development of Her-2-redirected t cell-based immunotherapeutic approaches in women with ovarian carcinoma.


may 23-26, 2010


Poster Abstracts

113IntRAMuSCuLAR dELIVERy OF ACRX-100 EnhAnCES AnGIOGEnESIS In A RABBIt MOdEL OF hInd LIMB ISChEMIAT. J. Miller1, J. Pastore1, R. Aras1, M. S. Penn2; 1Juventas Therapeutics, Cleveland, OH, 2Cleveland Clinic, Cleveland, OH.

Peripheral vascular disease (PVD) affects approximately 12 million americans and is associated with significant morbidity and mortality. PVD prevalence increases dramatically with age, affecting approximately 20% of americans age 65 and older. Patients with advanced PVD whom are ineligible for reconstruction or in whom prior revascularization has been unsuccessful have 6-month amputation and mortality rates as high as 40% and 20%, respectively. Despite its prevalence and cardiovascular risk implications, only 20 to 30 percent of PVD patients are undergoing treatment. aCrX-100 comprises a non-viral Dna plasmid engineered to transiently express human stromal cell-Derived Factor 1 (sDF-1). sDF-1 is a strong chemoattractant of endogenous organ specific and bone marrow derived stem cells and progenitor cells to the site of tissue damage, which promotes tissue preservation and blood vessel development. re-stimulating sDF-1 expression by gene transfer into ischemic muscle has a high therapeutic potential for treatment of PVD because, unlike current treatments that focus on alleviating symptoms, sDF-1 has the potential to regenerate vasculature and provides an opportunity to repair and retain function in degenerating limbs. We have previously demonstrated that injection of aCrX-100 into a large animal model of chronic heart failure increased heart function for at least 90 days post-injection. in this study, rabbits (n=5/group) underwent a unilateral femoral artery ligation and 10-days post ligation received 4 or 8 mg aCrX-100 via 8 intramuscular injections to the ischemic limb. blood flow was measured by angiography at 30 and 60 days post-treatment. aCrX-100 treated animals showed improved blood flow at both time points compared to vector injected control animals, which showed decreased blood flow. importantly, benefits were observed at 30 days with significant (p<0.05) benefits sustained at 60 days. these results indicate that patients with PVD may benefit from treatment with aCrX-100.

115ROBuSt InVO AntI-tuMOR FunCtIOn By huMAn t CELLS EnGInEEREd tO EXPRESS A 41-BB COStIMuLAtORy ζ-FOLAtE RECEPtOR-SPECIFIC ChIMERIC IMMunE RECEPtORS FOR OVARIAn CAnCERD. SONG1,2, Q. Ye1, C. Carpenito3, M. Poussin1, C. Ji2, S. Canevari4, C. June3,5, G. Coukos1, D. Powell Jr1; 1Ovarian Cancer Research Center, Philadelphia, PA, 2Department of Hematology, Qilu Hospital, Shandong University, Jinan, CHINA, 3Abramson Family Cancer Research Institute, Philadelphia, PA, 4Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, ITALY, 5Department of Pathology and Laboratory Medicine, Philadelphia, PA.

background: β-Folate receptor (Frβ) is a folate-binding protein overexpressed on 90% ovarian cancer. Here we sought to construct Frβ-specific single-chain fragment (scFv)-based Cir with or without novel costimulatory signaling motifs and test the reactivity of human t cells tansduced with Cir against ovarian cancer cell lines expressing Frβ in vitro and in vivo.methods: novel Cirs were constructed that contain a Frβ-specific scFv (mov19) coupled to either an inactive form of the CD3-β intracellular domain (mov19-Δβ), CD3 β chain signaling module (mov19-β) or in combination with the CD137 (4-1bb) costimulatory motifs(mov19-bb-β). Human t cells were transduced with the Cir using lentivirial vectors. in coculture assays, Cir transduced t cells were measured for reactivity against ovarian cancer cells expressing Frβ via iFn-g elisa and cytokine bead assay. Cytotoxicity was measured using a bioluminescence system in vitro and a Winn assay in nog mice with coinjection of t cells and ovarain cancer cells.results: Human t cells expressed cell surface scFv mov19 50% after lentiviral transduction. mov19-β and bb-β transduced t cells demonstrated target-specific release of iFn-g, tnF-β and il-2 cytokines and cytotoxicity function when cocultured with Frβ+ tumor cells, while t cells transduced with mov19-Δβ or with gFP did not. in an in vivo Winn assay, mov19-β transduced t cells were able to inhibit the out growth of Frβ+ ovarian cancer. in contrast, t cells transduced with the mov19-Δβ or with gFP had no effect on tumor growth. notably, in vivo lytic activity of mov19 Cir was improved through provision of CD137(41-bb) signaling.Conclusions: our results demonstrate direct and specific tumor recognition and killing of Frβ+ ovarian cancers cells by t cells genetically engineered to express mov19 Cir in vitro and in vivo and that incorporation of the CD137(41-bb) signaling domain in Cirs can enhance their antitumor activity.

114dEVELOPMEnt OF nOn VIRAL APPROAChES tO MEGAnuCLEASE EnGInEEREd CELLuLAR thERAPy FOR tREAtMEnt OF MOnOGEnEIC InhERItEd dISEASESL. Poirot1, C. Jacqmarcq1, J. Smith1, A. Gouble1, R. Galetto1, F. Paques1, C. Desseaux1, A. Viley2, C. Allen2, L. Li2, M. Peshwa2; 1Cellectis Genome Surgery, Romainville, FRANCE, 2Maxcyte, Gaithersburg, MD.

most current gene therapy approaches for monogenic inherited diseases rely on gene transfer, by random integration into the genome of a patient’s cells, of a functional copy of the mutated gene. However, the interest for the use of targeted approaches is rising as they allow the repair of a deleterious mutation precisely (“genome surgery” approach) as well as the insertion of a functional coding sequence in a chosen locus (“safe harbor” approach) using homologous recombination.meganucleases are endonucleases recognizing large cleavage sites (>12bp) with a high specificity and that can stimulate homologous gene targeting by a 1000-fold factor. redesigning their protein-Dna interface allowed us to engineer dozens of custom meganucleases with a chosen specificity and able to induce high level of gene targeting.We are investigating the use of non-viral vectorization methods in cell lines, primary cells and stem / progenitor cells, wherein the meganucleases can be delivered either in the form of plasmid Dna, messenger rna or as synthesized recombinant proteins. using a meganuclease targeting the human rag1 gene, targeting efficiency reaches 5 - 6% in adherent HeK293 cells using lipofectamine as transfection reagent or when using electroporation. in Jurkat cells, a CD4+ lymhocytic suspension cell line, whereas lipids and nucleofection permitted only limited success, maxcyte’s gt Flow transfection system consistently stimulated 50-fold higher gene targeting yielding homologous targeting efficiencies comparable to those obtained in HeK293 cells.blood cells are particularly amenable to ex vivo vectorization. our primary interests are in developing robust, scalable, cgmP and regulatory compliant processes for efficient vectorization of cellular therapeutics using immune cells, hematopoietic stem cells (HsCs) as they can differentiate into any blood-related cell type, and other primary and stem/progenitor cells. We are now optimizing the successive steps necessary to achieve gene targeting in HsCs: meganuclease expression, meganuclease activity and homologous recombination itself.

116FCGR3A-158V GEnE tRAnSduCtIOn AuGMEnt Cd16 EXPRESSIOn And AdCC ACtIVIty OF PERIPhERAL BLOOd MOnOnuCLEAR CELLS.T. Wakeda, Y. Hoshi, M. Kaida, Y. Yamaki, N. Takahashi, S. Yamagata, M. Shimada, N. Fukuda, Y. Takaue, Y. Heike; National Cancer Center Hospital, Tokyo, JAPAN.

background: it is well known that polymorphisms of Fcβ receptor iiia (158-V/F) show different clinical efficacies of monoclonal antibodies (mabs), such as rituxian and Herceptin. V-type Fcβriiia shows higher affinity for the Fc region than the F-type, which causes an increased antibody dependent cellular cytotoxicity (aDCC). Fc-engineering techniques (e.g., afucosylation and amino-acid alteration) have recently been developed to increase the efficacy of mabs. these approaches raised the affinity of antibodies for the Fcβ receptor and increased aDCC, however the resultant clinical efficacy is insufficient for F/F genotype patients. modification of effecter cells is another approach to enhance aDCC activity, so we evaluated the effect of V-type FCgr3a gene transduction into peripheral blood mononuclear cells (PbmCs) on aDCC activity, especially in 158-F/F carriers. in the present study, the FCgr3a-158V gene was transduced into PbmCs of volunteers and whether this transduction increased Herceptin-mediated aDCC against Her2 positive breast cancer cell lines was examined.method: FCgr3a cDnas (158-V and 158-F) were cloned from PbmCs of healthy volunteers by rt-PCr. after full sequencing, FCgr3a (158-V and 158-F)-expressing recombinant retroviruses were established using lngFr-containing shuttle plasmids. FCgr3a (158-V and 158-F) and lngFr genes were subsequently transduced into volunteers’ PbmCs. CD16 and lngFr expression were examined by flow cytometric analysis and aDCC against mCF-7-mediated Herceptin was tested by Calcein-acetyoxymethyl cytotoxicity assay.results and Conclusion: FCgr3a (158-V and 158-F) containing recombinant retroviruses were successfully made. in FCgr3a-158V gene-transduced PbmCs, CD16 expression increased as well as aDCC against Herceptin-mediated breast cancer cells. these results suggest that FCgr3a-158V gene transduction augments antibody-dependent cellular cytotoxicity. We conclude that cell therapy using FCgr3a-158V gene-transduced effecter cells is an effective approach to increase the efficacy of monoclonal antibodies.


Poster Abstracts

117PRE-CLInICAL EVALuAtIOn OF An OPtIMIZEd RnA ELECtROPORAtIOn SyStEM FOR t LyMPhOCytE BASEd CAnCER AdOPtIVE IMMunOthERAPy Y. Zhao1, E. Moon2, A. Cannon1, X. Liu1, C. Carpenito1, S. Jiang1, D. Maier1, A. Vogel1, Z. Zheng1, B. L. Levine1, S. M. Albelda2, C. H. June1; 1Abramson Cancer Center and Department of Pathology and Laboratory Medicine,University of Pennsylvania School of Medicine, Philadelphia, PA, 2Children’s Hospital of Philadelphia, Philadelphia, PA.

redirecting t lymphocyte antigen specificity by gene transfer can provide a large amount of tumor reactive t lymphocytes for adoptive immunotherapy. However, safety concerns and high viral vector production associated costs, have limited clinical application.t lymphocytes can be gene modified by rna electroporation without integration associated safety concerns. to establish a safe and cost effective platform for adoptive immunotherapy, we first optimized the vector backbone for rna in vitro transcription by adding non-translational regions (utr) or prolonged polya sequence. Different methods of producing capped rna to acheive high level transgene expression using less rna were tested. adding both 3’utr and longer polya could increase the transgene expression of the chimeric antigen receptors (Car) tested by 2-4 fold (mFi by flow cytometry). High quality rna, as defined by transgene expression and function of the rna electroporated t cells, and low cost could be produced by making rna using a capping enzyme system. Car expression of rna electroporated t cells could be detected up to 7-8 days post electroporation. nearly 100% transgene expression could be maintained for at least 2-3 days both in vitro and in vivo. the feasibility of treating large established tumors in a humanized mouse model was tested using rna electroporated human t lymphocytes with a large scale closed system electroporator (maxcyte), in a gmP facility. Dramatic tumor reduction was seen when the pre-existing intraperitoneal human-derived tumors that had been growing in vivo for over 50 days were treated by multiple injections of autologous human Pbls electroporated with Car rna. significantly prolonged survival and reduced tumor burden was observed in the treated mice compared to the control groups.this optimized rna electroporation system could be a novel and cost-efficient platform for the treatment of caner and infectious diseases without the associated safety concerns of integrating gene vectors.

119PARAMEtERS thAt IMPACt On tnC And Cd34+ CELL RECOVERy FOR uMBILICAL CORd BLOOd BAnKInGA. A. Carrasco-Yalán, C. Villanueva, J. Pando, P. Saenz, H. Rios, M. Marquez, I. Honda, I. Aranda, I. Perez, G. Vidal, J. Huamani, E. Obregón-Zegarra, P. Carrasco-Yalán, M. Vidurrizaga, J. F. Castillo-Aguirre; ICTC - PERU, Lima, PERU.

umbilical cord blood (uCb) has become an easily, available and viable source of hematopoeitic stem cells for transplant.the aim of our study was to compare mother´s age [ma], gestational age at delivery [ga], gender [g], birth weight [bW] and type of delivery [tD]; with tnC and CD34 + cells recovery.From may 2004 to Jun 2009, 4262 uCb were evaluated. 77.87% of the Cbu were collected after cesarean; median tCP was 30 hours 58 minutes. the mean Cbu volume and tnC count were 81.8 ml and 8.6 x 108 respectively. the volume was greater in cesarean delivery than vaginal delivery (p<0.001). tnC counts collected were directly correlated with ga: in preterm delivery (=37sem) was 9.93x108 and with bW (p<0.001) while the ma had inverse correlation (p=0.005); regarding tD there was a significant mayor tnC count in the vaginal vs. cesarean group (10.5x108 vs. 9.2x108, p<0.001); while it was a trend for major tnC count in females vs. males babies (9.8x108 vs. 9.1x108, p=0.059). CD34+ cells count was directly correlated with bW (p<0.001). the strongest correlation was with ga (in preterm: 62 CD34+/ul and in term delivery: 84.6 CD34+/ul, p=<0.001); moreover, there was association between CD34+ cells count with tD (vaginal: 87.8 CD34+/ul vs cesarean: 80.2 CD34+/ul, p=0.02). there was not association either with g (fem: 79.9 CD34+/ul vs mal: 83.48 CD34+/ul, p=0.197) neither ma (p=0.17). there was a significant difference between CD34+ viability cells among Cbu with less than 48 hours or more of tCP (99.6% vs. 99.4%, p=0.019); this difference was stronger when the CD45+ viability was evaluated (93.35% vs. 90.14%, p<0,001).it looks on time female with good weight, born from a younger mother and vaginal delivery reach highest tnC count. CD34+ cells count was directly correlated with bW and CD34+ viability is mainly influenced by tCP

118EStABLIShInG A CORd BLOOd SAMPLE REPOSItORy FOR A PERSOnALIZEd MEdICInE InItIAtIVEJ. Bullough1, V. Tahan2, M. Wilde1, J. Morris1, M. P. Henshaw1, P. Jacobson1, L. L. Kelley1; 1University of Utah, Salt Lake City, UT, 2University of Moron, Buenos Aires, ARGENTINA.

Personalized medicine is a dominant trend in health care and relies on a network of electronic records that link medical information to biosamples to help physicians make optimal treatment decisions. We sought to establish a repository of cord blood (Cb) samples as part of an institution-wide Personalized medicine initiative. Cb was collected from 96 healthy donors and processed using Ficoll-Hypaque of two different densities to separate plasma, mononuclear cells (mnC), granulocytes and erythrocytes (rbC) (n=40) or with a single Ficoll-Hypaque layer leaving rbC and granulocytes unseparated (n=56). total nucleated cell (tnC) and mnC recovery was consistently higher using the single layer technique (29±11% vs 14±8% for tnC and 41±18% vs 17±8% for mnC). mnC from a subset of frozen Cb samples (n=10) yielded 87±2% viability and 63±6% recovery upon thaw. CFu-gm, -gemm and bFu-e colonies were cultured in methylcellulose from thawed mnC and compared with %CD34+ cells as determined by flow cytometry from the same samples. excellent correlation was observed between total colony outgrowth and %CD34+ cells (r=0.83). Dna was isolated from either 60 µl of fresh whole Cb spotted onto genPlatetm membranes or from frozen granulocytes using gentra Kit. significantly more Dna was isolated from fresh Cb samples than from frozen granulocytes (10±3 vs 3±3 µg per 106 cells, respectively, p<0.005) and obviated the need for a two layer Ficoll-Hypaque procedure and for frozen storage. Dna isolated from the two techniques was further tested on microarrays and for Hla typing using molecular sequencing techniques and was found to perform comparably in all assays. in summary, we demonstrated that a simple, cost-effective, one layer Ficoll-Hypaque separation yields sufficient plasma, Dna and viable mnC for a wide variety of further testing with minimal requirements for frozen storage. the repository will be expanded to include disease-associated samples and made available for researchers.

120AntIGEn SPECIFIC huMAn AntIBOdy PROduCtIOn In huMAnIZEd nOd/SCId IL2R GAMMA C nuLL (nSG) MOuSE MOdEL, tRAnSPLAntEd huMAn FEtAL tISSuES WIth Cd34+ CELLSY. Chung1, J. Sohn2, B. Choi2, J. Kim3, M. Shin3, S. Song3, S. Kim4; 1Transplantation Research Center, Dept of Surgery, Samsung Medical Center, Seoul, KOREA, REPUBLIC OF, 2Transplantation Research Center, Samsung Medical Center, Seoul, KOREA, REPUBLIC OF, 3Dept of Surgery, Samsung Medical Center, Seoul, KOREA, REPUBLIC OF, 4Transplantation Research Center, Dept of Surgery, Samsung Medical Center, Seoul, KOREA, REPUBLIC OF.

Humanized mice would be valuable model for studies to understand the human immunobiology. However, functional human immune system in humanized mice still needs to be improved. in the current study, we report that transplantation of human fetal tissues and CD34+ cells leads to the development of improved and functional human immune system in supra-immunodeficient mouse line, noD/sCiD il2r gamma C null (nsg). in the human fetal tissues/CD34+ cells-nsg (Hu-nsg) mice, human b cells were fully differentiated, and significant number of human b cells was accumulated in the mouse spleen. the developmental stages of human b cell maturation were detected in the mouse spleen. the mice were evaluated for the ability to produce the antibody to the t cell specific antigen, 2, 4-dinitrophenyl hapten-keyhole limpet hemocyanin (DnP-KlH). DnP-KlH specific-igg and igm were successfully detected in the mouse sera, immunized with DnP-KlH. Hybridoma lines were successfully generated from spleen b cells in DnP-KlH immunized hu-nsg mice by fusing with human mouse chimeric myeloma cell line. one of these cell lines was DnP-KlH specific human igm and another one was DnP-KlH specific human igg. taken together, our human fetal tissues/CD34+ cells-nsg mice are even improved and achieved functional human immune system.


may 23-26, 2010


Poster Abstracts

121IMPACt OF ABO-MISMAtCh On tRAnSFuSIOn REQuIREMEntS In BOnE MARROW tRAnSPLAntAtIOnG. C. De Santis1, M. A. Madeira1, B. P. Simões2, J. C. Voltarelli2, D. T. Covas2; 1Hemocentro de Ribeirão Preto, Ribeirão Preto, BRAZIL, 2University of São Paulo-USP, Ribeirão Preto, BRAZIL.

abo-mismatch is not considered a barrier to bone marrow transplantation (bmt). it occurs in 30-40% of cases, owing to the fact that abo histoblood groups are inherited independently from human leucocyte antigens. some authors have suggested that abo mismatch could have a negative impact on survival, on disease recurrence, on the incidence of graft-versus-host disease and on transfusion requirements in bmt. there are 3 types of incompatibility: major (isoagglutinins in the recipient against donor red blood cell), minor (isoagglutinins in the donor against recipient red blood cell and bidirectional (a combination of both). our objective was to evaluate the impact of abo-mismatch on transfusion requirements in subjects submitted to bmt.material anD metHoDs: We recorded the number of units of red blood cell (rbC) and platelet (PC) concentrates transfused from days 0 to +100 on 152 consecutive patients submitted to related Hla-identical bmt between 1998 and 2008 in our institution. Patients transplanted with peripheral hematopoietic stem cells were excluded from the analysis.results: 42 of 152 (27.63%) patients presented abo-mismatch: 26 (61.90%) major, 14 (33.33%) minor and 2 (4.76%) bidirectional. the mismatch group received more units of rbC: 12 (0-47) x 5 (0-57) (P<0.0001) and PC: 83.50 (0-429) x 48 (0-756) (P<0.0184) than identical-abo group. Patients with major abo-mismatch received more units of rbC: 13 (0-47; P= 0.0002) and PC: 91.50 (0-429; P= 0.0476). Patients with minor abo-mismatch received more units of rbC: 9.5 (0-3-30; P= 0.0221) but not of PC: 83 (16-381; P= 0.1008). results are given in median (range) and percentage.ConClusion: We found in our population na inferior percentage of abo-mismatch in bmt than that reported in literature. the mismatch resulted in a higher requirement for rbC and PC transfusion, especially the major type, but even the minor type required more rbC transfusion.

123EXPRESSIOn OF AdhESIOn MOLECuLES On Cd34 CELLS FROM MOBILIZEd PERIPhERAL BLOOd, uMBILICAL CORd BLOOd And BOnE MARROWG. C. De Santis1, B. P. Prado1, K. L. Prata1, P. V. Palma1, D. T. Covas2; 1Hemocentro de Ribeirão Preto, Ribeirão Preto, BRAZIL, 2University of São Paulo-USP, Ribeirão Preto, BRAZIL.

introDuCtion: mobilization of CD34+ hematopoietic progenitor cells into peripheral blood is achieved by the administration of chemotherapy and/or hematopoietic growth factors like g-CsF. the mechanisms involved in this phenomenon may be related to the expression of adhesion molecules (am) on CD34+ cells. We analyzed the expression of some am on CD34+ cells from mobilized peripheral blood (Pb), umbilical cord blood (uCb) and from bone marrow (bm).material anD metHoDs: We analyzed by flow cytometry the percentage of CD34+ expressing the following am: CD11a, CD49d, CD54, CD117, CD62l, CD44 and CD31 from samples obtained from leukapheresis products of 10 patients with hematological malignancies submitted to CD34 cell mobilization (cyclophosphamide and g-CsF), from 10 healthy bone marrow donors and from umbilical cord blood of 27 fullterm newborns.results: CD11a, CD117, CD44 and CD31 were equally expressed on CD34+ cells from the 3 sources (data not shown). CD49d and CD62l were less expressed on CD34 cells from Pb related to bm (68.7 x 97.2%; P= 0.001) and to uCb (23.9 x 49.1%; P=0.0079), respectively. However, a lesser percentage of CD34 cells from bm expressed CD54 related to the 2 other sources (49.0 x 70.7-75.5%; P= 0.002).ConClusion: CD34 cells from Pb expressed lower levels of CD49d and CD62l, a fact that could be related, at least in part, to their migration from bm into Pb. these findings suggest that the down-regulation of some ams could be an alternative to mobilize CD34 into PF in order to permit their harvest, especially in patients considered ‘poor mobilizers’.

122EFFECt OF WAShInG CELL SuSPEnSIOnS On AdVERSE REACtIOnS ASSOCIAtEd WIth InFuSIOn OF CRyOPRESERVEd hEMAtOPOIEtIC StEM CELLG. C. De Santis1, L. C. Oliveira1, M. D. Orellana1, K. L. Prata1, B. P. Simões2, J. C. Voltarelli2, D. T. Covas2; 1Hemocentro de Ribeirão Preto, Ribeirão Preto, BRAZIL, 2University of São Paulo-USP, Ribeirão Preto, BRAZIL.

introDuCtion: infusion of cryopreserved peripheral hematopoietic stem and progenitor cell (HsPC) has been associated with a number of related toxicities which are attributed to cellular debris, to free hemoglobin and mainly to dimethyl-sulphoxide (Dmso) present in the thawed cell suspension. the frequency and severity of the toxicities are considered to be related to the volume infused. the most frequent ones are nausea and vomiting but fatalities have been reported.material anD metHoDs: 110 consecutive patients with hematological malignancies were submitted to autologous HsPC transplantation in our institution. nineteen of them (17.27%) received washed HsPC suspensions, as requested by the medical assisting team. ninety one subjects were infused with unmodified cell suspensions. the adverse reactions during the infusion were recorded. HsPC were harvested from peripheral blood by apheresis, cryopreserved with 10% of Dmso in a controlled-freezing system and stored in liquid nitrogen. the cell suspensions were thawed and washed manually (2x) with a solution of albumin 2% before infusion. Patients received diphenidramine before infusion.results: the group that received unmodified HsPC suspensions presented a higher frequency of adverse reactions: facial flushing: 56/91 x 1/19 (P<0.0001); nausea/vomiting: 49/91 x 2/19 (P=0.0007) and abdominal cramps/diarrhea: 27/91 x 1/19 (P=0.0392). the frequency of severe reactions was not statistically different between groups: 9/91 (encephalopathy 2, acute renal failure 2 and respiratory distress 5) x 1/19 (respiratory distress). all patients recovered. neutrophil recovery (> 500/µl) was not different between the groups: on days 10 (unmodified) and 11 (washed).ConClusion: manual washing of HsPC suspensions is a save procedure but labor-intensive. However, it is a powerful means to prevent adverse reactions associated with the infusion of cryopreserved cells. We believe that this procedure should be adopted by the institutions that perform autologous HsPC transplantation, at least for the subjects considered to be at higher risk.

124FIBROnECtIn And StRESS GROWth FACtORS EnhAnCE EX VIVO EXPAnSIOn And MAtuRAtIOn OF MEGAKARyOCytE And hEMAtOPOIEtIC PROGEnItORS FROM uMBILICAL CORd BLOOdV. R. Deutsch, E. Hubel, S. Kay, E. Naparstek, D. Grisaru; Tel Aviv Medical Center, Tel Aviv, ISRAEL.

Protracted thrombocytopenia remains a serious clinical problem following cord blood transplantation due to the paucity of the stem and progenitor cells. administration of thrombopoietin (tPo) was not clinically effective post bmt due inadequate numbers of mK-p. ex-vivo expanded mk-p could supply the appropriate target cells to maximize the effect of naturally elevated tPo in these patients to facilitate thrombopoiesis. While isolated Cb CD34+ cells can be expanded in-vitro, this is not practical because the limited number of stem cells in the Cb units cannot be sacrificed. additionally, mK expansion from purified stem cells requires long culture periods. We propose a novel strategy to expand mK-p and HsPC from Cb mononuclear cells (mnC) in short term 10 day cultures using 20 ml of cord blood. Progenitors enriched mnC (1) were expanded on fibronectin (Fn) coated dishes with autologous plasma and stress growth factors. these included r-hu-tPo (10 ng/ml) and arP a peptide derived from the stress variant of acetylcholinesterase (aChe-r), recently discovered to have potent hematopoietic stem cell and mK growth factor activity (2). application of high definition flow cytometry using multiple gates, enabled clear resolution of the expansion of HsPC and mk-p and their early subsets in culture. Fn increased viability and expansion of all CD34+ HsPC (7-fold), early CD34+/CD41low (90-fold) and mk-P(ssClow/CD45dim/neg/CD41high) (4-fold). the addition of arP to Fn dramatically expanded the CD34+/CD41low/ HsPC (400 fold) but not the CD41high/mk-p due to accelerated maturation. Fn+tPo+arP expanded thrombopoietic precursor(ssClow/CD45dim-neg/CD41high) and the very early myeloid (ssClow/CD33+/CD34highCD41low), increased the proliferation of mK, myeloid and multilineage colony forming progenitors and supported mk maturation as measured by ploidy and gPiib/iiia expression. these engineered growth conditions which supported the expansion of early mk precursors without sacrificing the coveted stem cells in the unit, may enable improved cell therapy modalities to facilitate earlier platelet production post-Cbt.


Poster Abstracts

125An AutOMAtEd uMBILICAL CORd BLOOd unIt (CBu) WASh PROCEduRE uSInG thE SEPAX® CELL PROCESSInG SyStEM PROVIdES hIGh QuALIty POSt-thAW yIELdS FROM CRyOPRESERVEd CBuR. Dornsife1, A. Reese2, O. Waridel2, A. Kaestner3, M. Reese3, S. Avrutsky3, J. Kurtzberg4; 1Carolinas Cord Blood Bank, Duke University Medical Center, Durham, NC, 2Biosafe America, Houston, TX, 3Stem Cell Laboratory, Duke University Medical Center, Durham, NC, 4Carolinas Cord Blood Bank & Stem Cell Laboratory, Duke University Medical Center, Durham, NC.

recovery of viable and functional cells from thawed cord blood units (Cbu) undergoing preparation for transplantation can be critical to the engraftment outcomes of a cord blood transplant (Cbt). Currently there is no consensus around a standardized method to thaw, dilute, and/or wash these cells. a standard procedure using an automated device to thaw and wash cryopreserved Cbu that reproducibly results in high quality yields (recovery of potent cells) for infusion, would greatly enhance the field of Cbt. to date, clinical transplant laboratories have primarily used labor intensive, manual washing methods that may not always result in high quality yields. automation is one approach to standardize this procedure with the goal of processing in a closed system and resulting in a reproducible, high quality product. an automated procedure would also reduce the amount of hands-on time needed to prepare units for infusion. accordingly, we tested a new, 35 minute, automated wash program using the sepax® cell processing system (biosafe) with a sterile, closed-system kit that incorporates a controlled dilution with dextran 40 plus 5% human serum albumin (Da) solution and produces a 50 ml final volume product for infusion. in preliminary studies, thawing and automated washing of 8 Cbu resulted in a mean recovery of total nucleated cells of 91% ± 10% (± standard deviation) with 95% ± 2% viability. the wash procedure tested incorporates two rinse cycles of the cryobag and offers great promise in meeting quality goals. testing to determine the optimal number of rinse cycles that maximizes cell recovery without significantly impacting cell recovery or viability of stem and progenitor cells is in progress. Validation of this automated Cbu wash program will be presented including additional quality measurements enumerating Cbu stem and progenitor cells (CD34+, alDH bright, CFus).

127nOVEL uSE OF MAtuRE EnuCLEAtEd REd BLOOd CELLS AS A VIRAL tRAP/SInK FOR hIV And OthER MAMMALIAn VIRuSES.L. F. Glaser; Public, Fairfax Station, VA.

this is a Patented and novel approach proffering a completely new basis for cell therapy utilizing red blood cells as viral traps or viral sink(s).all viruses must pass from host to host and gain entry into choice cells to then replicate and continue cycling. Viruses leverage a choice receptor/coreceptor of a cell to gain entry into cells. aside from receptors/coreceptors, there are additional factors viruses leverage to further the probability that cellular access will be granted frequently enough to perpetuate and propagate a given viral-based disease.if this cycle is sufficiently interrupted through direct attenuation of the virus the disease condition the virus normally generates will cease to manifest.my approach is detailed in my us Patent grant # 7462485, with improvements outlined in further filings, including a recently filed PCt international filing. i proposed the use of red blood cells modified to include receptors and coreceptors which will cause HiV to bond, fuse, and inject the viral nucleocapsid into the red blood cell, as a decoy or misdirection. once inside the rbC, the viral content is not capable of escape, nor can it advance its replication cycle. there can be additional elements either natural, or placed by us, to disable the HiV content and lastly, when the cell ages to approximately 90 days, erythrophagocytosis will utterly eliminate these rbCs and their viral content without risk of releasing any valid variant or form of HiV.the method has been shown to function in niH funded research using lethal viruses and live mammals.i asked for a Patent grant with certainty that i teach a valid method of therapy and with the hope and belief this will lead to a broad-based cure for viral-driven disease in mammals. i am promoting experimentation now, to advance this model to simians and humans.

126AutOMAtIOn OF A RAPId SEVEn-dAy COLOny-FORMInG CELL ASSAy FOR MEASuRInG hEMAtOPOIEtIC PROGEnItORS In CORd BLOOd uSInG MEthOCuLt® EXPRESSO. Egeler, N. Yuan, B. Wognum, S. Woodside, T. Thomas, A. Eaves; Stemcell Technologies, Vancouver, BC, CANADA.

the colony forming cell (CFC) assay is the most widely accepted method for quantifying the frequency of functional hematopoietic progenitors in cell products such as cord blood (Cb), mobilized peripheral blood and bone marrow. While providing a powerful predictive measure of hematopoietic recovery and survival following clinical transplantation, the usefulness of the CFC-assay for routine evaluation of graft quality and potency is impeded by the long assay duration and subjectivity of the analysis. to address these limitations, a semisolid medium (methoCult® express) was formulated to enable the quantification of total colonies after only 7 days of culture. automated colony enumeration was achieved with an image-based analysis system, in combination with cultureware designed to improve image quality and colony distribution.the 7-day assay was validated in Cb cell culture by comparison to the standard 14-day assay using methoCult® H4034. total colony number was quantified manually using standard light microscopy, as well as by automated imaging and analysis. the total colony counts after 7 days in methoCult® express were closely correlated with the counts in methoCult® H4034 after 14 days (r2 = 0.9, p = 0.02), demonstrating that the shorter assay accurately estimates progenitor numbers in Cb samples. in addition, the methoCult® express assay exhibited greater reproducibility, with a significantly smaller coefficient of variation between results obtained by multiple assay operators (6+3% vs. 11+5% for express vs. standard assays, p=0.02). the automated colony counts in methoCult® express were highly correlated to, and not significantly different from manual counts (r2 = 0.96, p<0.01).We conclude that methoCult® express allows for a shorter and more reproducible CFC assay when counting total colonies derived from hematopoietic progenitors in Cb. Furthermore, automated quantification of the 7-day assay colonies is equivalent to the manual method and is a reliable substitute for microscope counting.

128uLtRASOund tREAtMEnt EnhAnCES PROLIFERAtIOn OF hEMAtOPOIEtIC StEM/PROGEnItOR CELLS: IMPLICAtIOnS FOR CLInICAL tRAnSPLAntAtIOn, GEnE And CELLuLAR thERAPIESH. Gul-Uludag1,2, P. Xu1, W. T. Ang2, M. Huang1, X. Yang1, J. Xing2,3, J. Chen1,4; 1University of Alberta, Edmonton, AB, CANADA, 2IntelligentNano Inc., Edmonton, AB, CANADA, 3Cross Cancer Institute, Edmonton, AB, CANADA, 4National Institute for Nanotechnology, Edmonton, AB, CANADA.

background: low-intensity pulsed ultrasound, a form of mechanical energy that is transmitted through and into living tissue as acoustic pressure waves, has been shown to enhance proliferation of human skin fibroblasts by activating the erK1/2 pathway. in addition, the optimization of the cord blood (Cb) mesenchymal stem cell growth was recently established using ultrasound. in this work, we investigated the effect of ultrasound on the proliferation and differentiation of human hematopoietic stem/progenitor cells (HsPC). methods: the human CD34+ cells from Cb and leukophresis product (lP) were stimulated by ultrasound signals at a frequency of 1.5 mHz and intensity of between 40mW/ cm2 and 70 mW/ cm2 for 10 minutes for 4 days. Cell proliferation and viability was assessed at day 5 by trypan blue exclusion and mts cytotoxicity assay. to determine whether differentiation was induced in ultrasound-stimulated cells, the surface antigen expressions of CD34 and CD14 were analyzed by fluorescence-activated cell sorting (FaCs) and morphologically by giemsa staining. Furthermore, the effect of ultrasound-stimulation on the colony formation of HsPC was investigated by colony forming unit (CFu) assay. results: We found that (i) ultrasound-stimulation increases proliferation of HsPC up to 2-fold relative to unstimulated cells, (ii) the expression of CD34 or the percentage of CD14-expressing cells are not affected by ultrasound-stimulation, (iii) both control and ultrasound-stimulated cells retained immature blast-like morphology, and (iv) ultrasound-stimulated cells gave rise to more CFu-gm (colony formation unit granulocyte-macrophage) and bFu-e (burst-forming unit-erythrocyte ) colonies after 14 days in comparison to unstimulated cells. Conclusions: these findings underscore the potentiality of our novel ultrasound-stimulation approach to enhance amplification of HsPC for the clinical transplantation, gene and stem cell therapies.


may 23-26, 2010


Poster Abstracts

129dIRECt IntRA-CARdIAC dELIVERy OF MOBILIZEd BLOOd StEM CELLS In PAtIEntS WIth SEVERE MyOCARdIAL InFARCt: An ALtERnAtIVE FOR hEARt tRAnSPLAntAtIOn?P. R. HENON1, S. Pasquet1, H. Sovalat1, Y. Arkam2, M. Ojeda-Uribe2, N. Bischoff3; 1I.R.H.T., MULHOUSE, FRANCE, 2Department of Hematology, Hôpital E. Muller, MULHOUSE, FRANCE, 3Department of Cardiac Surgery, Hôpital E. Muller, MULHOUSE, FRANCE.

starting from experimental data proposing hematopoietic stem cells as candidates for cardiac repair, we postulated that human peripheral blood (Pb) CD34+ cells mobilized by hematopoietic growth-factor (g-CsF) would contain cell subpopulations capable to regenerate post-ischemic myocardial damages.in a Phase-i clinical assay enrolling seven patients with acute myocardial infarct, we directly delivered to the injured myocardium autologous Pb-CD34+ cells previously mobilized by g-CsF, collected by leukapheresis, and purified by immunoselection. in parallel, we looked for the eventual presence of cardiomyocytic and endothelial progenitor cells in leukapheresis products of these patients and of controls, using flow-cytometry, reserve transcription-quantitative (rtQ)-polymerase chain reaction (PCr), cell cultures, and immunofluorescence analyses.the whole clinical process was feasible and safe. all patients were alive at an average follow-up of 49 months (range 24-76 months). improvement of heart function parameters became obvious from the third month following cell reinjection. left ventricular ejection fraction values progressively and dramatically increased with time, associated with Petscan demonstration of myocardial structure regeneration and revascularization and new york Heart association (nyHa) grade improvement. Furthermore, we identified Pb-CD34+ cell subpopulations expressing characteristics of both immature and mature endothelial and cardiomyocyte progenitor cells. in vitro CD34+ cell cultures on a specific medium induced development of adherent cells featuring morphologies, genes expression and immunocytochemistry characteristics of endothelial or cardiac muscle cells.mobilized CD34+ cells contain stem cells committed along endothelial and cardiac differentiation pathways, which would play a key role in a proposed two-phases mechanism of myocardial regeneration after direct intracardiac delivery, probably being responsible for the long-term clinical benefit observed.

131MInOR ABO MISMAtChEd ALLOGRAFtS FROM dOnORS WIth hIGh ISOhEMAGGLutInIn tItERS dO nOt PROduCE MORE InFuSIOn REACtIOnS OR LOSS OF RECIPIEnt ERythROCytE MASS thAn dO ABO MAtChEd GRAFtS OR GRAFtS FROM dOnORS WIth LOWER tItERSA. Ribickas, L. Meyer, J. Colwell, R. C. Smilee, W. E. Janssen; Moffitt Cancer Center, Tampa, FL.

HPC allografts are frequently administered across minor abo mismatches wherein donors have serum antibodies against recipient rbC. to reduce risk of infusion reactions it is recommended that minor mismatched grafts be washed of plasma if donor serum antibody titer exceeds 64. our facility follows this practice, except for unrelated donor grafts >24 hours old, which are not washed. We reviewed infusion reactions and Hct changes associated with unwashed product infusion. 15 products were from donors with serum antibody titers >64. 21 were from donors with titers of 64 or less. 12 were from abo matched donors. We eliminated products given to recipients who received post-infusion blood transfusions from review of Hct changes. We compared pairwise the results of infusing products from donors with >64 antibody titers with either products from donors with lower titers, or with products from abo matched donors. mann-Whitney u-statistic was employed to compare Hct changes and Fisher’s exact test was employed to compare infusion reaction frequency. Findings are summarized in the table. Products from donors having high antibody titers produced comparable infusion reactions to products from donors having low titers (p=0.48) or that were abo matched (p=0.20). Products from high-titer donors produced no greater Hct decrease than products from donors having low titer (p=0.70) or who were matched (p=0.18). We conclude that washing of allografts obtained from donors having isohemagglutinin titers >64 may be of little benefit. We propose pursuit of larger scale studies directed at changing of practice.

abo match antibody titer <= 64 antibody titer > 64

infusion reactions 6/12 7/21 4/15

Hct change (±std) -1.73±1.86 -2.09±4.53 -2.03±2.87

130APhERESIS-RELAtEd EnRIChMEnt OF Cd26BRIGht t LyMPhOCytES In hEMAtOPOIEtIC PROGEnItOR CELL tRAnSPLAntS IS PREdICtIVE FOR An unFAVOuRABLE OutCOME In AutOLOGOuS tRAnSPLAntAtIOnD. Dijkstra1, K. Daemen1, M. Stevanovic-Meyer1, H. Gollasch2, W. Ludwig2, M. Hildebrandt1; 1IFB-Tx, GMP Development Unit, Medizinische Hochschule Hannover, Hannover, Germany, 2Robert-Rössle-Klinik, Helios Klinikum Berlin-Buch, Berlin, Germany.

the lymphocyte surface glycoprotein CD26 is involved in various signalling and costimulatory events. CD26 possesses dipeptidyl peptidase iV (DPP iV) activity, a serine protease known to inactivate chemokines such as rantes/CCl5, eotaxin/CCl11 and sDF-1alpha/ CXCl12, a key mediator of stem cell homing and engraftment. in this project, we determined the numbers of a distinct subset of CD26bright lymphocytes in autologous hematopoietic progenitor cell transplants (HPCt). Further, the phenotype of CD26bright lymphocytes was investigated in more detail.Forty-two patients (multiple myeloma, n=31; Hodgkin’s Disease, n=3; nHl, n=6; Pnet, n=1; aml, n=1), receiving a HPtC were enrolled.Distinct subpopulations in the peripheral blood and in the apheresis product were determined. these included: CD34+, CD26+, CD26bright, CD4+, CD8+ and CD45ro+ respectively. Possible correlations between the cell numbers, kinetics of engraftment and the progression-free survival were investigated.the numbers of CD26bright t lymphocytes in the autograft correlated inversely with progression-free survival (PFs, p=0.013). in regression analyses, CD26bright/CD45ro+ t lymphocytes transfused per kg body weight were the only variable predictive for the occurrence of disease progression or relapse (p=0.006). importantly, the numbers of CD26bright cells in the autograft showed a highly variable degree of enrichment in the apheresis products irrespective of their numbers in the peripheral blood. CD26bright lymphocytes had a relative expression of CD45robright, CD127bright, CD49dbright, CD278+, CD28+, CD62ldim, CD184dim and CD56-, CD25low, correlating with a memory t lymphocyte phenotype that possesses proliferative capacity and tissue homing characteristics.Conclusion: CD26bright lymphocytes are a subset of memory t cells. their numbers were associated with an adverse outcome in autologous HPC transplantation. the numbers of these cells appeared to be artificially elevated by the apheresis procedure, and not patient-, or disease- related. because of the potential risk of high numbers of CD26bright lymphocytes, the apheresis procedure should be optimized to reduce the enrichment of CD26bright lymphocytes.

132LABOR EFFICIEnCy MAnAGEMEnt uSInG SIMuLAtIOn SOFtWARE LEAdS tO SIGnIFICAnt COSt SAVInGS In A hEMAtOPOIEtIC StEM CELL LABORAtORyN. E. Omer, P. Jacobson, L. L. Kelley; University of Utah, Salt Lake City, UT.

academic hematopoietic stem cell (HsC) laboratories are often requested to justify resource utilization and budgeting requests in the context of other comparable HsC laboratories; however, benchmark data for the industry is not readily available. We sought to undertake a proof-of-concept analysis to determine our baseline labor costs as related to workload and reimbursement by 3rd party payers. Data were collected on 19 vital labor activities associated with collection, processing, testing, storing and distributing autologous HsC apheresis products. Variables included activity times, daily workload, payroll data (staff salary/hr), billing records (billable activities), staff and equipment capacity (number of hrs available/wk). Direct activities, those associated with the manufacture of the product i.e. processing, cryopreservation, etc., were included. indirect activities, those associated with billing, quality assurance, etc., were calculated separately. a simulation software program, arena®, was used to create activity and process models by altering variables associated with each task. the first model simulated activities and processes as they actually existed. the second model altered variables described above to predict greater efficiency. Validations of the models were performed using patient charts, beta-tests and cross-checks to verify model accuracy. the study revealed that direct and indirect activities accounted for 55% and 45% of technologist time, respectively. seventy-five percent of direct activities were reimbursable by 3rd party payers. multiple simulations in the second model demonstrated the potential to reduce labor costs by 40%, primarily by eliminating overtime hours, while maintaining the same workload and staff. three changes accounted for the majority of cost savings including 7on/7off scheduling, flex scheduling and daily assignment of non-urgent activities, i.e. validation studies, quality assurance, document review, inventory control etc., regardless of workload. the changes were implemented over a 14-month time-frame and resulted in $112,000 savings in labor costs, allowing those resources to be utilized for new project development.


Poster Abstracts

133SELF-SECREtEd huMAn IntERLuKIn-6 And SOLuBLE IntERLIKIn-6 RECEPtOR ALPhA InItIAtEd thE JAK1/StAt3 And PI3K/AKt SIGnALS And InduCEd MAIntAIn StEMnESS duRInG EX VIVO EXPAnSIOn WIth huMAn CORd BLOOd Cd34+ And AFt024 CELLS.B. Choi1,2, M. Kim3,2, S. Joo1,2, H. Lee1,2, Y. Chung2, H. Yang2, J. Kim4, J. Moon4, M. Shin4, S. Song4, J. Joh4, K. Lee3, S. Kim4,2; 1Sungkyunkwan University, Seoul, KOREA, REPUBLIC OF, 2Samsung Biomedical Research Institute, Seoul, KOREA, REPUBLIC OF, 3Sungkyunkwan University, Suwon, KOREA, REPUBLIC OF, 4Samsung Medical Center, Seoul, KOREA, REPUBLIC OF.

the purpose of in this study, we demonstrate for main factors and mechanisms of maintains stemness, and improves of stem cell proliferation, survival, and anti-apoptotic ability during ex vivo expansion human umbilical cord blood (huCb) CD34+ cells. We were performed ex vivo expansion with aFt024 cells under cell-cell contact condition. We found that effectively maintained stemness and reduced of apoptotic activity through overexpressed of anti-apoptotic regulators in expanded cells. interestingly, we identified that significantly self-secreted human interlukin-6 (hil-6; 22.284 ± 0.905 pg/ml, p < 0.01) and soluble interlikin-6 receptor alpha (hsil-6rβ; 193.103 ± 5.732 pg/ml, p < 0.01) in cultured supernatant. moreover, we detected that initiates Janus kinease 1 (Jak1)/signal transducers and activators of transcription 3 (stat3) and Phsphoinositide 3 kinase (Pi3k)/akt signal pathways by self-secreted hil-6 and hsil-6rβ. Furthermore, we demonstrated that these signal pathways plays important roles that contributes to stem cells proliferation, maintaining stemness, and continuous self-secreting of hil-6 and hsil-6rβ during ex vivo expansion under cell-cell contacted condition. We provide evidence that the expanded huCb CD34+ cells were effectively maintained stem cell characters in consequence of interaction with huCb CD34+ and aFt024 cells by cell-cell contact during ex vivo expansion bring about the self-secretion of hil-6 and hsil-6rβ, and initiating of the Jak1/stat3 and Pi3k/akt signal pathways.

135SuCCESSFuL EStABLIShMEnt OF huMAnIZEd MICE MOdEL hAVE FunCtIOnAL huMAn hEMAtOPOIEtIC CELLS thROuGh tRAnSPLAntEd OF uMBILICAL CORd BLOOd Cd34+ CELLS IntO BuSuLFAn SOLutIOn InJECtEd nEOnAtAL nOd/SCId/IL2RζnuLL MICE LIVER.B. Choi1,2, M. Kim3,2, S. Joo1,2, H. Lee1,2, Y. Kim2, Y. Chung2, H. Yang2, J. Kim4, J. Moon4, M. Shin4, S. Song4, J. Joh4, K. Lee3, S. Kim4,2; 1Sungkyunkwan University, Seoul, KOREA, REPUBLIC OF, 2Samsung Biomedical Research Institute, Seoul, KOREA, REPUBLIC OF, 3Sungkyunkwan University, Suwon, KOREA, REPUBLIC OF, 4Samsung Medical Center, Seoul, KOREA, REPUBLIC OF.

the humanized mice models have complete functional human immune cells are very useful tools for studies of human immune systems. in this report, we established humanized mice models by injected busulfan via a facial vein, and intrahepatic injected of human umbilical cord blood (huCb) CD34+ cells in neonatal noD/sCiD/il2rβnull (nsg) mice liver. in all mice injected with 2 x 105 cells, successfully engrafted and reconstituted human CD45 hematopoietic cells were detected in peripheral blood mononuclear cells (PbmCs). 24 weeks after transplantation, we sacrificed humanized nsg (hu-nsg) mice and assessed of repopulated human cells in hu-nsg mice lymphoid organs, bone marrow (bm), spleen, liver and lymph nodes. moreover, we observed that expanded huCb CD34+ cells complete differentiated to mature human blood cells, including CD3, CD4 and CD8 t lymphocytes; CD19 and CD20 b lymphocytes; CD3-CD56+ natural killer (nK) cells; CD11b and CD68 monocytes/macrophages; CD11c and CD21 dendritic cells (DCs). the result of mixed lymphocyte reaction (mlr), we observed that treated of mitogenic stimulator, PHa but also non-treated hu-nsg spleen cells responded to allogenic human cells. moreover, significant approximately 40-fold increased of human CD3 t lymphocytes in Hu-nsg spleen cells. Furthermore, these hu-nsg mice produced of human immunoglobulin m (igm) and igg. thus, we suggest that the newly completed hu-nsg mice are very valuable models for analyses of human hematopoiesis, immune responses and producing human specific antibodies for human antigens. in addition, these mice can also utility to with preclinical investigates model, such as diverse human specific diseases and infections studies.

134EStABLIShMEnt OF COMPLEtE RECOnStItutEd huMAn LyMPhOCytES And PROduCEd AntIGEn-SPECIFIC AntIBOdIES AFtER BuSuLFAn tREAtEd OF nOd/SCId/IL2RζnuLL MICE.B. Choi1,2, M. Kim3,2, S. Joo1,2, H. Lee1,2, Y. Kim2, Y. Chung2, H. Yang2, J. Kim4, J. Moon4, M. Shin4, S. Song4, J. Joh4, K. Lee3, S. Kim4,2; 1Sungkyunkwan University, Seoul, KOREA, REPUBLIC OF, 2Samsung Biomedical Research Institute, Seoul, KOREA, REPUBLIC OF, 3Sungkyunkwan University, Suwon, KOREA, REPUBLIC OF, 4Samsung Medical Center, Seoul, KOREA, REPUBLIC OF.

the humanized mice were very useful in vivo model for human immune system. During humanized mice modeling almost treated whole body irradiation before human hematopoietic stem cells (HsCs) engraftment. However, established humanized mice by radiotherapy were very short life span, therefore limited long-term study for human immune system. We are performing for overcomes this problem that injected chemotherapeutic reagent, busulfan solution. in this study, we determined of optimal concentration of injected busulfan, before transplantation human umbilical cord blood (huCb) CD34+ cells in noD/sCiD/il2rβnull (nsg) mice. From 12-week to 20-weeks after transplantation, furthermore, we performed boosted for observed that secreted antigen specific antibody used human t-cell dependent antigen, 2,4-dinitrophenyl hapten-keyhole limpet hemocyanin (DnP-KlH) for every 2 weeks and final injected into mice spleen. Here, we detected that injected 30 mg/kg body weight group achieved significantly higher levels of hCD45+ in peripheral blood than other group. moreover, hCD19+ b and hCD3+ t lymphocytes were higher reconstituted in peripheral blood and secondary lymphoid tissues. although incomplete, we detected that lymphoid follicle-like structure in 30 mg/kg injected group mice spleen by immunohistochemistry. Furthermore, we detected that human total igm and igg, as well as DnP-KlH-specific igm and igg were significantly secreted in 30 mg/kg group. reconstituted human t lymphocytes in these group mice (DnP-KlH immunization in 30 mg/kg injection mice group) showed strong antigen-specific human t lymphocytes proliferation and responses in vitro. in these results, we were successful established complete reconstitute human lymphocytes and produced antigen-specific antibodies used 30 mg/kg busulfan and these humanized mice model can also utility to with preclinical investigates model, such as diverse human specific disease and infections researches.

136InStEM tRIAL - IntRAOPERAtIVE, AutOLOGOuS Cd133+ CELL ISOLAtIOn And tMLR-SuPPORtEd tRAnSPLAntAtIOn duRInG CABGH. Klein, A. Assmann, A. Lichtenberg, M. Heke; Clinic for Cardiovascular Surgery, Heinrich-Heine-University Medical School, Duesseldorf, GERMANY.

introductiontransplantation of CD133+ cells with the potential to improve myocardial function is a promising alternative treating chronic cardiac ischemia. the instem trial aims at evaluating safety and feasibility of transepicardial application of CD133+ cells during coronary artery bypass grafting due to end-stage heart failure.methods & resultsPatients suffering from severe ischemic cardiomyopathy (ejection fraction > 15% and < 35%) are enrolled in the study. bone marrow is harvested from the iliac crest. CD133+ cells are purified employing the ClinimaCs device, yielding purities of up to 99%. the myocardial region of interest is pretreated by transmyocardial laser revascularisation, particularly to support cell homing. autologous bone marrow CD133+ cells (up to 30 x 106 cells) are injected into predefined regions. neither have we observed any perioperative mortalities nor did we detect any major adverse cardiac events during the follow up. left ventricular ejection fraction - assessed by echocardiography - has improved from 25% ± 5% preoperatively to 40% ± 8% after 6 months. Quality of life has been enhanced notably for all patients.Conclusionthe intraoperative isolation of CD133+ cells while performing coronary artery bypass grafting and subsequent transepicardial cell transplantation proves feasible and safe. no perioperative major adverse cardiac events have occurred thus far. although the follow up has not yet been completed for all patients the data clearly indicates this procedure to significantly improve left ventricular function and thus, might be a promising causal therapy for severe ischemic cardiomyopathy.


may 23-26, 2010


Poster Abstracts

137GSK-3ζ InhIBItIOn InduCES EnGRAFtMEnt OF EX VIVO EXPAndEd hEMAtOPOIEtIC StEM CELLSK. Ko1, T. Holmes1, P. Palladinetti1, E. Song1, R. Nordon2, T. A. O’Brien1, A. Dolnikov1; 1Sydney Children’s Hospital, Randwick, AUSTRALIA, 2The University of New South Wales, Randwick, AUSTRALIA.

glycogen synthase kinase-3β (gsK-3β) has been recently identified as an important regulator of stem cell function We have earlier demonstrated that gsK-3β inhibition preserves the regenerative function of human haematopoietic stem cells during the ex-vivo expansion process. Here we show that treatment of cytokine-expanded CD34+ cells with the gsK-3β inhibitor 6-bromoindirubin 3’-oxime (bio) prior to transplantation significantly increases bone marrow repopulation in the non-obese, severe combined immunodeficient (noD-sCiD) mouse model. in vitro studies show that gsK-3β inhibition delays proliferation of human haematopoietic progenitor cells while increasing numbers of slowly dividing multipotent progenitors. gene expression analysis revealed that gsK-3β inhibition modulates the expression of a relatively small subset of genes that are transcriptional targets for cytokines. gsK-3β inhibition antagonised the down-regulation of cdki p57 and up-regulation of cyclin D1 by hematopoietic cytokines, providing a possible mechanism for the bio-induced delay in differentiation and cell cycle progression. surprisingly, inhibition of gsK-3β, a negative regulator of Wnt/β-catenin signalling, was not sufficient to activate β-catenin target gene expression in haematopoietic stem cells. therefore alternate mechanisms were responsible for enhancing engraftment of ex vivo expanded stem cells. the data supports a clinical role for gsK-3β inhibition to improve engraftment efficiency of ex vivo expanded stem cells.

139EVALuAtIOn OF CELL COnCEntRAtE PREPAREd FROM BOnE MARROW ASPIRAtE uSInG thE RES-Q™ 60 BMC SyStEMV. Kumar1, R. Suzuki2, H. Pedrozo3, R. Johson4, C. Meyer5; 1Thermogenesis Corp., Rancho Cordova, CA, 2Spine Smith LP, Austin, TX, 3Spine Smith LP,, Austin, TX, 4Methodist Hospital, San Antonio, TX, 5Foundation Surgical Hospital, Houston, TX.

background: the res-Q system is a point of care device that allows for the efficient and rapid collection of bone marrow concentrate (bmC) from bone marrow aspirate (bma). the fluid path within the disposable is sterile and non-pyrogenic. the goal of this study was to evaluate the res-Q for its performance in preparing cell concentrates from human bone marrow aspirate in the intra-operative point of care setting.methods: bone marrow aspirate was collected in the presence of the anticoagulant (aCD-a) and concentrated at the point of care as part of the procedure for making an inductive graft material to be used for spinal fusion. small samples of marrow prior to and after processing were collected and sent to the laboratory for cell analysis. Complete blood counts were measured using sysmex Xe-2100 cell counter and CD34+ cells were measured by flow cytometry using stemKit (beckman Coulter, brea, Ca).results: a total of 10 bone marrow aspirates were processed. time to process each sample was less than 15 minutes. the cell counts for pre- and post-res-Q samples, cell enrichment factor and cell recovery are reported in the table below.

WbC (x10^6/ml)

mnC (x10^6/ml)

Platelet (x10^6/ml)

CD34+ Cell (x10^4/ml)

Pre- res-Q 24+/-15 7+/-5 163+/-66 18+/-15

Post- res-Q 142+/-86 41+/-14 995+/-647 163+/-72

mean enrichment Factor

6x 6x 4x 9x

Cell recovery (%) 69+/-27 83+/-22 70+/-33 90+/-37

Conclusion: the res-Q bmC system demonstrated an efficient, consistent, rapid and easy method to prepare cell concentrates from human bone marrow in an intra-operative setting and at the point of care.

138MEthOd FOR EVALuAtIOn OF tEFLOn OVERWRAP BAGS uSEd tO PREVEnt MICROBIAL SuRFACE COntAMInAtIOn OF CRyOPRESERVEd CORd BLOOd unItS V. Kumar, J. Chapman; Thermogenesis Corp., Rancho Cordova, CA.

baCKgrounD: the contamination of the surface of cryopreservation bags with pathogens is a very low but present risk during cryostorage using either nitrogen vapor or liquid nitrogen freezers. to mitigate this risk, teflon overwrap bags can be used to seal cord blood units prior to the cryopreservation step. to characterize the physical integrity of overwraps a surrogate for microbial pathogens, horse radish peroxidase (HrP) enzyme -substrate system was used as the means to detect microleaks not visible to the eye. HrP is much smaller than any viral particle.metHoDs: twenty-four freezing bags were filled with HrP enzyme solution. in test and positive control samples, a single puncture was created in the freezing bag to simulate an improperly sealed freezing bag. after undergoing a freeze/thaw cycle, freezing bags containing the enzyme were exposed to substrate and observed for blue coloration. the visible evidence of enzyme substrate reaction was recorded. results: results for this test are shown in below.

group/iDHrP enzyme in the product bag

yes/no

Product bags exposed to

Freeze/ Cycle yes/no

HrP substrate added to the test

sandwich bag yes/no

Color of substrate bag after 15 min incubation

negative Control bags( n=4)

no yes yes Clear

test bag (n=20) yes yes yes Clear

*Positive Control bags(n=4)

yes yes yes blue

Clear substrate: no leak in overwrap bagblue substrate: leak in overwrap bag*Positive Control: Hole was created in product & overwrap bag ConClusions: no leaks were observed in overwrap bags after the liquid nitrogen freeze/ thaw cycle using an ultra-high sensitivity test system. it is concluded that the teflon overwrap bags employed provide an effective physical barrier to prevent ingress/egress of potentially biohazardous fluid. overwrap bags should be employed by cord blood banks using either liquid or vapor nitrogen storage to protect against the risk of surface contamination during cryopreservation.


Poster Abstracts

140uSE OF PLERIXAFOR In COMBInAtIOn WIth G-CSF And ChEMOthERAPy yIELd SuFFICIEnt nuMBERS OF Cd34+ CELLS tO OFFER “POOR MOBILIZERS” hIGh dOSE thERAPyD. Josefsen1, H. Holte2, K. Parto3, G. Kvalheim1; 1Dep of Cellular Therapy, Dep of Oncology, Oslo University Hospital, Oslo, NORWAY, 2Dep of Oncology, Oslo University Hospital, Oslo, NORWAY, 3Dep of Pediatric Hematology, University Hospital of Tampere, Tampere, FINLAND.

mobilization of autologous hematopoietic stem cells from non Hodgkins lymphoma and myeloma patients is routinely performed by g-CsF in combination with chemotherapy. to obtain a quick and sustained tri-lineage engraftment after high dose therapy, a minimal number of 2x106 CD34+ cells/kg are desirable. However, 20% of our patients do not reach this number of CD34+ cells in spite of 3 consecutive days of leukapheresis. these patients, characterized as poor-mobilizers, will either be remobilized, or can not be offered high dose therapy even if it can be curative. recently, new mobilizing agents including plerixafor, a CXCr4 antagonist, have been developed. a limited number of poor-mobilizers have so far been studied, and most investigators have remobilized the patients with g-CsF and plerixafor. remobilization with this regimen requires treatment-free interval of 4-6 weeks before a new mobilization can be attempted. in lymphoma patients such chemotherapy free treatment interval might have clinical implications prior to high dose therapy.in this study five poor-mobilizers were mobilized with plerixafor + chemotherapy/g-gsF.(bold text indicates plerixafor given)in conclusion, our preliminary experiences show that perlixafor can be successfully used in addition to chemotherapy and g-CsF in patients when the CD34+ cell concentration of 5-11 x 106/l in blood is reached. CD34+ cell concentrations below this threshold appear not to give any additional effect of Plerixafor. importantly, plerixafor (pat2+3) can also be added upfront to mobilize sufficient PbPCs in poor-mobilizers. more patients are currently recruited to further examine the effect of plerixafor upfront together with chemotherapy+g-CsF in poor-mobilizers.

Day 11 Day 12 Day 13 Day 14 Day 15 Day 16

Patient 1

leukocytes x 106/l 0,7 1,3 3,4 16,0

CD34 x106/l 0,6 1,8 6,5 22,5

CD34 x 106/kg 1,24 4,87

Patient 2

leukocytes x 106/l 4,9 5,9 10,1 26,6

CD34 x106/l 6,4 10,0 11,1 34,6

CD34 x 106/kg 1,25 2,62

Patient 3

leukocytes x 106/l 1,9 3,0 8,5 28,6

CD34 x106/l 0,6 0,9 6,8 62,9

CD34 x106/kg 9,57

Patient 4

leukocytes x 106/l 0 0,1 1,0 4,3 7,1

CD34 x106/l 0 0 0,5 1,9

Patient 5

leukocytes x 106/l 0,3 0,9 4,3 6,1 7,6

CD34 x106/l 0 0 1,3 0,4 0,6

141EVALuAtIOn OF InCIdEnCE OF MICROBIOLOGICAL COntAMInAtIOn In thE CORd BLOOd unItS COLLECtEd By PRIVAtE uMBILICAL CORd BLOOd BAnK In BRAZIL.J. J. Machado, K. P. Urago, V. B. Melo, T. Lima, M. H. Nicola, E. Cruz; Cryopraxis Cryobiologia Ltda, Rio de janeiro, BRAZIL.

Cryopraxis® Criobiology was established in 2001 as the first private umbilical cord blood bank (uCbb) in brazil, with the objective of collection, shipping, processing and cryopreservation of autologous cord blood cells. We have already collected more then 14000 cord blood units (Cbu) during the last 6 years. and we were recently accredited by american association of blood bank (aabb).the aim of this study was to analyze the incidence of microbiological contamination and prevalence of microorganisms present in the Cbu collected. according to national regulatory agency (anVisa) and aabb, a sterility test shall be done on all Cbu bags after processing, and before cryopreservation. Despite of a stricter disinfection protocols, Cbu carry a greater risk of being contaminated by low level of bacteria. Cbu are routinely screened for presence of microorganism as aerobic, anaerobic bacteria and fungus by bact/alert® system. among 14914 Cbu collected during this period, positive cultures were identified in 343 units (median 1.8%). most of isolated bacteria in this serie were nonpathogenic, mostly from normal skin flora (62.5%), predominantly Coagulase-negative staphilococci (Cns). nine Cbu (0.06 %) were contaminated with two different organisms. escherichia coli, enterococcus, Coagulase-negative staphilococci, Corynebacterium spp., Klebsiella pneumonia, bacteroides sp., bacteroides fragilis, and bacteroides vulgates were the most frequently microorganisms founded. no fungus contamination was founded. regular quality monitoring of collection and processing indicates that the major source of contamination is at collection. a higher contamination rate of the Cb units was noted after vaginal delivery (61%) compared to caesarian section (39%).extensive training in Cb collection, a closed collection system, good procedures and protocols, monitoring contamination rates, have promoted a lower contamination rate, thereby maximizing efficiency of procedures realized in our bank.

142COntRAStInG EFFECtS OF thE COMPLEMEnt COMPOnEntS C1Q And C5A On hSPC tRAFFICKInGL. A. Marquez-Curtis1, N. Shirvaikar1,2, A. Jalili1,2, Y. Qiu1,2, A. R. Turner2, M. Z. Ratajczak3, A. Janowska-Wieczorek1,2; 1Canadian Blood Services, Edmonton, AB, CANADA, 2University of Alberta, Edmonton, AB, CANADA, 3University of Louisville, Louisville, KY.

the complement system and innate immunity have emerged as important yet underappreciated modulators of hematopoietic stem/progenitor cell (HsPC) retention within the bone marrow (bm) and their mobilization. in this study we further investigated the roles of complement C1q, the initiator of the classical pathway of complement activation, and C5a, a downstream product of the activation cascade, on the trafficking of HsPC. CD34+ HsPC isolated from cord blood (Cb), bm and mobilized peripheral blood (mPb) and ex vivo-expanded progenitors were evaluated for expression of the receptors C1qrp and C5ar using rt-PCr and flow cytometry. Chemotactic responses and chemo-invasiveness across reconstituted basement membrane towards bm-produced stromal cell-derived factor (sDF-1), as well as C1q and C5a were examined. the expression of matrix metalloproteinase (mmP)-9 and C5a levels in Pb were also evaluated. C1qrp was found on bm, Cb and mPb CD34+ cells and on more mature myeloid and megakaryocytic precursors. C1q itself was not a chemoattractant for HsPC, but enhanced i) the chemotactic response of CD34+ cells to a low sDF-1 gradient, ii) their chemoinvasion across matrigel, and iii) secretion of mmP-9. on the other hand, C5ar was not detected on CD34+ cells, but appeared on more mature myeloid precursors, monocytes and granulocytes. C5ar was more highly expressed in mnC and Pmn from mPb compared to non-mobilized Pb, and plasma levels of C5a were significantly higher in patients who were good mobilizers. moreover, C5a stimulation of granulocytes decreased chemotaxis towards an sDF gradient and increased secretion of mmP-9. in conclusion, C1q was able to prime responses of HsPC to an sDF-1 gradient, and could have an enhancing effect on HsPC retention and homing. in contrast, C5a induced a highly proteolytic microenvironment in the bm reducing HsPC retention, and also chemoattracted granulocytes, thus promoting their egress into Pb and the subsequent mobilization of HsPC.


may 23-26, 2010


Poster Abstracts

143COMPARAtIVE In VItRO AnALySIS OF dIFFEREnt CELL POPuLAtIOnS FROM huMAn uMBILICAL CORd BLOOd: In SEARCh OF thE BESt OPtIOn FOR CELL EXPAnSIOn.P. FLORES-GUZMAN, V. FERNANDEZ-SANCHEZ, G. ALARCON-SANTOS, H. MAYANI; National Medical Center, IMSS, Mexico City, MEXICO.

background: During the last several years, significant efforts have been made in order to develop the most convenient culture conditions for the clinically-oriented ex vivo expansion of hematopoietic stem and progenitor cells (HsC/HPC) from umbilical cord blood (uCb). one issue of controversy, however, refers to the cell population that should be used to establish such cultures. in order to contribute to the development of a reliable culture system for uCb cell expansion, in the present study we have compared the expansion potentials of five different cell populations. materials & methods: Five cell populations were obtained from human uCb: mononuclear cells (mnC); a cell fraction enriched for CD34+CD38+lin- cells; a cell fraction enriched for CD34+CD38-lin- cells; purified CD34+CD38+lin- cells; and purified CD34+CD38-lin- cells. all populations were seeded in serum-free medium, with a combination of 8 recombinant stimulatory cytokines. medium change was performed every 7 days, and the numbers of total cells (tC), HPC and long-term Culture-initiating Cells (ltC-iC) were determined. results: in relative terms (fold-increase), all four enriched and purified cell populations showed statistically similar proliferation and expansion capacities; only mnC showed significanlty reduced potentials. in terms of absolute numbers, the cell fraction enriched for CD34+CD38+lin- cells was the one that showed the greatest levels of both tC and HPC, without loss of ltC-iC. interestingly, only the fraction enriched for CD34+CD38-lin- cells showed a significant increase in ltC-iC levels. Conclusions: under our culture conditions, the cell fraction enriched for CD34+CD38+lin- cells seems to be the most adequate cell population to establish clinically-oriented cultures for ex vivo expansion of uCb cells, since we were able to obtain, in a short-term period (14 days), the greatest numbers of tC and HPC, without loss of ltC-iC.

145VALIdAtIOn OF MILtEnyI CRyOMACS FREEZInG BAGS FOR hEMAtOPOIEtIC CELL PROduCtSK. McGee, L. Stiles, K. Annandale, N. Ponweera, S. Olchesky, K. Tran, E. Shpall, J. D. McMannis; The University of Texas M. D. Anderson Cancer Center, Houston, TX.

our laboratory has been using freezing bags from one manufacturer for Hematopoietic Cell Products (HPC) for the past 15 years. Due to availability issues, we decided to validate another manufacturer (miltenyi CryomaCs). the validation study was divided into two parts. First, we evaluated three different size bags (50ml, 250ml and 1l) over a range of volumes for each bag. HPC,apheresis collected two separate days were used for this set of experiments. Products were diluted to 3.2X1010nC/ml and divided into six cryobags (3 sizes/2 volumes). the bags were frozen in a control rate freezer (CrF) and stored in vapor phase liquid nitrogen (ln2) tanks. the second validation was intended to increase the “n” value for the 250 ml bag. HPC,Cord units were pooled and then aliquoted into two Cryocyte bags and six CryomaCs bags. the bags were frozen in a CrF and half were stored in ln2 and half in vapor phase. the total nucleated cell (tnC) count, CD34+ cells, colony forming unit (CFu) and viability post-thawed were compared to pre-cryopreservation values. since the total number of runs was small, we normalized the data to avoid run to run variation. Cryocyte bags are considered our laboratory standard, so the recovery for these bags was given a value of one. the difference in recovery between CryomaCs and Cryocyte (maCs/Cyte *100) was calculated and presented below.

% tnC recovery

% CD34+ recovery Viability CFu assay

HPC-a 100 102 101 91

HPC-C 102 102 100 82 overall, there was no difference in tnC, CD34, and CFu recovery with regards to size bag, volume or storage condition. We believe the bags to be comparable.

144POSt-thAW StABILIty OF dILutEd/RECOnStItutEd uMBILICAL CORd BLOOd PROduCtSD. M. McCarter1, B. Wang1, C. Connor1, R. Bolanos1, A. Lin2, L. Hsiao2, E. Lan2, Z. Wu2, F. Chen2, A. Chu2, R. Chow1, L. Petz1; 1StemCyte International Cord Blood Center, Covina, CA, 2StemCyte Taiwan National Cord Blood Center, Linkou, TAIWAN.

baCKgrounD: the objective was to determine the stability of post-thaw diluted/reconstituted umbilical cord blood products(Cb) over 6 hours, and to determine optimally how rapidly Cb products should be infused after thawing & dilution. Design/metHoDs: in a pilot study, with 2 Cb diluted 2.5:1 v:v with 10% Dextran-40 and 25% 12.5g Human serum albumin within 10 minutes of thawing, 10 replicate measurements were made measuring nucleated cell count(tnC), viability, CD34+ cell count and colony forming unit content(CFu) in order to derive the respective coefficients of variation for this laboratory. based on these results, it was determined that triplicate measurements of 5 Cb would be sufficient to detect a 10% difference in tnC, CD34+ count, viability and CFu in our setting [sample size determination based on 95% confidence(β=0.05) and 90% power(β=0.1)]. Four time points were chosen; immediately post-thaw(t=0), and 1-hour, 3-hours, and 6-hours thereafter at 4°C and at room temperature(rt).results: For both conditions, CFu and Viability decreased significantly by t=3 hours (p≤0.03). since viability was unaccounted for, tnC and CD34 results were not significantly decreased until t=6 hours rt for CD34.ConClusion: Despite a rapid 2.5:1 dilution/reconstitution, significant reductions in viability and CFu occurred after the first time point (t=1 hour). With no significant reductions on all four parameters at one hour, diluted/reconstituted (and by extrapolation, diluted/washed) Cb can be safely infused within one hour of thawing. if tC is contemplating a thaw-to-infusion time exceeding one hour, significantly higher dilutions (e.g.7:1 v:v) should be tested and validated prior to implantation.

146EVALuAtIOn OF thE COBE 2991 And thE SEPAX InStRuMEnt FOR VOLuME REduCtIOn OF hEMAtOPOIEtIC PROGEnItOR CELLS (hPC,APhERESIS)M. Belton, A. Harris, B. Toth, B. Spencer, K. Annandale, S. Olchesky, E. Shpall, J. McMannis; The University of Texas M. D. Anderson Cancer Center, Houston, TX.

HPC,apheresis often exceed ideal volumes for cryopreservation resulting in, especially after multiple collections, a large number of bags for infusion. Volume reduction of the HPC,apheresis product after leukapheresis reduces storage space needed and the likelihood of further processing on day of infusion. our laboratory has been using a manual method (sorvall centrifuge) for HPC,apheresis volume reductions (Ponce-lomboy, et al, aabb 2008). this method requires continuous user interaction, additional steps to reconstitute product to final volume, and is limited to starting volumes less than 600ml.We evaluated two systems for automated volume reduction, Cobe 2991 (Caridian, lakewood, Co) and sepax (biosafe america, Houston, tX). total nucleated cell (tnC), and CD34+ cells recoveries were compared. a total of 58 HPC,apheresis products were volume reduced on the Cobe 2991. Cells were centrifuged at 1000rpm for 5 minutes and the supernatant removed. leukapheresed products (n=54) were volume reduced on the sepax per manufacturers’ recommended conditions. the sepax separates the erythrocyte and leukocyte fraction into a bag connected to the processing set and uses supernatant waste to bring the reduced product to the user specified final volume accurately within ±2 ml. Procedural data is simultaneously saved to the sepax memory card. results from these runs were compared to 857 runs performed with the manual procedure. results were comparable for the three methods. the manual method was the least expensive to perform. However, if training time, automation with documentation, and closed system technology are considered, the sepax is the system of choice.

Pre Post

Volume tnC CD34 Volume tnC tnC recovery CD34 CD34

recoverymanual Centrifuge (n=857)

233 44447 276.10 124 44754 99.6 ± 7 269.36 98.30 ± 18

Cobe 2991 (n=58) 249 50228 438.77 136 50984 101 ± 7 458.05 101 ± 17

biosafe sepax (n-54) 227 42963 318.02 126 42171 97 ± 4 289.51 91 ± 9


Poster Abstracts

147PLERIXAFOR MOBILIZAtIOn LEAdS tO A LOWER RAtIO OF Cd34+ CELLS tO tOtAL nuCLEAtEd CELLS WhICh RESuLtS In GREAtER StORAGE COStSY. Tanhehco, J. Adamski, M. Sell, K. Cunningham, D. Magee, E. A. Stadtmauer, U. O’Doherty; University of Pennsylvania, Philadelphia, PA.

background: Plerixafor (mozobil, amD3100) with granulocyte-colony stimulating factor (g-CsF) mobilizes more CD34+ cells/kg compared to g-CsF alone. given that plerixafor enhances mobilization of multiple WbC lineages, we determined if more storage space is required for products collected from patients mobilized with plerixafor.methods: a review of the medical records of 31 patients mobilized with plerixafor and g-CsF and 15 patients mobilized with chemotherapy and g-CsF was performed. Data on demographics, baseline characteristics, CD34+ cells/kg, total nucleated cells, total mononuclear cells, total apheresis sessions and total bags for storage were collected. mean values were determined and compared using students t-test.results: We found that more nucleated cells and mononuclear cells were mobilized with plerixafor plus g-CsF compared to chemotherapy plus g-CsF (P=0.0029 and P=0.0005). However, total CD34+ and CD34+ cells/kg was similar between the plerixafor group and the control group. the proportion of CD34+ cells among total nucleated cells was less in the plerixafor group compared to the control group (P=0.001). more storage bags were required for the plerixafor group compared to the control group (mean, 18 vs 9, P=0.0017). We also analyzed our plerixafor group after dividing them into 2 categories, those who failed prior mobilization and those who had never been previously mobilized. both groups had statistically fewer CD34+/total nucleated cells (P=0.0037), but the effect was greatest in those who failed prior mobilization.Conclusion: Plerixafor plus g-CsF mobilized a similar number of CD34+ cells compared to chemotherapy and g-CsF, but the proportion of CD34+ cells among the total nucleated cells was lower. more mononuclear cells were mobilized resulting in an increased number of storage bags required. an increase in the number of bags required for stem cell storage may be logistically problematic and will also lead to increased costs for storage of stem cells.

149IMPACt OF AntICOAGuLAnt And tIME FROM COLLECtIOn tO PROCESSInG On CORd BLOOd CELL VIABILIty And COunt.M. Praught1, T. Turner2, H. Brown1; 1Scientific and Medical Affaris, Cbr Systems Inc., San Bruno, CA, 2Laboratory and Processing Facility, Cbr Systems Inc., Tucson, AZ.

background: umbilical cord blood (uCb) cell dose is an important determinant of treatment outcomes. the collection anticoagulant and time lapsed from collection to processing (transit time) may affect the uCb cell count and viability.objective: to evaluate the impact of transit time and anticoagulant on uCb collection cell count and viability prior to processing and storage.methods: uCb was collected from consenting mothers and transported to a processing facility. upon receipt, collection volume, transit time, cell viability, total nucleated cell (tnC) and mononuclear cell (mnC) counts (x106 cells) were accessed preceding processing. this analysis compared a group of uCb collections (n=92) with 35cc of liquid citrate phosphate dextrose (CPD) to a subset of collections with (dry) lyophilized heparin (lH) matched by blood volume and transit time (n=92) to the CPD collections. Collections were grouped according to transit time into those arriving <24, 24-36 or >36hrs.results: the matched lH collections showed equivalent mean blood volume (p=ns) and transit time to the CPD collections (p=ns), confirming the collections were well matched. transit time did not impact lH collections with no significant differences found in cell viability, mnC or tnC between lH collections arriving <24, 24-26 and >36hrs to the facility. Whereas CPD collections arriving >36hrs showed significantly decreased cell viability and mnC than CPD collections arriving 24-36hrs (-12.5±3.6%, p<0.001; -105±35, p<0.004) and <24hrs (-12.6±4.2%, p<0.004; -160±41, p<0.0002). additionally CPD collections arriving >36hrs had significantly decreased tnC compared to <24hrs CPD collections (-305±111, p<0.008).Conclusion: the anticoagulant used to preserve uCb from collection to processing can significantly impact the quality of the uCb. our findings show that uCb collections with lH are well preserved maintaining cell counts and viability regardless of transit time, whereas collections with CPD show a decrease in cell viability and count 36 hours after collection.

148Cd34+ CELLS FREQuEnCy And VIABILIty EVALuAtIOn OF hPC-C PRE-CRyOPRESERVAtIOn K. L. Prata, G. C. De Santis, M. D. Orellana, A. C. Silva, N. C. Gonçalves, P. V. Palma, C. C. Menezes, S. M. Quintana, D. T. Covas; National Institute of Science and Technology in Stem Cell and Cell Therapy, Center for Cell Therapy and Regional Blood Center, Faculty of Medicine, University of São Paulo, Ribeirão Preto, São Paulo, BRAZIL.

FDa has recently proposed that additional quality measures should be considered for the placental/umbilical cord blood (HPC-C) in hematopoietic reconstitution. Possibly, the most important one seems to be the CD34+ viability evaluation by flow cytometry, pre-cryopreservation. However, FDa does not recommend the erythroblasts quantification, as FaCt does. in brazil, our agency recommends the viability evaluation only for tCn and most of the umbilical cord blood banks (uCbb) use the tripan blue dye to perform this assay. in our public uCbb we evaluate the tCn and CD34+ viability by flow cytometry with 7aaD. Here we describe consecutive data obtained during our 1st 18 months. the results are shown as median (range).results: there were cryopreserved 235 units. tCn (x107)/unit: 90.8 (45.2-269.8); CD34+ (x106)/unit: 2.28 (0.36-13.13); %CD34+ (of the tCn): 0.26 (0.07-1.19); CD34+ viability (%): 91.1 (57.5-100); tCn viability (%): 97.5 (68.4-100); time until the volume reduction (hours): 19 (2-46). 113 (48%) units presented % CD34+ <0.25% tCn; 56 (24%) units presented CD34+ viability <85% and 17 (7%) units presented tCn viability <85%. there was no correlation between the CD34+ and tCn viability (r=0.3504; spearman rank Correlation) or between CD34+ viability and time between the collection and the volume reduction (r=-0.1948).Discussion: our data corroborate with FDa and FaCt recommendations. the low frequency of CD34+ in relation to tCn is probably secondary to the erythroblasts cells counted as tCn. We believe that tCn and CD34+ viability (by more sensible tests) and erythroblasts count need to be evaluated pre-cryopreservation, informed to the transplantation center and considered during the selection of the HPC-C unit in association with the tCn count.


may 23-26, 2010


Poster Abstracts

150ChARACtERIStICS OF thAWEd AutOLOGOuS uMBILICAL CORd BLOOd E. H. Rosenau1, M. W. Sugrue1, M. Haller1, D. Fisk2, S. S. Kelly3, M. Chang1, W. Hou1, L. Eldjerou1, W. Slayton1, J. R. Wingard1; 1University of Florida, Gainesville, FL, 2Shands Hospital at the University of Florida, Gainesville, FL, 3MD Anderson Cancer Center, Houston, TX.

baCKgrounD autologous umbilical cord blood (autouCb) has historically been cryopreserved for potential use in the hematopoietic transplant arena. a novel therapy using autologous cord blood infusions for the treatment of pediatric type-1 diabetes (Phase 1) afforded our institution an opportunity to analyze characteristics of autouCbs obtained from multiple banks.metHoDstwenty patients were infused with autouCbs collected within a 9 year period (1996 – 2005) from 9 aabb-accredited cord banks and assessed for collection, processing, and cryopreservation parameters. nine patients opted to retain a portion of their autouCb for future use. all thaw/wash procedures employed a modified Coblt method using standardized conditions. units were assessed post-wash for viable total nucleated Cell (tnC) and CD34+ recoveries, HCt (%), mnC (%) and CFu-gms. the post-wash viable tnC recoveries were compared against pre-freeze processing variables including length of storage, processing method, and cryopreservation volume and container.resultsanalysis showed a wide range in autouCb collection volumes (19.9 ml -170 ml) and cryopreserved tnC (7.6 x 107 - 3.34 x 109), with %tnC processing recoveries ranging from 39-100%. Post-wash viable tnC recoveries ranged from 58-100%. length of storage did not affect post-wash viable tnC recoveries. However, autouCb processing methods appeared to affect tnC recoveries, with “in-totos” having the highest recoveries, followed by rbC depletion and density gradient separation, respectively. Viable tnC recoveries were lower in products stored in vials when compared to bags.ConClusionsmarked heterogeneity of autouCb characteristics exist. the type of processing method, volume, and type of cryopreservation container may influence tnC recovery.

autouCb Characteristics n mean median range

Collection Volume ml (with anticoag.)

20 81.34 66.35 19.9 - 170

total tnC Collected (x108) 15 10.12 5.90 2.9 -

42.7

total tnC Cryopreserved (x108)

18 6.99 5.04 0.8-33.4

total %tnC Processing recovery

13 72.3 78 39-100

Post-Wash %Viable tnC recoveries

total tnC 18 81.1 84 58-100

Processing method: in toto 2 94 94 88-100

rbC Depletion 6 79.5 83.5 67-87

Density gradient separation 8 77.4 81 58-99

Cryopreservation Volume: <25 ml 12 77.1 80 58-99

>25 ml 6 89.2 90 75-100

Cryopreservation Container: Vial 11 76.6 77 58-99

bag 6 86.3 85.5 75-100

151EnRIChMEnt OF PRIMItIVE hEMAtOPOIEtIC StEM CELLS WIth POLyPLOIdIZAtIOn OF PROLIFERAtInG CELLS By SMALL MOLECuLE InhIBItIOn OF MyOSInJ. Shin1, D. E. Discher2; 1University of Pennsylvania School of Medicine, Philadelphia, PA, 2University of Pennsylvania, Philadelphia, PA.

investigation of mechanisms behind hematopoietic stem and progenitor cell (HsPC) division is of clinical importance, including identification of optimal ex vivo culture conditions for maintenance of functional HsCs and differentiation into specific lineages. Contributions of cytoskeletal machinery to HsPC division and population dynamics remain largely uninvestigated. one important cytoskeletal component of cell division is myosin ii which mediates furrow ingression during cytokinesis. in the present study, contributions of myosin to HsPC division were evaluated under ex vivo culture conditions that were previously used to expand human CD34+ cell number in clinical studies. this is followed by pharmacological modulation of myosin activity, combined with subpopulation analysis using flow cytometry and defined surface markers for HsPCs. Cultures in the exponential phase of growth were treated with different doses of inhibitor for 2~3 days. Dose-dependent suppression of total cell growth is due to selective reduction in total viable g0/g1 cell number of both committed and multipotent progenitor cells, while primitive HsCs remained unchanged in number. this leads to relative enrichment of phenotypic primitive HsCs and also coincides with generation of viable polyploid cells with up to 16n Dna content. a narrow window of drug dose is shown to maximize the numbers of viable polyploid cells, progenitors, and primitive HsCs, and the polyploid cells will adhere to substrates, extend processes and fragment, reminiscent of platelet generation by megakaryocytes - which are known to be modulated by myosin. Functionality of primitive HsCs and polyploid cells generated by ex vivo myosin inhibition is under investigation using both in vivo xenotransplantation mouse models and in vitro differentiation assays.

152dEVELOPMEnt OF A SEGMEnt-BASEd ALdEhydE dEhydROGEnASE ASSAy tO dEtERMInE uMBILICAL CORd BLOOd unIt (CBu) POtEnCyK. Shoulars1, J. Sun1, K. Page1, R. Dornsife1, T. Gentry2, A. Balber2, J. Kurtzberg1; 1Duke Univeristy, Durham, NC, 2Aldagen Inc., Durham, NC.

after uCb transplantation (uCbt), up to 20% of patients experience primary graft failure, highlighting a need for rapid assays to assess Cbu potency. Preliminary clinical studies demonstrated that post-thaw colony forming units (CFu) predict engraftment and overall survival, but time (14 days) and variability limit their usefulness. CFus in fresh cord blood are highly correlated with the number of hematopoietic stem cells expressing aldehyde dehydrogenase (alDHbr). retrospective studies of transplanted Cbus indicated that engraftment success could be accurately predicted by the number of alDHbr cells infused per kilogram. therefore, we developed a modified flow cytometric assay to enumerate alDHbr cells in attached frozen Cbu segments to measure potency for uCbt.blood was withdrawn from the segment with a needle and 3 drops of blood were spotted onto a Fta card for Hla confirmatory typing. the remainder of the blood was washed with 5% dextran/albumin to remove the Dmso. Following the removal of 100,000 white blood cells for CFus, the segments were assayed for alDHbr [aldecount ], viability (7-aaD), expression of CD34, CD45 and glycophorin a [flow cytometry] and total nucleated cell (tnC) count [sysmex], and total number of each cell population in the segment was calculated. this method reproducibly assays alDHbr cells ( 85% CD34/45 positive) from a single segment in less than a day while allowing testing of routine parameters of the Cbu (Hla matching, tnC, viable CD34) currently used to select units for transplantation. Furthermore, the assay produces reliable results when performed by people from different laboratories (Duke vs. mD anderson) and on different cytometers (accuri C6 vs. bD bioscience’s Facscalibur). therefore, this method will be suitable for further prospective studies on the predictive value of the alDHbr assay as an indicator of a Cbu’s ability to confer neutrophil and platelet engraftment and overall survival after uCbt.


Poster Abstracts

153EXPERIEnCE WIth dIVERSE CELL COnCEntRAtIOn, REtEntIOn, And WASh APPLICAtIOnS FOR uSE In StEM CELL PROCESSInG And CELL thERAPy PROCESSInGR. Speziale; Invetech, Guilford, CT.

Current cell separation and wash technologies have limited applications in cell therapy processing and manufacturing as they are optimized for either simple centrifugation, blood processing, or bone marrow aspirates. For example, most cell harvest processes are efficient in removing cells but not in maximizing viable cell density, viability, and recovery; parameters that are most important for stem cell processing or cell therapy manufacturing. Frequently, cells are retained by either centrifugation or filtration based devices. these approaches are often problematic. Conventional centrifugation based devices cause stress and nutrient deprivation to the cells in the pellet which results in low viability, while filtration based devices often suffer from issues such as clogging, retention of cell debris, and shear stress on cells.invetech in working with various partners over the past 10 years has been tasked with helping to develop commercial scale stem cell and general cell separation and wash apparatus. the problem has been that existing technology either stress the cells, killed cells, resulted in poor recovery from wash or could not be integrated into a functionally closed processing system. invetech has successfully adapted several existing technologies and recently helped Kbi bioPharma, inc. design and develop a centrifugal fluid flow counter-force device for disposable cell recovery. the results of our efforts and examples of these apparatus will be exemplified, including the kseptm by Kbi biopharma, inc

155A nEW CLOSEd COntAInER SyStEM FOR CLInICAL CRyOPRESERVAtIOn OF BIOMAtERIALSS. Thirumala1, R. Helman1, L. Larson1, M. Byers1, W. S. Goebel2, E. J. Woods1; 1General Biotechnology LLC, Indianapolis, IN, 2Indiana University School of Medicine, Indianapolis, IN.

the use of closed systems for processing/storage of cell therapy products is necessary for good manufacturing practices (gmP). We recently evaluated a new closed system sterile device (Cellseal*tm cryogenic vials, Vialco, llC, in, usa) for secure cryopreservation and storage of cell therapy products at cryogenic temperatures (Figure 1). Vials were tested for durability and integrity utilizing a 1-meter drop test. in addition, frozen, sealed vials were transported and tested using pharmaceutical packaging tests including dye ingress and microbial challenge. the results of all tests indicated container closure integrity of the vials with no failures.

Clonogenic capacities of post thawed uCb cryopreserved in Cellseal vial & Pall medical freezing bagFurther, human umbilical cord blood hematopoietic progenitor cells from 3 donors were used to establish the performance of the vials. For each donor, a 30ml concentrated uCb was prepared after adding 10% Dmso as cryoprotectant. next, 10ml of this was cryopreserved using two 5ml vials with the remaining 20ml cryopreserved in standard Pall® medical cord blood freezing bags as a control. For both Cellseal vials and bags, an integral test segment was retained. all samples were analyzed for total nucleated cell count, colony-forming unit assay and viability using 7-aaD (table 1). the post-thaw results suggest no significant variation in performance of Cellseal vials compared to bags.

154QuALIty OF AutOLOGOuS uMBILICAL CORd BLOOd unItS InFuSEd IntRAVEnOuSLy In ChILdREn WIth ACQuIREd nEuROLOGICAL COndItIOnSJ. Sun1, J. Allison1, C. McLaughlin1, L. Sledge2, B. Waters-Pick1, S. Wease3, J. Kurtzberg1,2; 1Pediatric Blood & Marrow Transplant Program, Duke University, Durham, NC, 2The Carolinas Cord Blood Bank, Duke University, Durham, NC, 3The EMMES Corporation, Rockville, MD.

background: numerous animal models demonstrate neurological and survival benefits of bone marrow or umbilical cord blood (Cb) infusion in the setting of brain injury. based on these data, we conducted a pilot study to determine the safety and feasibility of intravenous (iV) administration of autologous Cb in young children with acquired neurological disorders. most units were electively stored in private Cb banks at the request and expense of the children’s parents. unlike public banks, which utilize specific criteria for banking, private banks generally store all collected units regardless of size or cell content.study Design and methods: Patients were self-referred or referred by their treating physicians. Cb units (Cbu) containing >1x10e7 cells/kg were shipped to Duke from the banks of origin after confirming identity by Hla typing. on the day of infusion, Cbus were thawed and washed in dextran/albumin and infused intravenously. Patients were premedicated with tylenol, benadryl and solumedrol. Data regarding patients, infusions, and Cbus were collected retrospectively. Characteristics of Cbus were compared to existing data from Cbus publicly banked at the Carolinas Cord blood bank.results: From 3/2004 to 12/2009, 184 children received 198 Cb infusions. three patients had infusion reactions, all responsive to medical therapy +/- stopping the infusion. median pre-cryopreservation volume (60 vs 89ml, p<0.0001), tnC (4.7 vs 10.8x10e8, p<0.0001), and CD34 (1.8 vs 3.0x10e6, p<0.0001) were significantly lower than publicly stored Cbus. Post thaw sterility cultures were positive in 7.6% of infused Cbus, but no clinical infections were seen.Conclusion: iV infusion of autologous Cb is safe and feasible in young children with neurological injuries. Quality parameters of privately banked Cbus are inferior to those stored in public banks. if efficacy of autologous Cb is established clinically, the quality of autologous units should be held to the same standards as those stored in public banks.

156StRAtEGIES tO MInIMISE thE RISK OF ERROR In tRAnSPLAnt COndItIOnInG PROtOCOLSA. E. Trickett1,2, F. Wright3, T. A. O’Brien3,2; 1BMT Network NSW, Darlinghurst, NSW, AUSTRALIA, 2University of New South Wales, Sydney, NSW, AUSTRALIA, 3Sydney Children’s Hospital, Randwick, NSW, AUSTRALIA.

Currently many transplant centres prescribe conditioning regimens for haematopoietic progenitor cell transplants by manually typing details into a basic Word template. this approach is both laborious and prone to error, since individual drug doses and modifications (e.g. for small patients or drug interactions) need to be thoroughly checked. Hence a strategy for semi-automated preparation of conditioning regimens was sought.a template was created in excel that contains dropdown lists and locked formulas to ensure consistency. the formulas were created to calculate individual drug doses and incorporate dose rounding / adjustment according to the specific drug, patient’s height, weight, body surface area and/or age. transplant type, cell dose and virology automatically determine the majority of supportive care. each draft protocol was independently validated to ensure that all formulas functioned correctly in different scenarios. Protocols were then authorised, document controlled and given a unique document identifier and version number. each template was password protected and locked to prevent unauthorised changes.For each patient, the appropriate conditioning regimen template is selected, patient parameters entered and graft type selected to automatically populate daily drug doses, standard supportive care and gvHD prophylaxis. other supportive care (e.g. antifungal prophylaxis) can be selected from drop down boxes. once complete, the excel protocol is converted to pdf format, checked, digitally dated & signed by the transplant Director and Pharmacist then distributed.Previously, the preparation of a conditioning regimen was a lengthy process with manual drug calculations and entry into a Word document. the new templates automatically perform dose modifications and ascribe gvHD prophylaxis according to in-built algorithms. this approach has significantly reduced the time taken to create a patient protocol, reduced the risk of errors in dosing calculations and standardised the approach to viral and gvHD prophylaxis.


may 23-26, 2010


Poster Abstracts

157thE GROWth OF CORd BLOOd uSEF. Verter1, J. Nietfeld2; 1Parent’s Guide to Cord Blood Foundation, Brookeville, MD, 2University Medical Center Utrecht, Utrecht, NETHERLANDS.

in Fig. 1 we plotted autologous use of cord blood (Cb) units versus time, cumulatively, based on the only international database of autologous medical treatments with Cb (ParentsguideCordblood.org). by the end of 2009, over 200 autologous treatments were performed, of which 73% took place in the last two years. the overwhelming majority of the treatments (89%) could be categorized as regenerative medicine: for diagnoses including cerebral palsy (60%), other forms of brain disorder or injury (16%), type 1 diabetes (11%), or other (2%). the rising rate of autologous therapy exceeds exponential growth.in Fig. 1 we also plotted nmDP data for allogeneic use of Cb units versus time, cumulatively. more than half of nmDP facilitated transplants cross international borders. the growth of nmDP Cb transplants has been about linear since 2006. Worldwide, the cumulative number of allogeneic Cb transplants is 20,000 by the end of 2009 (rocha et al. 2009, bJH 147:262-274).assuming that the rate of the worldwide growth of allogeneic transplants is not different from that of nmDP facilitated allogeneic transplants, a comparison of both plots shows that the growth rate of autologous transplantation is much faster than that of allogeneic transplantation.We conclude that medical societies which issue opinions on Cb storage that ignore the fast growing autologous use of Cb for regenerative medicine are not making evidence-based recommendations.

159thE SWISS MuLtICEntER IntRACOROnARy StEM CELLS Study In ACutE MyOCARdIAL InFARCtIOn (SWISS-AMI): A CELL-BASEd CLInICAL PROtOCOL FOR PAtIEntS WIth ACutE MyOCARdIAL InFARCtIOn. Study dESIGn And PRELIMInARy dAtAG. Astori1, R. Corti2, P. Erne3, P. Jamshidi3, U. Landmesser2, V. Lo Cicero1, T. Lüscher2, R. Manka4, A. Moschovitis5, S. Plein6, S. Soncin1, S. Windecker5, G. Soldati1, D. Sürder1,2, T. Moccetti1; 1Cardiology and Cell Therapy Unit, Cardiocentro Ticino, lugano, SWITZERLAND, 2Department of Cardiology, University Hospital, Zurich, SWITZERLAND, 3Cardiology, Cantonal Hospital, Luzern, SWITZERLAND, 4Institute for Biomedical Engineering, University and ETH, Zurich, SWITZERLAND, 5Department of Cardiology, University Hospital, Berne, SWITZERLAND, 6Academic Unit of Cardiovascular Medicine, Leeds, UNITED KINGDOM.introDuCtion: recent studies report that intracoronary administration of autologous bone marrow mononucleated cells (bm-mnC) may improve remodeling of the left ventricle after acute myocardial infarction (ami). subgroup analysis suggest that early treatment is probably most effective; however, the optimal time point of cell administration has never been addressed.materials anD metHoDs: We report preliminary data of the trial sWiss-ami (nCt00355186). 180 ami patients treated by PCi are randomized to one control and two treatment groups. treatment groups received bm-mnC at 5-7 days or 3-4 weeks after ami. left ventricular function, scar size, transmural extension and regional wall motion are assessed by cardiac magnetic resonance (Cmr) at baseline and after 4 and 12 months.50 ml of bm are subjected to density gradient centrifugation. Cells are resuspended in 10 ml X-ViVo 10 medium ±20% v/v of autologous serum, and filled in syringe. FaCs analysis for CD34/CD45/CD133 is performed and cell viability assessed by 7-aad. sterility is assessed by using the bact/alert 3D system. bm-mnC cells are released if cell number is between 5x107 and 5x108 and cell viability ≥80%.Cell potency is assessed “in vitro” by quantifying the invasion capacity of the cells and “in vivo” by using a nude mice model of myocardial infarction. For the latter, bm-mnC will be injected in the infarct border zone 60 minutes after ligation of the left anterior descendent artery.results: so far 4 active centres have included 128/180 patients. a mean of 180±110x106 bm-mnC (mean±sD, n = 51) have been infused (viability 93.8 ± 4.5%). mean number of CD34+ cells was 2.5±2.0x106. invasion was 30.26±15.31% (n=28). in-vivo testing is ongoing. after the 50 patients a safety committee has validated the clinical outcome.ConClusions: We aim to determine the optimal time point of iC administration of autologous bm-mnC after ami on lV remodeling.

158tWO CASES OF ELEVAtEd LIVER FunCtIOn tEStS (LFtS) FOLLOWInG InFuSIOn OF CRyOPRESERVEd hEMAtOPOIEtIC PROGEnItOR CELL APhERESIS (hPC, APhERESIS) PROduCtSR. S. Weinberg1, B. Mehrotra2, B. Greco1, Y. Galperin1, C. Johnson2, M. Diaz2, K. Grima1; 1New York Blood Center, New York, NY, 2Long Island Jewish Medical Center, New Hyde Park, NY.

two adult male non-Hodgkin’s lymphoma patients each received autologous cryopreserved HPC, apheresis products. the cryopreservative consisted of 5% dimethylsulfoxide (Dmso) and 4% human serum albumin (Hsa) and lot numbers of each were different in each patients’ HPC, apheresis products. microbial cultures were negative. Patient 1’s alkaline phosphatase, ast and alt were 28, 22, and 30 pre-treatment and increased to 47, 504, and 549, respectively within several hours post-infusion. similarly, Patient 2’s values pre-treatment were 53, 21, and 29, and post-infusion were 71, 554, and 765, respectively. in both instances, lFts began to decrease within 24 hours and returned to pre-treatment values. engraftment (anC ≥500) occurred on days 10 and 9, respectively. elevated lFts were not reported in 22 additional patients who received HPC, apheresis products containing the same Dmso and Hsa lot numbers. other diagnoses: neuroblastoma (2), medulloblastoma (1), epyndemoma (2), glioma (1), Hodgkin’s lymphoma (2), non-Hodgkin’s lymphoma (3), amyloidosis (1), burkitt’s lymphoma (1), multiple myeloma (9). the only additional infusion adverse event reported was mild nausea in 7 patients. engraftment occurred in all patients. Patients’ HPC products are compared in the table below. We conclude that elevated lFts are observed in some patients shortly after infusion of HPC, apheresis products cryopreserved with Dmso and Hsa. However, elevated lFts could not be specifically attributed to volume of Dmso, or tnC/Kg, or CD34+ cells/Kg infused.

infused HPC, apheresis ProductsVolume infused

(ml)

tnC X 109/Kg

CD 34+ Cells X 106/Kg

Day anC ≥ 500

Day WbC ≥ 1000

Day Plts ≥ 20 K

Patient 1 236 8 3 10 10 10Patient 2 122 5 5 9 9 1022 patients mean ±1 sD 128±93 7±5 6±5 9±1 9±1 16±9

160MAXIMIZInG StABILIty OF huMAn StEM CELLS thROuGh OPtIMIZEd BIOPRESERVAtIOn SOLutIOnSD. Clarke, J. Doolittle, A. Mathew; BioLife Solutions, Inc., Bothell, WA.

background: stem cells offer great therapeutic potential to treat a diverse array of diseases. With the number of applications and variety of stem cell lineages continually expanding, optimized biopreservation methods are critical. Development of effective preservation methods permits extended stability for collection, transportation, processing, testing, and long-term cell banking.methods: Human mesenchymal stem cells (hmsC) and dental pulp stem cells (DPsC) were used to characterize optimal biopreservation conditions. Cryopreservation: cells were cryopreserved in traditional freezing media (culture media with/without serum and Dmso) or the commercially available serum- and protein-free Cryostor™. standard cryopreservation methods were used. Hypothermic storage: cells were plated and exposed from 1-7 days to refrigerated (2-8°C) temperatures in commercially available solutions including culture media (with serum), Celsior®, Viaspan®, normosol®-r, Plasma-lyte®, and Hypothermosol®-Frs. recovery was assessed 24-hours post-preservation.results: overall recovery following cryopreservation was similar for both stem cell populations. the inclusion of serum to the media improved recovery by 10-15% in comparison to media solutions without serum, but none were as effective as the serum-free Cryostor solutions (15-20% improvement compared to media without serum; 5-10% to media with serum). For hypothermic storage, no viable hmsC or DPsC were recovered following 1 day storage in culture media, Viaspan, or Celsior solutions. normosol-r and Plasma-lyte solutions offered a 40-60% recovery after 1 day storage, but no extended storage was possible. in contrast, an ~100% recovery of cells was observed following 1 day storage in Hypothermosol and extended storage was achievable to 7 days.Discussion: Current methods for preservation of stem cells are limited and improvements are required to ensure effective transport and long-term storage. unlike traditional home-brew or leading commercial products, Hypothermosol and Cryostor are formulated to balance the intracellular state at low temperature resulting in broad based improvement in preservation stability, while offering a robust quality and regulatory footprint.


Poster Abstracts

161EnhAnCEMEnt OF MyOGEnIC dIFFEREntIAtIOn OF huMAn AdIPOSE tISSuE dERIVEd StROMAL CELLS In VItRO S. Han1, J. Kim1, J. Kang1, J. Kim1, Y. Park2, C. Kim3, B. Do1; 1HurimBioCell Inc., Seoul, KOREA, REPUBLIC OF, 2Dept of Rehabilitation, Yongdong Severance Hospital, Seoul, KOREA, REPUBLIC OF, 3Dept of Life Science, Hanyang University, Seoul, KOREA, REPUBLIC OF.

adipose tissue-derived stromal cells (asCs), one type of mesenchymal stem cells, are easily to obtain and have multipotency and highly expansion potency. in various approaches to develop a cell-based therapy, asCs are an attractive candidate for inherited myogenic disorder like as muscular dystrophy (mD). this study was investigated to determine an effective induction method of myogenic differentiation of human asCs. the experimental groups were divided into skeletal muscle-extract treated group, dedifferentiation group, and co-culture group with myoblasts. asCs treated with muscle extracts from mouse skeletal muscle were not observed a significant efficiency of muscle differentiation. We induced asCs into dedifferentiation state to elevate the rate of myogenic differentiation. undifferentiated asCs was cultured with dedifferentiation medium for 3 days before myogenic induction. after dedifferentiation step, the enhanced myogenic differentiation was confirmed by mrna expression of myogenic regulatory factor including mrF4, mrF5, and sKa. While the expression of desmin was up-regulated than that of un-dedifferentiation groups, the expression of myoD was not detected. Co-culturing with human skeletal muscle precursor cells (myoblast) was designed to check myogenic influences of in vitro interaction between asCs and myoblasts. asCs were labeled with Pe-conjugated nanoparticle before co-cultured. myogenic differentiation in the co-culture group was determined by up-regulated myoD and the muscle fiber-like structures, composed of labeled asCs and myoblasts, were observed in 4 weeks after induction. these data suggested method of dedifferentiation or co-culture in myocyte differentiation was more effective than the result of induction medium alone. Further study for determine an effective method of myocyte differentiation would be provide a clue to therapy in mD patient.

163tRAnSPLAnt OF AutOLOGOuS BOnE MARROW StEM CELLS tO PARKInSOn’S dISEASE PAtIEntS VIA MuLtIPLE MInIMALLy InVASIVE ROutES IS SAFE And MAy IMPROVE thEIR QuALIty OF LIFE: PRELIMInARy RESuLtSL. Geffner1,2, J. Bernabe1, F. Ramirez1, C. Mariscal3, X. Montenegro4, M. Pinos5; 1University Hospital SHDUG, Guayaquil, ECUADOR, 2San Francisco Hospital, Guayaquil, ECUADOR, 3Cedic, Guayaquil, ECUADOR, 4Cerid, Guayaquil, ECUADOR, 5Roberto Gilbert Hospital, Guayaquil, ECUADOR.

to date there is no cure or definitive treatment for Parkinson`s disease (PD). studies in PD patients show that for a treatment to be effective it must primarily improve their quality of life, avoid adverse side effects and attenuate the progression of the disease. many studies support that stem cells may be a possible treatment for different disease and trauma states. autologous CD34+ stem cells have been used to treat leukemia, symptoms of cardiomyopathy, spinal cord injury, diabetes, and several neurodegenerative diseases including multiple sclerosis and amyotrophic lateral sclerosis. We report 8 non-surgical, minimally invasive transplants to PD patients who were administered autologous bone marrow stem cells (bmsCs) via multiple routes. Preliminary evaluations show improvements in uPDrs, Hoehn & yahr scale, schwab & england scale, reduction in current medication total dosage and prolonged span between doses. no motor complications or impairment were seen. to date, we have administered autologous bmsCs into 135 patients including this group of PD cases. no tumor formation, pain, infections or rejection reactions were seen up to 5 years follow up. this study suggests bmsCs may enhance dopamine synthesis probably triggering some paracrine effect which may explain the better response to standard drug therapy. bmsCs administration via multiple routes is feasible, safe, and may improve the quality of life for patients living with PD. this study meets the Position statement regarding the use of embryonic and adult Human stem Cells in biomedical research of the american academy of neurology and american neurological association.

162PROLIFERAtIOn And ChARACtERIZAtIOn OF huMAn uMBILICAL CORd dERIVEd StROMAL PROGEnItOR CELLS undER CuLtuRE COndItIOnH. Park1, J. Kim1, S. Han1, M. Kim1, S. Park1, C. Kim2, Y. Yoon2, B. Do1; 1HurimBioCell Inc., Seoul, KOREA, REPUBLIC OF, 2Dept of Life Science, Hanyang University, Seoul, KOREA, REPUBLIC OF.

many studies have been reported multipotency and safety of mesenchymal stem cells are accelerated in cell-based therapies. recent report has introduced effective multipotency of mesenchymal stem cells isolated from the human placenta-related materials; amniotic membrane, amniotic fluids, umbilical cord vein or artery, Wharton’s jelly and so on. this study was investigated the characterization for proliferation and gene expression of human umbilical cord derived stromal progenitor cells (uC-sPCs) under culture condition. the cells were incubated in three type medium (basal medium with Fbs, msC-qualified or endothelial cell (eC)-qualified medium). During the culture period, population growth of uC-sPCs increased in msC-qualified medium and eC-qualified medium compared to that of basal medium. the cells cultured in eC-qualified medium showed typical phenotype of uC-sPCs. Phenotypic analysis of the cells by flow-cytometry was resulted positive expression for CD73 and CD90 but negative expression for CD31, CD34, and CD45. the uC-sPCs in immunocytochemistry were expressed collageni, tra-1-60, Hla class i, thy-1, and VegFr2, but not expressed CD31 and Hla class ii. also we examined mrna expression profiling according to passage during the culture period. the profiling related gene was determined a well-known stem cell marker and the expression did not show a significant changes. these results showed uC-sPCs are able to incubate a stable expansion and phenotypic profiles, uC-sPCs will consequently become a good candidate for cell therapy.

164CELL thERAPy FOR AMI tREAtMEnt uSInG EX-VIVO EXPAndEd BM Cd34 POSItIVE CELLSM. Gunetti1, I. Ferrero1, D. Rustichelli1, F. Molla2, E. Errichiello1, L. Staszewsky2, M. Salio2, N. De Angelis2, M. Berger1, R. Latini2, F. Fagioli1; 1Stem Cell Transplantation and Cellular Therapy Unit; Pediatric Onco-Hematology Division, Regina Margherita Children’s Hospital, Turin, ITALY, 2Pharmacological Clinical Research Unit, Department of Cardiovascular Research, Mario Negri Institute, Milan, ITALY.

background. Cell therapy is a new approach to treat acute myocardial infarction, above all in those cases in which the best mechanical and pharmacological therapies do not lead to the functional repair of a damaged myocardium. However, the following still needs to be clarified: what the best cell lines to use for therapy are; when the best time after the acute event to inject is.aims. on these basis we validated pre-clinical studies, in vitro and in vivo, of an ex-vivo bone marrow (bm) CD34+ stem cell expansion protocol to use in ami patients.methods. the isolation and expansion protocol of CD34+ hematopoietic stem cells followed the requirements of the current cell therapy rules. CD34+ bone marrow cells were one week expanded using sCF+Fl+tPo+il6 growth factors. the expanded cells were carefully characterized in vitro and in vivo. We performed timing and dose in vivo studies:- single dose of purified bm CD34+ cells;- single dose of purified bm CD34+ cells;- single dose of 1 week ex-vivo expanded bm CD34+ cells;- double dose of cells: purified CD34+ bm cells + 1 week ex-vivo expanded CD34+ cells.results. the expansion procedure committed CD34+ cells to endothelial lineage. Quality controls on cell therapy products were satisfactory.intramiocardial injection experiments in scid beige mouse model, with permanent legation of left descendent coronary artery, demonstrated that expanded bm CD34+ cells are able to survive in the damaged organ, compared to unmanipulated bm CD34+ cells. echocardiography analysis demonstrated that there is a trend of heart functional recovery in mice injected with double dose of CD34+ cells.Conclusions. based on these pre-clinical results we can suppose the use of ex-vivo expanded bm CD34+ cells for clinical trials in ami patients.


may 23-26, 2010


Poster Abstracts

165SAFEty And EFFICACy OF huMAn FEtAL LIVER dERIVEd StEM CELL tRAnSPLAntAtIOn FOR thE MAnAGEMEnt OF dECOMPEnSAtEd LIVER CIRRhOSIS- 2 yEAR FOLLOW uP StudyC. M. Habibullah1, A. A. Khan1, N. Parveen1, M. V. Shaik1, A. Rajendraprasad1, M. A. Aleem1, A. M. Habeeb1, G. Srinivas2, T. R. Avinash2, K. Kumaresan3, J. Venkateswarlu1, G. Pande2; 1Deccan College of Medical Sciences, Hyderabad, INDIA, 2Centre for Cellular and Molecual Biology, Hyderabad, INDIA, 3KK Scan Cente, Hyderabad, INDIA.

background: extensive experimental data have shown that hepatocyte transplantation may be useful in bridging some patients to olt. a major limiting factor has been the shortage of mature functioning human hepatocytes. the liver stem/progenitor cell therapy provides great potential to manage the end stage liver diseases. this study is an extension of our data published of 25 cases followed for 6 months wherein we reported the safety and efficacy of the procedure . in order to assess the long term safety and efficacy, herein we are reporting the data extended of 15 cases followed up for 2 years. therefore, the present study was aimed to study the safety and efficacy of hepatic progenitor/stem cells in patients with end stage liver cirrhosis via the hepatic artery followed for 2 years.method: 15 patients with liver cirrhosis of different etiologies were infused epCam positive liver cells through hepatic artery. `results: no complications were observed in any of the patient up to 2 years and there was marked improvement in all the biochemical and physiological parameters. the two years follow up showed a decrease in mean melD score (p<0.01) observed in two years follow up in all patients.Conclusion: thus human fetal liver is the richest source of progenitor cells. the study proved the safety and efficacy of the cells in the liver disease patients followed for 2 years.

167IntRAOPERAtIVE StEM CELL tREAtMEnt In PAtIEntS SuFFERInG FROM CRItICAL LIMB ISChEMIAR. R. Kolvenbach1, C. Kreissig2; 1Catholic Clinics Duesseldorf, Duesseldorf, GERMANY, 2DRK-Blutspendedienst West, Ratingen, GERMANY.

introduction: in a prospective trial we wanted to test whether adjunctive intraoperative stem cell treatment in patients with critical limb ischemia can be performed safely in combination with bypass surgery and/ or interventional treatment to improve the outcome in end stage disease. endpoints of our study were limb salvage and integrity of the novel point of care system used.material and methods: We included only patients with critical limb ischemia and tissue loss according to the rutherford categories 4 - 6. the Harvest bone marrow aspirate Concentrate system (bmaC) consists of an automated, microprocessor controlled dedicated centrifuge with decanting capability and the accessory bmaC Pack for processing a patient’s bone marrow aspirate (bma). the centrifuge is portable and enables bma to be rapidly processed in the or to provide an autologous concentrate of nucleated cells for immediate injection. the surgeon aspirated 120 ml bma from iliac crest.results: a consecutive number of 8 patients was treated according to the study protocol. the mean follow up period was 9.2 months (2 - 18). stem cells were always injected during the last and final revascularization attempt. one minor amputation and two major amputations were required.in five of eight patients there was a discreet increase in the ankle brachial index post stem cell treatment. the dose of stem cells after centrifugation was 17.2 (13.8 - 54.2) x10e6 CD34-positive cells and 7.8 (1.8 - 35.9) x10e6 CD133-positive cells. the injected dose of VegFr-2-coexpressing stem cells was 0.5 - 5.7 x10e4.Conclusion: We were able to show, that the buffy coat preparation using a point of care system is a simple and fast method to enrich stem cells from bone marrow aspirates. this automated system gives high recovery rates and good reproducebility.

166thE EFFECt OF VEGF On thE MyOGEnIC dIFFEREntIAtIOn OF AdIPOSE tISSuE dERIVEd StEM CELLS WIth In VIVO GEL FORMInG MPEG-PCLM. Kim1, M. Kim2, S. Kim3, G. Kang3, C. Park1, S. Kwon1, H. Hong1, J. Hong1; 1University of Ulsan, Seoul, KOREA, REPUBLIC OF, 2Asan Institute for Life Sciences, Seoul, KOREA, REPUBLIC OF, 3Chonbuk National University, Chonju, KOREA, REPUBLIC OF.

We investigated the combination of human adipose tissue derived stem cells (aDsC) and in vivo gel forming methoxy poly (ethyleneglycol)-poly (3-caprolactone) (mPeg-PCl) as a muscle regeneration matrix, with and without inclusion of vascular endothelial cell growth factor (VegF).soft-tissue engineering solutions, those used to repair defective muscle, require a soft muscle conductive vehicle that differs from that used for hard tissue. in vivo gel-forming, degradable scaffolds exhibit temperature-dependent phase-change properties, converting from a solution to a gel at approximately body temperature. since these matrices provide for the proliferation and differentiation of delivered cells without the need for additional surgery, these have been used as effective tools for targeted cell delivery.Complex tissues, such as muscle tissues, are embedded with blood vessels, which are critical for supplying necessary nutrients and removing toxic metabolites and enhanced angiogenesis may thus improve muscle function in ischemic tissue. VegF is known to be a critical and specific growth factor for blood vessel formation. in particular, the VegF-a subtype, which includes VegF165 used in the current study, is a dominant and potent member of the VegF family.VegF165-treated stem cell grafts showed significant proliferation and differentiation into muscle tissue in vivo. importantly, the inclusion of VegF enhanced vascularization. this scaffold supported preconditioned aDsC, and allowed them to differentiate into mature muscle tissues in vivo, indicating that aDsC of human origin and mPeg-PCl scaffolds provided an appropriate environment for cellular growth and expansion. our results thus provide a potential solution to the major obstacle encountered in the engineering of thick complex tissues, which require an adequate blood supply to maintain cell viability during tissue growth and to induce appropriate structural organization. therefore, the combination of aDsC and in vivo gel forming mPeg-PCl with VegF165 might serve as a suitable non-invasive biomaterial for clinical muscle regeneration applications.

168ChARACtERIZAtIOn OF CELL COnCEntRAtES PREPAREd At POInt OF CARE FROM BOnE MARROW ASPIRAtE uSInG thE MARROWXPRESS™ SyStEMJ. Li1, M. Nguyen1, J. Chapman1, R. Suzuki2, J. Poser2; 1Thermogenesis Corp, Rancho Cordova, CA, 2SpineSmith LP, Austin, TX.

background: the marrowXpress Platform (mXP) is intended for the preparation of cell concentrates from bone marrow aspirate (bma) in a functionally closed disposable at the point of care or in a laboratory. the system delivers an operator-selected volume of the concentrate in 40min. the goal of this study was to characterize the cell concentrates prepared by mXP system from bma under intended use conditions. methods: bma was collected in an operating room using heparin and aCD-a as anticoagulants. bma (50 to 120 ml) was concentrated by the mXP process to a final volume of 10 to 20 ml selected by the operator. Complete blood counts were measured using the sysmex Xe-2100 cell counter for pre-mXP processed and post-mXP processed samples. CD34+ cells and cell viability were measured by flow cytometry using stemKit (beckman Coulter, brea, Ca). results: a total of twenty-five human bmas were processed and evaluated from september 1 to october 15, 2009. the cell counts for pre-mXP and post-mXP samples, cell enrichment factor and cell recovery are reported below.

WbC(x10^6/ml)

mnC(x10^6/ml)

Platelet(x10^6/ml)

CD34+ Cell (x10^4/ml)

Pre-mXP 21+/-10 4.9+/-2.2 100+/-35 15+/-13

Post-mXP 89+/-43 27+/-13 426+/-222 89.3+/-75

enrichment Factor 4.2x 5.5x 4.3x 5.9x

Cell recovery (%) 69+/-12 89+/-19 71+/-24 95+/-15

the percent rbC depletion was 91.0+/-3.7% with a total packed rbC of 2.1+/-0.4ml remaining in the cell product. the percent cell viability was unchanged by the processing (91+/-3% vs. 93+/-5%).Conclusion: the mXP system was demonstrated to provide a reliable and reproducible method to reduce bma volume for the preparation of cell concentrates including WbC, mnC, platelets and CD34+ cells. the stem cells and platelets were concentrated on average 5.9x and 4.3x, respectively above baseline by mXP processing.


Poster Abstracts

169GROWth FACtOR StIMuLAtIOn OF CARdIAC StEM CELLS (CSCs) tO GEnERAtE CELLS WIth In VIVO POtEntIAL I. McNiece, B. Oskouei, K. Chatzistergos, J. Hare; University of Miami, Miami, FL.

stem cells have been identified and isolated from heart tissue based upon expression of c-kit. However, little is known about the control of proliferation and differentiation of CsC. We hypothesized that cardiac stromal cells could secrete growth factors that stimulate CsCs and further that sCF could be a key regulator of CsCs. using clonal assays in semi solid media we evaluated the gF stimulation of c-kit+ cells isolated from human heart tissue. large colonies (diameter >1.0mm) formed with the combination of stem cell factor (rhsCF) plus media conditioned (Cm) by human heart stromal cells (HuHstr). Colonies contained primitive cells that formed 20 colonies upon replating. Human c-kit+ cells were also grown in liquid culture in teflon bags with rhsCF and HuHstr Cm. the cells proliferated over a two week period and formed spheres of c-kit+ cells that differentiated to a cardiac phenotype expressing nkx2.5 and gata-4. the cultured human c-kit+ cells (30,000/mouse) were also injected into the hearts of noD/sCiD mice following mi. 4- weeks after injection the mice were sacrificed and iH demonstrated extensive human myocytes (alu+ cells) and human cells in vessel walls. in conclusion, these studies demonstrate that sCF and cardiac stromal cell derived factors stimulate proliferation of CsCs that have the capacity to generate cells capable of engrafting ischemic cardiac tissue.

171huMAn AutOLOGOuS LIVER CELL tRAnSPLAntAtIOn FOR thE tREAtMEnt OF CIRRhOSISA. Schwarz1, W. Pieken2, T. Lindl3, C. Höhneke2; 1Kreisklinik Biberach, Biberach, GERMANY, 2HAC Biomed GmbH, Munich, GERMANY, 3Institut für Angewandte Zellkultur, Munich, GERMANY.

We report here the application of autologous liver cell transplantation on a polymer scaffold for the therapy of human liver cirrhosis.a liver tissue sample and a pancreas biopsy were harvested from cirrhotic patients. Vital liver and pancreas cells were isolated from these tissue samples, seeded onto a poly-l-lactic acid matrix and re-implanted into the mesentery of the same patient.the autologous matrix assisted co-transplantation of liver cells together with a small amount of pancreatic cells was applied to 57 individual treatments of liver cirrhosis. the average survival rate following one year after transplantation was 75 % for all patients. For those with melD <= 10, the one year survival was 91 %. the average melD score stayed constant for 37 patients for whom 12 or 24 months follow up data was available. this is in contrast to the literature, which reports a significant degradation in melD score for patients treated conventionally. For a majority of patients, the liver related blood values remained stable or improved 12 months post treatment, except for the gamma gt value, which in most cases did not improve. the majority of patients also reported improved quality of life one year post treatment.the autologous matrix-seeded transplantation of liver cells warrants further controlled clinical study for the stabilization and possibly bridging to orthotopic liver transplantation of cirrhotic patients.

170hIGh ShEAR RAtES nEGAtIVELy AFFECt CELL VIABILIty And FInAL PROduCt QuALIty In tFF PROCESSInG OF LARGE SCALE thERAPEutIC CELL hARVEStSJ. G. Pattasseril, J. Rowley; Lonza Walkersville Inc., Walkerville, MD.

Currently, therapeutic cells are harvested and purified using open centrifugation technology. open centrifugation is time consuming, introduces high process risk and cost prohibitive to process high volume batches. We have developed and characterized a scalable, closed system technology based on tangential Flow Filtration (tFF) that can process high volume batches in 1-3 hours while maintaining >90% cell viability, >85% product recovery and cell functionality. the tFF system includes standard hollow-fiber filters, processing bags and tubing sets, and control pumps. msCs and fibroblasts were processed through the tFF system at varying shear rates (1800-5500 sec-1) and samples were obtained at varying processing times and assayed for cell concentration, viability, bsa residual levels, and delayed onset of cell death.shear rates in the range of 1800-3000 sec-1 resulted in aviability drop up to 3%, while 3100-3500 sec-1 resulted in a viability drop of 5-7%. shear rates >3500 sec-1 resulted in viability drops from 12-20% in the processed cells. Viabilities of >90% and product recoveries of >85% were consistently achieved in multiple tFF runs in which shear rates were maintained <3000 sec-1. Vialight assay results proved that the processed cells had equivalent viability and metabolic activity after 24 hours of culture as unprocessed cells. scale-up runs at 25l (10-20 billion cells) were successfully performed with >90% cell viability and >85% product recovery. the diafiltration (cell washing) performed during the tFF process decreased bsa residuals approximately 6000 fold to less than 100 ng/ml, well below the current FDa requirement of 1 ppm.in conclusion, the fluid shear rate in tFF should be maintained at a narrow range (1800-3000 sec-1) to achieve acceptable product quality parameters such as >90% cell viability and >85% product recovery. this system has been designed as a completely closed system for aseptic processing.

172BIOACtIVE, SEMI-dEGRAdABLE hydROGELS FOR CARtILAGE tISSuE EnGInEERInGK. L. Spiller, A. Lowman; Drexel University, Philadelphia, PA.

Poly(vinyl-alcohol) (PVa) hydrogels have long been investigated as replacement materials for articular cartilage, but their lack of bioactivity has impeded their utility. We have prepared bioactive PVa hydrogels by incorporating a degradable phase of poly(lactic-co-glycolic acid) (Plga) that enhances porosity and controls the release of growth factors. Hydrogel properties such as porosity, pore size, and degradability were dependent on the hydrogel composition and fabrication conditions. Porosity and pore size increased over time in physiological conditions as the Plga phase degraded within the nondegradable PVa hydrogels. Cell-seeding efficiency and tissue formation in vitro were proportional to the amount of Plga. When insulin-like growth factor-1 (igF-1) was incorporated into the degradable Plga phase, release was controlled over 6 weeks, with no burst effect. Hydrogels containing a low or high dose of igF-1 were wrapped in Pga fibers, seeded with chondrocytes and implanted subcutaneously into nude mice. after 6 weeks, the amount of cartilage tissue formation and integration with the hydrogels were higher compared to controls without igF-1, although there were no differences in mass, proteoglycan content or compressive modulus between hydrogels with low and doses of igF-1. these simple modifications to PVa hydrogels may finally make them suitable as cartilage replacements.


may 23-26, 2010


Poster Abstracts

173FunCtIOnAL tEndOn EnGInEERInG And ItS APPLICAtIOn In REPAIRInG MOnKEy tEndOn dEFECtB. Wang; Shanghai 9th People’s Hospital, Shanghai, CHINA.

Poor mechanical property of in vitro engineered tendon is a major challenge to clinical application. We developed a novel composite scaffold based on networks of varying Pga and Pla fiber ratios, thus to enhance early mechanical property. the obtained scaffolds in combination with fibroblasts as the seed cells were applied to in vitro construction of tendon tissues. it was found the Pga/Pla=4:2 scaffold possessed good cytocompatibility, suitable degradation rate and good tissue formation capability. the ultimate tensile strength of the tendon engineered with this scaffold reached 55.3±5.2n, which displayed sufficient strength for in vivo implantation. Further application of this engineered tendon to repair flexor tendon defect in monkey exhibited that active finger motion could be achieved with less adhesion to surrounding tissues. after 6-month of implantation, proper cell/collagen ratio was observed and collagen superstructure displayed regular D-band periodicity, which confirmed the maturation of the engineered tendon. in conclusion, functional tendon could be engineered with a novel scaffold and dermal fibroblasts.

175InCREASEd EXPRESSIOn OF SIdE POPuLAtIOn MARKERS In huMAn AMnIOtIC EPIthELIAL StEM CELLS CuLtuREd WIth A COMMERCIAL SERuM REPLACEMEntD. L. ZANETTE1,2, K. L. PRATA3, C. M. KANETO1, P. V. PALMA3, C. C. MENEZES3, G. DUARTE1, D. T. COVAS1, W. A. SILVA-JR1; 1Medical School of Ribeirão Preto, University of São Paulo, Ribeirao Preto, BRAZIL, 2National Institute of Science and Technology in Cell Therapy, Ribeirão Preto, Ribeirão Preto, BRAZIL, 3Regional Blood Center of Ribeirão Preto and National Institute of Science and Technology in Cell Therapy, Ribeirao Preto, BRAZIL.

Human epithelial cells from placental amniotic membrane (haeC) display great differentiation potential, are poorly immunogenic and have immunomodulatory properties. easy access and isolation of these cells suggest they can be a great source of stem cells. to achieve this, culture systems must be free of animal components, such as fetal bovine serum (Fbs). so, we aimed to characterize phenotypic markers of haeCs cultured in serum-free conditions, with a commercial serum replacement (Ksr, Knockout serum replacement, invitrogen). term placentas were obtained after cesarean sections and amniotic membrane was peeled off from the chorion and further digested with trypsine-eDta. We then compared the same samples cultured with Ksr and Fbs. Flow cytometry was performed to address the expression of stem cells/embryonic markers. Cells in Ksr system grew slower, reaching the first confluence (P1) two days later than cells cultured with Fbs. at this time point, flow cytometry revealed an increased percentage of cells expressing side-population markers (abCg2 and PgP) in the Ksr system. embryonic stem cells markers nanog, soX and ssea-1 were also more expressed in these cells, although other markers were expressed in lower levels (oCt-4, ssea-4, tra1-81 and tra1-60). embryonic and side population expression profiles described here for the cells grown with Ksr may indicate the preservation of the pluripotent state. the main argument is the increased expression of side population markers, abCg2 and PgP, which reflect the efflux pump activity, an exclusive functional feature of stem cells. the slower growth of the haeC cultured with Ksr also corroborates this hypothesis. although it is well known that Fbs stimulates commitment of some primitive stem cells, to our knowledge it is the first report of an increased expression of side population markers in haeC cultured under serum free conditions. Future studies may investigate the mechanisms and effects of these findings.

1743d-PRIntEd SCAFFOLdS FOR tISSuE EnGInEERInG And CELL-BASEd GEnE thERAPyH. Zachary, M. Janssen, A. Cornick, A. McCormick, Q. Tan, V. Leszczak, A. M. Yousefi; Miami University, Oxford, OH.

introduction: maintaining adequate nutrient and oxygen diffusion to cells is an essential requirement for the success of the scaffold-based tissue engineering and cell-based gene therapy. Pore size, overall porosity, and material used are all critical factors that influence the biology of ingrown tissue. the goal of this work is to design 3D-printed scaffolds with mechanical properties mimicking the stiffness of target biological tissues while providing maximum porosity and highest effective surface for cell attachment.materials and methods: the 3D-printed scaffolds were fabricated using three different polymeric powders. the compressive young’s modulus and equilibrium modulus were estimated for the samples.1 the effective surface of the 3D-printed constructs was determined as a function of scaffold topological parameters (strand diameter D, pore size l, layer thickness h; see Fig. 1) and scaffold geometry.2 a similar model was developed to relate the scaffold stiffness to its topological parameters and the stiffness of the nonporous matrix.results and Discussion: Figure 1 shows a typical input CaD file to the 3D-printer. the photomicrograph of the scaffold is given in Fig. 2. this work explores the potential of the new computational approach and presents the performance modeling results for the designed scaffolds.

176nAIVE tuMOR-SPECIFIC Cd4+ t CELLS dIFFEREntIAtEd In VIVO ERAdICAtE EStABLIShEd MELAnOMA In LyMPhOPEnIC hOStSP. A. Antony (P.I.), K. Harris, Y. Xie; U of Maryland School of Medicine, Baltimore, MD.

in vitro differentiated CD8+ t cells have been the primary focus of immunotherapy of cancer with little focus on CD4+ t cells. immunotherapy involving in vitro differentiated t cells given after lymphodepleting regimens significantly augments antitumor immunity in animals and human patients with cancer. However, the mechanisms by which lymphopenia augments adoptive cell therapy and the means of properly differentiating t cells in vitro are still emerging. We demonstrate that naïve tumor/self-specific CD4+ t cells naturally differentiated into t helper type 1 (th1) cytotoxic t cells in vivo and caused the regression of established tumors and depigmentation in lymphopenic hosts. therapy was independent of vaccination, exogenous cytokine support, CD8+, b, nK and nK-t cells. Proper activation of CD4+ t cells in vivo was important for tumor clearance as naïve tumor-specific CD4+ t cells could not completely treat tumor in lymphopenic common gamma chain deficient (βc-/-) hosts. βc signaling in the tumor-bearing host was important for survival and proper differentiation of adoptively transferred tumor-specific CD4+ t cells. lastly, removal of nK cells enhanced t cell therapy and autoimmunity resulting in increased chemokines and cytokines in vivo which appeared to be dependent on surface-bound il-15 to mHC class ii+ cells. thus, these data provide a platform for designing immunotherapies that incorporate tumor/self-reactive CD4+ t cells.


Poster Abstracts

177thERAPEutIC CELL EnGInEERInG uSInG SuRFACE-COnJuGAtEd SynthEtIC nAnOPARtICLESM. T. Stephan, J. J. Moon, S. Um, A. Bershteyn, D. J. Irvine; Massachusetts Institute of Technology, Cambridge, MA.

a major limitation of cell therapies is the rapid decline in viability and function of transplanted cells. We devised a facile and generalizable strategy to robustly augment the therapeutic potential of cytoreagents, based on the conjugation of adjuvant drug-loaded nanoparticles to the surfaces of therapeutic cells. using this method to provide sustained pseudo-autocrine stimulation to donor cells, we elicited dramatic enhancements in tumor elimination in a model of adoptive t-cell therapy for cancer and increased the in vivo repopulation rate of hematopoietic stem cell grafts, using very low doses of adjuvant drugs that were ineffective when given systemically. this approach is a facile and generalizable strategy to augment cytoreagents while minimizing systemic side effects of adjuvant drugs. in addition, these results suggest therapeutic cells are promising vectors for actively targeted drug delivery.

179ABStRACt WIthdRAWn.

178A nOVEL SCREEnInG MEthOd FOR thE IdEntIFICAtIOn OF tuMOuR AntIGEn SPECIFIC scFv’s FOR uSE In AdOPtIVE IMMunE thERAPy OF CAnCERG. Lipowska-Bhalla, R. E. Hawkins, D. E. Gilham, D. G. Rothwell; Cancer Research UK, Medical Oncology Group, Paterson Institute for Cancer Research, University of Manchester, Manchester, UNITED KINGDOM.

adoptive transfer of chimeric immune receptor (Cir) expressing autologous t lymphocytes is a promising strategy in cancer immunotherapy. a fundamental component of the Cir is the single chain variable fragment (scFv). the scFv defines Cir’s antigen specificity and plays a pivotal role in mHC-independent recognition of tumour antigen. Developing a reliable screening method for the identification of novel scFv’s is a potent avenue to improve Cirs function and broaden their clinical application. Conventional phage display techniques, although proven effective in prokaryotic systems, do not ensure expression or functionality of identified scFv’s in mammalian cells. therefore, we have developed an alternative protocol in which the screen is performed in an eukaryotic system and the tumour antigen specific scFv’s are identified in the context of Cir structure. the protocol involves expression of a scFv library on the surface of t lymphocytes as a fusion with the Cir signaling domain CD3z using retroviral transduction. such a system allows linking of the scFv specificity with the functional capacity of the Cir to activate t lymphocytes. Following effective interactions between the Cir and the tumour antigen, cells expressing antigen specific scFv-Cirs become activated and rapidly up-regulate the expression of the early activation marker CD69 that acts as a selection marker for FaCs based isolation of cells expressing antigen-reactive Cirs. the initial proof of principle experiments were performed using mini-libraries consisting of mFe, a scFv specific for a carcinoembryonic antigen (Cea) and anti-CD19, a non-Cea specific scFv. using selection on immobilized Cea antigen and CD69 as a selection marker we have achieved 11-193 fold enrichment of Cea-specific Cir following two rounds of selection using real-time PCr quantification. We have also screened a partially pre-screened Cea-specific scFv library, from which mFe was identified and demonstrated almost five-fold enrichment of mFe Cir during one selection round.

180thE VALIdAtIOn Study OF MyCOPLASMA dEtECtIOn MEthOdS FOR thE QuALIty COntROL OF CELL thERAPEutIC PROduCtS In hOSPItALSD. Wang1, C. Chou1, K. Lee1, S. Weng1, W. Chang1, M. Yeh2, C. Yen2; 1Food and Drug Administration, Department of Health, Taipei, TAIWAN, 2Industiral Technology Research Institute, Hsinchu, TAIWAN.

Cell therapeutic products are composed of or contain living human cells, tissues and/or their derivatives. the major manipulation of cell products is based on the aseptic cell culture techniques. the mycoplasma contamination is one of the major potential contaminations in many laboratories. therefore, the detection and monitoring of mycoplasma during processing steps of cell therapeutic products are important and need to be established. typical method for mycoplasma detection needs almost 4 weeks incubation. it is not adequate for living cell therapeutic products release. the PCr method has the characteristics of high sensitivity and fast testing. it is a commercialized kit and easy for cell laboratories to use in hospitals. relatively, the result from PCr method is easily affected by environmental factors. the european Pharmacopeia asked the manufacturers to have appropriate validation procedure on the PCr method before use. However, analytical method validation is difficultly to be done by laboratories in most hospitals. in this study, we collected over 80 cultured medium specimens to validate two commercialized mycoplasma PCr kits and compared the sensitivity and specificity with the growth-based fluorescent-dye staining method. these results confirmed the PCr methods had the 97~100% of specificity and the 68~76% of sensitivity. our results showed the necessity of two-stage mycoplasma tests should be done before cell therapeutic product release. We suggest that cell therapeutic product with PCr test negative has to do the second round high sensitivity testing to eliminate the false negative noise of mycoplasma detection.


may 23-26, 2010


Poster Abstracts

181thE VALIdAtIOn Study OF GROWth-BASEd RAPId BACtERIAL dEtECtIOn SyStEM FOR thE QuALIty COntROL OF CELL thERAPEutIC PROduCtSD. Wang1, C. Chou1, K. Lee1, S. Weng1, W. Chang1, M. Yeh2, C. Yen2; 1Food and Drug Administration, Department of Health, Taipei, TAIWAN, 2Industiral Technology Research Institute, Hsinchu, TAIWAN.

Cell therapeutic products are one of the advanced therapeutic medicinal products in 21 century. since these products are composed of or contain living human cells, tissues and/or their derivatives, they can not be treated by terminal sterilization. the sterility of these cell products can only rely on the fully aseptic processing, periodic environmental/microbial monitoring and the final product sterility test before release. typical pharmacopeia sterility testing method needs 14-day incubation and well-controlled testing environment. However, the cell therapeutic products have too short expiration “hours” (within 24-48 hrs). the long-term incubation method is not adequate for cell product release. therefore, establishment of rapid bacterial detection method is an important issue for cell therapeutic product release testing. in this study, we established an automatic growth-based rapid bacterial detection system for cell culture condition. the validation study of this new method is based on the comparison of performance between growth-based rapid bacterial detection method and membrane filtration sterility testing method. our results showed that validated growth-based rapid bacterial detection system has lower detection limit (30 CFu) within 24 hrs determination. We recommended that the validated automatic growth-based rapid bacterial detection system can be adequate for cell therapeutic product releasing test.

183ELECtROnIC SyStEM FOR MAnAGInG thE dEVIAtIOn PROCESSG. O. Majkowski1, S. Locke1, C. Elliott2; 1G2MQ Corp, Alexandria, VA, 2CT Auditing and Compliance Services, Sugar Hill, GA.

it is a fact that a mechanism to capture, investigate, and when applicable, report deviations is essential in the manufacture of cell therapy products. it is both a regulatory and accreditation requirement. Failure to understand and manage deviations may affect the safety, potency and purity of cell therapy products, compromise patient and donor safety. it also exposes organizations to legal and compliance actions, the potential for significant financial loss, and waste time and resources.Deviations may be errors, incidents, accidents, non conformances, sentinel events, near misses, complaints, and adverse events. not only is it important to capture deviations but there must be a process to track and trend daily occurrences that have yet to reach the stage of a deviation and/or become the focus of compliance inspections.a well-designed, implemented, and manageable “Deviation management system” offers a mechanism for capturing and investigating events in a timely manner. it also facilitates monitoring, tracking, trending, review and on-going analysis allowing for quick response to failures, early warning of potential failures, redeployment of resources to problematic areas, and, when applicable, reporting to customers and regulatory agencies. in addition serve as a critical tool for the measurement, analysis and continuous improvement of operations and organizational performance.not every organization can afford expensive systems or platforms to manage deviations. Finding smart and creative savings strategies in this challenging economic environment is the quest of every organization. this abstract introduces the use of microsoft™ application (excel and PowerPoint) to capture, document, and report, track and trend, analyze, monitor and manage deviations. the system design also aim to demonstrates a mechanism to prioritize deviations for corrective and preventive actions, follow-up and performance improvement. organizations can leverage readily available programs such as microsoft to develop, validate, and implement a Deviation management system.

182FIVE-yEAR dEVELOPMEnt OF REGuLAtIOn FOR CELL thERAPy In tAIWAn - A REGuLAtORy MOdEL OF CELL thERAPy In hOSPItALS FOR SMALL COuntRIESD. Wang1, C. Yen2, C. Chou1, C. Chang2, C. Lin1, T. E. Suen1; 1Food and Drug Administration, Department of Health, Taipei, TAIWAN, 2Industiral Technology Research Institute, Hsinchu, TAIWAN.

Cell therapeutic technology/products are very different from small molecule drug. the regulation became a new challenge for national authorities. united states FDa and european Commission had enforced the new regulations and directives for cell therapeutic products in recent 5 years, respectively. the new rules gave the definitions and brought highly manipulated products into regulatory framework for biologics and medical devices. However, these regulations were not suitable for applying in all countries. at least, this two- or three-tiered classification was not adequate for some cell therapy medical technology in hospitals. taiwan scientists and physicians in the medical centers developed many emerging cell therapy technology for clinical treatments in recent 10 years. these new technologies were hardly brought into the existing pharmaceutical regulations. taiwan Department of Health (DoH) established the good tissue Practice (gtP) in 2002 and enforced for laboratories in hospitals in 2004. our DoH temporally decided the cell therapy as a kind of new medical technologies at that time. based on the medical affair act, all new medical technologies for cell therapy have to proof the safety and efficacy via clinical studies. in addition, all applications of cell therapeutic clinical trials have to be inspected before they get the approval since 2007. the bureau of Food and Drug analysis (bFDa) had proceed the gtP conformity inspection for 16 cases of phase i studies and 4 cases of phase ii studies from 2007 to 2009. in this stage, bFDa and industrial technology research institute (itri) collaborated to facilitate the quality control of the cell laboratories in hospitals via a series education and training courses. bFDa and other agencies were reorganized into taiwan Food and Drug administration (tFDa) since Jan 1st 2010. We can expect our pharmaceutical acts and new cell regulations will be harmonized soon in next regulatory stage.

184PRELIMInARy REPORt On IMMunOMOduLAtIOn OF MESEnChyMAL StEM CELLS In PAtIEntS WIth tuBERCuLOSIS InFECtIOnL. K. Chelluri1, P. Chelluri2, P. Vennela3, G. AGK1, V. Adavi1, R. KS1, R. K1; 1Global Hospitals, Hyderabad, INDIA, 2Gandhi Medical College, Hyderabad, INDIA, 3Osmania University, Hyderabad, INDIA.

background: tumor necrosis factor-alpha (tnF-β) and interferon-gamma (iFn-β) are two important pleiotropic inflammatory cytokines that perpetuate tissue damage in response to stress and in the presence of excess glucocorticoids during tuberculosis (tb) infection. Hence, modulating cytokines will be an important step to contain inflammation and scarring during infectionaim: the aim of this study is to measure the ex-vivo cytokine responses when co-cultured with various cellular concentrations of mesenchymal stem cells (msCs) with peripheral blood mononuclear cells (PbmCs) obtained from tb infected patients.materials & methods: short-term co-culture experiments were performed using PbmCs obtained from five tb patients and allogeneic bone marrow derived msCs at three different cellular concentrations. initial serum levels and 5-day co-culture supernatant levels of tnF-β and iFn-β was analyzed by elisa.results: at 104 cells/µl and 105 cells/µl of msCs, there was a significant dose-dependent down modulation of tnF-β (p=0.20 at 80% confidence interval level, t< 0.90), whereas, there was no changes in iFn-β levels were observed.Conclusion: the outcome demonstrates that exposure of msCs to tb-infected PbmCs in an ex-vivo setting has a profound impact in modulating the tnF-β levels that plays a pivotal role in infection mediated inflammation.


Poster Abstracts

185IdEntIFICAtIOn OF thE MESEnChyMAL StEM CELL BOnE nIChE uSInG nOVEL CELL SuRFACE MARKERS: CAn FGF RECEPtORS dO thE tRICK?D. L. Coutu, M. François, J. Galipeau; McGill University, Montreal, QC, CANADA.

bone-derived mesenchymal stem cells (msCs) are somatic stem/progenitor cells endowed with with mesodermal plasticity as well as pro-angiogenic, pro-regenerative and immuno-modulatory properties. these attributes make them attractive candidates for cell therapy, tissue engineering and regenerative medicine. However, the fundamental study and clinical use of msCs are impeded by the absence of known markers for their prospective isolation and the lack of a functional definition for these intriguing cells. based on the experimental paradigm developed for hematopoietic stem cells, a single prospectively isolated msC should be assayable by transplantation in vivo and shown to repopulate all non-hematopoietic lineages in bones. in this study, we took a step further to characterize msCs and their potential in vivo niche by using novel cell surface markers of msCs. because of the importance of FgF signaling during bone ontogeny and since FgF receptors (FgFrs) are developmentally regulated in bone cells, we studied how this system is involved in msCs biology. We first demonstrated that C57bl/6 murine msCs are dependent on FgF signaling for self-renewal ex vivo, by a mechanism that involves Pi3K-mediated phosphorylation of mDm2 and inhibition of cellular senescence. We next show that culture-expanded, undifferentiated primary msCs express three FgF receptors (FgFr1(iiib), FgFr1(iiic), FgFr2(iiic)) that are consistent with the role of msC in development and tissue repair. Confocal microscopy demonstrated that FgFr2(iiic) expressing cells localized mainly to the perichondrium whereas FgFr1+ cells were mainly perivascular in the periosteum and cortical bone, although some were also detected in the marrow cavity. this again is consistent with the role of msCs in development and homeostasis, as well as with previous studies. at the time of submitting this abstract, we have prospectively isolated genetically-marked FgFr-expressing msC-like cells and are performing transplantations without prior ex vivo expansion to assess the lineage contribution of those various progenitor cell populations.

187dEFInInG thE BESt SOuRCE OF huMAn MuLtIPOtEntIAL MESEnChyMAL StROMAL CELLS FROM CORd BLOOd, FAt tISSuE And BOnE MARROW.Volchkov S.E., Tyumina O.V., Boriskin P.V., Trusova L.M., Toropovskiy A.N.Clinical Center, Moscow, RUSSIAN FEDERATION. Clinical Center (Samara, Russia)

regenerative medicine is in need of high amount of regenerative cells at minimum time, with low invasiveness and cost. so we tested three easily available sources of mesenchymal stromal cells (msCs): bone marrow, cord blood, lipoaspirate. We have tested and compared the proliferative potential, differentiation capability including adipogenesis, chondrogenesis, osteogenesis, phenotype, cytokine synthesis, ability to form combined transplant with matrixes. Proliferative potential was examined by ViCell Xr (beckman Coulter). Differentiation was performed with commercially available media from miltenyi biotec, using manufacturer’s protocol. Phenotyping was performed using recommended antibody panel on FaCs Canto cytometer (becton Dickinson). Cytokines testing was performed on luminex 100 is platform, using biorad™ 27-plex assay. to estimate ability for establish combined transplant we seed cells on different types of matrixes, including lyophilized bone, titanium, nickel titanium and nickel titanium with hydroxyapatite. to estimate cells on surfaces we used scanning electron microscopy by Fei.We estimated that fat-derived cells has the lowest doubling time (0,0088 /hr), but the highest CFu-F frequency (0,25% per 1x106 mononuclear cells), leading to maximum yields of regenerative cells at minimum time. the cells from all three sources successfully differentiated into all three lineages. all cells were positive for mesenchymal and negative for hematopoietics markers. We didn’t find any statistically significant differences in cytokine expression within cells from various sources. all cells were successfully established mixed transplant with matrixes. our study shows that the cells from lipoaspirate are the best candidate for regenerative medicine due to having identical properties with cells from other sources, accessibility to collect big volumes, a maximum yields at lowest culture time.

186An InVEStIGAtIOn IntO thE KInEtICS OF MSC-InduCEd IMMunOSuPPRESSIOn duRInG thE ALLO-REACtIVE PhASE OF An MLRM. C. Rae; Newcastle University, Newcastle upon Tyne, UNITED KINGDOM.

Key mechanisms of msC immunosuppression (indoleamine-2,3, deoxygenase (iDo), inducible nitric oxide synthetase (inos) and Hla-g) were studied using mixed lymphocyte reactions (mlr) blocking experiments and FaCs analysis.to investigate whether the mechanisms described above act in a synchronised manner, levels of activity for each mechanism were measured and compared.results demonstrated that iDo and inos showed a bi-phasic reciprocal activity during the lag phase of the alloresponse. Hla-g expression was demonstrated to be acting independently of both inos and iDo. Priming msCs with iFnβ and tnFβ altered the kinetics of iDo and inos expression by prolonging the activity of iDo and inos during the lag phase of an allo-reaction but had no significant effect upon Hla-g. this may explain the augmentary effects of these cytokines on msC function. msCs significantly reduced the graft versus Host reaction (gvHr) in the in vitro human skin explant model. the graft versus Host type alloreactivity (gvHr) seen in this model is analogous to graft versus host disease (gvHD) in a clinical transplant setting. briefly, msCs and responder cells were taken from a mlr then co cultured with a skin biopsy from the stimulator for 3 days. Various conditions were tested, and msCs showed significant immunomodulatory effects (p=0.018). blockade of inos also altered the inhibitory effects of msCs in this context. Hla-g was expressed on msCs (n=4) and it was shown that Hla-g is shed from the msC cell surface and taken up and expressed by responder cells via a process described as trogocytosis during an mlr (p=0.053) using elisa and FaCs. msC immunomodulatory activity is multi-phasic -indicative of an important homeostatic in vivo role which can be manipulated using cytokines. this may have relevance in their future use in transplantation.

188tROPhIC MOLECuLES dERIVEd FROM huMAn MESEnChyMAL StEM CELLS EnhAnCE SuRVIVAL, FunCtIOn, And AnGIOGEnESIS OF ISOLAtEd ISLEtS FOLLOWInG tRAnSPLAntAtIOnS. Kim, J. Kim, M. Shin, C. D. Kwon, J. Joh, S. Lee, S. Kim; Samsung Medical Center, Seoul, KOREA, REPUBLIC OF.

mesenchymal stem cells (msCs) release several factors that support cell survival and enhance wound healing. We hypothesized that msC-secreted molecules would induce trophic effects in pancreatic islet cultures.mouse and human pancreatic islets were cocultured with msCs. aDP/atP ratios, glucose stimulated insulin secretion, Dna fragmentation were evaluated to measure islet quality and viability in vitro. the induction of signal molecules related to the control of survival, function, and angiogenesis was analyzed. using msC-conditioned medium (msC-Cm) we also evaluated for enhancing islets qualities. We identified a change of soluble molecules within msC-Cm.mouse and human islets cocultured with msCs or msC-Cm demonstrated lower aDP/atP ratios, and higher gsis indexes and viability.Cocultured islets revealed higher levels of anti-apoptotic signal molecules (XiaP, bcl-xl, bcl-2, and HsP-32), and demonstrated increased VegFr2 and tie-2 mrna expression and increased levels of phosphorylated tie-2 and FaK protein. Diabetic mice that received islet transplants cultured in msC-Cm for 48hr demonstrated significantly lower blood glucose levels and enhanced blood vessel formation.il-6, il-8, VegF-a, HgF, tgF-beta were detected at significant levels in msC-Cm, which affect the survival, function and angiogenesis/revascularization of islets.results suggest that the trophic factors secreted by human msCs enhance islet survival and function following transplantation.


may 23-26, 2010


Poster Abstracts

189VALIdAtIOn OF GOOd MAnuFACtuRInG PRACtICE (GMP) COMPLIAnt QuALIty COntROLS OF Cd133+ CELLS FOR thE tREAtMEnt OF End StAGE LIVER dISEASE.G. Andriolo, M. Viganò, T. Montemurro, E. Montelatici, F. Chelli, G. Spaltro, D. Casale, R. Giordano; CELL FACTORY, Fondazione IRCCS Policlinico, MILAN, ITALY.

Preclinical data and preliminary clinical experiences suggest that CD133+ cells might reduce fibrosis and improve liver regeneration in end-stage liver disease. in our hospital-based gmP facility, we validated gmP CD133+ cell production from apheresis and the analytical methods for the quality control of the final products (sterility, endotoxins, purity, viability and cell dose). the sterility method was validated following eu. Ph. 2.6.27 with both the standard microbial strains and those isolated during environmental monitoring. the endotoxin method (eu. Ph. 2.6.14) was validated at different dilutions of the final product to exclude the presence of inhibitors. For the validation of the immunophenotypic analysis, different concentrations (from 100% to 0.5%) of CD133+ cells isolated from cord blood (Cb) were prepared and analysed (CD133 clone 293C3; CD45 clone J33). Furthermore, for cell viability different concentrations (from 100% to 5%) of freshly isolated and heat-treated dead Cb CD133+ cells were prepared and analysed with annexin V/P.i. the sterility assay was able to relieve in the final product both standard and environmental microbial strains. the validation of endotoxin assay excluded the presence of inhibitors in the final products. For cell counting, an automated method (nucleoCounter, Chemometec) showed high accuracy (r2 >0.9) in the range of the expected cellular concentration in the final product (1000, 1500, 2000 cells/µl). Finally, immunophenotypic characterization for both CD133 antigen expression and viability demonstrated high accuracy (r2= 0.9 and r2= 0.9, respectively), specificity (absence of false positives) and sensitivity (absence of false negatives). We are currently applying this methods for the quality control of cell products in the context of a phase i dose escalation clinical trial.

191APPROAChES tO AVOId AdVERSE EVEntS In thE GEnEtIC MOdIFICAtIOn OF hEMAtOPOIEtIC CELLS C. Baum; Dept. Experimental Hematology, Hannover Medical School, Hannover, GERMANY.

adverse events related to insertional mutagenesis or ectopic transgene expression may compromise the therapeutic prospects of gene therapy in the hematopoietic system. over the past years, major progress has been made in the understanding of the underlying mechanisms, opening rational approaches to increase the therapeutic index. We have established sensitive nonclinical models, in transplanted C57bl/6J mice or using cultured cells, to reveal the transforming capacity of insertional activation of cellular proto-oncogenes such as evi1 or Prdm16, and developed integrating gene vectors designed to reduce the risk of insertional transformation by altering the insertion pattern and the expression cassette of the transgene. in a murine model of mpl deficiency, major adverse events related to ectopic protein expression could be controlled by transcriptionally targeted vectors. two important principles emerge to ensure undisturbed long-term hematopoiesis from gene-modified cells. the first is the establishment of polyclonal hematopoiesis using vectors with an untargeted integration profile and “physiological” transgene cassettes. the second is the generation of clonally defined hematopoiesis derived from induced pluripotent cells with genetic modification in bona fide safe harbours.

190PRE-CLInICAL dEVELOPMEnt OF ALLOGEnEIC tuMOR dERIVEd LyMPhOCytE (tdL) IMMunOthERAPy PROduCtV. S. Fellowes, F. T. Hakim, J. J. Rose, N. M. Hardy, M. R. Bishop; Experimental Transplantation and Immunology Branch, NCI/NIH, Bethesda, MD.Donor leukocyte infusion (Dli) is the primary treatment for relapse after allogeneic hematopoietic stem cell transplantation (HsCt); however Dli are successful in < 50% of patients. We theorized that lymphocytes from post-HsCt tumor tissue (tt) are “primed” to recognize tumor but are inadequately co-stimulated for tumor cell killing. ex-vivo stimulation and expansion may increase lymphocyte potency without significant gVHD. We developed a method to produce a donor derived t-cell product from post-allogeneic sCt patient’s tt .to liberate t cells from tt, enzymatic digestion (enZ; liberase® collagenase blend) was compared to mechanical disruption (meCH;b-D™ medimachine and later miltenyi gentlemacs) . meCH resulted in a cell suspension containing more debris and was more labor intensive; however, enZ resulted in transient loss of some cell surface markers which rendered identification of the starting cell population difficult.liberated cells were placed into culture containing X-Vivo 20 (bioWhittaker) with 5% normal donor ab plasma and 100iu/ml il-2 (Chiron). t lymphocytes were stimulated with a 3:1 (bead:t) ratio of anti-CD3/anti-CD28 (a3/28) coated beads. meCH cultures had lower t-cell expansions than enZ (582 vs 1761 fold and 24 vs 30). However, one culture showed the reverse (30 and 10 fold). both a3/28+ selections and two layer ficoll hypaque separations resulted in a slight enrichment of t cells, however, purities were poor.We have expanded t cell populations from pleural fluid, lymph node and bone marrow on 19 patients post-allosCt. We have consistently observed expansion of t cells to the point where >95% of the culture was CD3 positive, and a loss of tumor cells when these were present at the start of culture. Furthermore, in replicate cultures, we have observed consistent phenotypes in the expanded cell population.Donor t-lymphocytes can be consistently isolated and expanded from post-HsCt tt while meeting all safety testing required by FDa.

192EVALuAtIOn OF PROCESSInG tEChnOLOGIES FOR unRELAtEd uMBILICAL CORd BLOOd (uCB)J. Wofford, C. Henderson, K. Fortune, D. Regan; St. Louis Cord Blood Bank @ SSM Cardinal Glennon Children’s Medical Center, St. Louis, MO. Evaluation of Processing Technologies for Unrelated Umbilical Cord Blood (UCB) St Louis Cord Blood Bank at SSM Cardinal Glennon Children’s Medical Center St Louis, Missouri (SLCBB)an evaluation of three uCb processing technologies was performed to compare product purity and potency as defined by characterization markers. the technologies were PrepaCyte-Cb (bioe, minnesota), aXP autoXpress Platform (ge Healthcare, new Jersey), and sepax (biosafe, switzerland). these technologies were compared to the manual Hespan method.PrepaCyte-Cb is a reagent based two-step manual method requiring centrifugation. the aXP is an optically controlled device using two-step centrifugation with operator interaction between steps. biosafe’s sepax instrument performs automated cell processing through centrifugation and optical sensor controlled separation.to maintain validity of the data comparison, uCb utilized in this study was harvested in 35 ml CPD anticoagulant, less than 48 hours old, had a minimum volume of 45 ml neat cord blood, and a minimum tnC of 0.9 x 10e9. Characterization analysis was performed on pre-processing, post-processing, and post-thaw samples. testing conditions and methodology were consistent for all samples. results are presented below.table 1: median Post Processing and Post thaw Comparisons

Post Processing CharacteristicsHesPan PrepaCyte-Cb sePaX aXP

tnC recovery (%) 86.0 84.0 83.0 78.0tmnC reovery (%) 85.5 83.5 84.0 90.5CD34+ x 106 3.2 4.1 3.5 2.2CFu x 105 12.0 10.3 11.7 8.7trypan blue (%) 94.0 97.5 98.0 95.07-aaD (%) 95.5 97.1 90.6 95.1Post thaw Characteristics

HesPan PrepaCyte-Cb sePaX aXPtnC recovery (%) 81.8 89.9 85.8 79.7tmnC reovery (%) 92.0 87.0 93.0 80.0CD34 recovery (%) 68.5 76.1 80.8 67.1CFu recovery (%) 52.9 80.2 62.7 47.0trypan blue (%) 68.0 72.0 73.0 78.07-aaD (%) 48.0 48.2 50.0 95.0

*n=10 for all processing methodsConclusion: Prompted by significant CFu and tnC recoveries post thaw, and minimum impact to operations and capital budget, the slCbb has initiated a trial with PrepaCyte-Cb.


Poster Abstracts

193AutOLOGOuS BOnE MARROW dERIVEd StEM CELLS tRAnSPLAntAtIOn FOR REtInItIS PIGMEntOSAR. C. Siqueira1,2, A. Messias3, J. C. Voltarelli3, I. Scott4, R. Jorge3; 1Retina Cell, São José do Rio Preto-SP, BRAZIL, 2Catanduva Medicine School, Catanduva, BRAZIL, 3São Paulo University, Ribeirão Preto-SP, BRAZIL, 4Department of Ophthalmology and Health Evaluation Sciences., Hershey, PA.

Purpose: to evaluate the short-term (10 months) safety of a single intravitreal injection of autologous bone marrow stem cells (abmsC) in patients with retinitis Pigmentosa (rP).methods: a prospective, phase i, nonrandomized, open-label study was carried out . the study was approved by the local and national institutional review board (Clinicaltrials.gov identifier: nCt01068561).Five patients with rP were included, age-gender: 31-F; 35-m; 23-F; 33-m; and 35-m. evaluations were performed at baseline, and 1, 7, 13, 18, 22, and 40 weeks after intravitreal injection of 10 x 106 autologous bone marrow stem cells (0.1 ml). Patients presented best-corrected etDrs visual acuity (bCVa) of 20/200 or worse at baseline. in addition to comprehensive ophthalmological examination, patients yielded: standard full field electroretinography (erg - isCeV standard); kinetic visual field (goldman); fluorescein and indocyanine-green angiography; and optical coherence tomography (oCt).results: no adverse side effect due to the injection was observed. a slightly improvement on bCVa (1 line) was measured in 4 patients 1 week after injection and kept during follow-up. three patients showed undetectable erg responses in all visits, while one patients showed residual responses for dark-adapted standard flash stimulus (a-wave amplitude around 35 µV), which was kept recordable during the entire follow-up. one patient showed a small response (a-wave amplitude around 20 µV) recordable only at weeks 7, 13, 22 and 40. Visual fields showed no reduction on visible area (goldman standard V5e) for any patient, at any visit. no other changes were observed whatsoever on oCt or fluorescein and indocyanine green angiograms.Conclusions: intravitreal injection of abmsC in eyes with advanced tapetoretinal dystrophies was followed by absence of detectable structural or functional retinal worsening over a period of 10 months. Further studies are required to investigate the role, if any, of bone marrow stem cell therapy for the management of retinal dystrophies.

195CELLuLAR thERAPy FOR REGEnERAtIVE MEdICInE: ACtIVIty In thE uS duRInG 2008M. C. Pasquini, S. Pirog, H. Baldomero, K. Soboncinski, A. Keating, J. Hare, K. Loper, M. Laughlin, E. Horwitz, H. Heslop; CIBMTR Cellular Therapy Working Committee, Philadelphia, PA.

the Center for international blood and marrow transplant research (Cibmtr) launched an initiative to develop a cellular therapy database and compile cases to study outcomes following cellular therapy for regenerative medicine (Ctrm). the first step of this initiative was to understand the activity of these therapies in the us. a total of 333 centers from 295 institutions in the us were surveyed after being selected among transplant centers involved with the Cibmtr (n=338) and centers with open clinical trials in Ctrm in 2008. the survey instrument distributed focused on center activity, disease indications, cell and donor type, and information on cell manipulation. a total of 22 responses related to 27 centers were received. six centers reported no activity for 2008 and one center reported pancreatic islet cell transplant activity. among 20 centers that replied, there were 126 recipients of Ctrm. the most common indication was cardiovascular diseases (n=68), with 44, 13, 7 and 4 patients receiving Ctrm for acute myocardial infarction, peripheral vascular disease, chronic coronary artery disease and congestive heart failure, respectively. neurologic indications included treatment of traumatic brain injury (n=4), chronic pain (n=3) and cerebral palsy (n=2). other indications included Crohn’s Disease (n=18), diabetes mellitus type i (n=8), wound healing (n=3), renal failure (n=18), retina repair (n=1) and traumatic joint injury (n=1). sixty-three percent of patients received an autologous product and among 108 patients with cell source information, 51, 34, 13 and 10 patients received unmanipulated bone marrow, mesenchymal cells, mobilized peripheral blood progenitor cells and umbilical cord derived cells, respectively. annual activity surveys of Ctrm are a resource to the community and demonstrate the indications and types of cells under investigation. this will also assist in the development of the cellular therapy registry for future studies of outcomes of cellular therapy applications.

194SuRGICAL GRAFtS FOR REPAIRInG ChOndRAL dEFECtSF. Lin1, S. Lee2, C. Yen3, H. Liu4; 1Institute of Biomedical Engineering, National Taiwan University, Taipei, TAIWAN, 2EMO Biomedicine, Taipei, TAIWAN, 3Center for Measurement Standards, Industrial Technology Research Institute, Hsinchu, TAIWAN, 4Department of Orthopaedic Surgery, National Taiwan University Hospital and Taiwan Adventist Hospital, Taipei, TAIWAN.

articular cartilage is difficult to heal after injury. a cell/matrix complex obtained by culturing mesenchymal stem cells (msCs) in a collagen medium, which can be easily filled in the chondral defect of the patient and provides an efficacy in repairing the full-thickness cartilage defects after transplantation.by following the gtP regulation in taiwan, bone marrow msCs were cultured in a collagen-based special culture medium for a sufficient time to obtain a cell-matrix complex which comprises chondrocyte-like cells secreting glycosaminoglycan (gag) but without lacunal. We implanted the tissue-engineered cartilaginous tissue on ten patients who had osteochondral defect from osteonecrosis on the medial femoral condyle.significant improvement was found 6 months and 12 months after operation. Four patients were followed up more than 12 months and received arthroscopy. the chondral defects were found to have been completely filled with cartilage without gap between the recipient site and the graft (Fig.1).Conclusion: stem-cell induced novel chondral graft has satisfactory healing effect for chondral defect which was caused by osteonecrosis.


may 23-26, 2010


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