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Stem Cells and Regenerative Medicine
Geoffrey C. Gurtner, MD, FACSStanford University School of Medicine
Lucile Salter Packard Children’s Hospital
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Human Response to InjuryHuman Response to Injury
Regeneration
Injury
Repair with Scar
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Human Response to Injury Human Response to Injury
Injury
Regeneration
Repair with Scar
Regenerative Medicine
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Is Regeneration Possible?Insights from Developmental Biology
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Regenerative MedicineRegenerative Medicine
STEM CELLS
TISSUE ENGINEERING
REGENERATIVE MEDICINE
Nanosciences/devices
Developmental biology
Material Science
Bioengineering
Genomics
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What is a Stem Cell?What is a Stem Cell?
1. Cells that can be grown indefinitely (self renewal)
2. Can be differentiated into different specialized cells
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Stem vs. Progenitor CellStem vs. Progenitor Cell1. Stem Cell
• Asymmetric Division to produce itself (self renewal)
• Differentiate into specialized daughter cells
2. Progenitor Cell• Cannot self renew• Differentiate into
specialized daughter cells
http://www.nih.gov/news/stemcell/scireport.htm
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Stem Cells: From Embryo or AdultStem Cells: From Embryo or AdultTotipotent: stem cells capable of giving rise to an intact organism
Pluripotent: stem cells capable of giving rise to cells of all (3) germ layers
Multipotent: stem cells capable of giving rise to cells that comprise a germ layer or organ
http://www.nih.gov/news/stemcell/scireport.htm
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Different Stem Cell Types
? Immunogenic, pluripotent
Pluripotent
Immunogenic, ? Pluripotent vs
multipotent
Immunogenic,multipotent
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Embryonic Stem Cells
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Embryonic Stem CellsEmbryonic Stem Cells
•Derived from inner cell mass of blastocyst
•Pluripotent
•Can differentiate into all adult cell types
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Human Embryonic Stem CellsHuman Embryonic Stem Cells
• Human ESCs can differentiate into myocardium
• Human ESCs can produce insulin secreting endocrine cells
Eur J Cardiothorac Surg. 2006 Jan;29(1):50-5
Nat Biotechnol. 2006 Oct 19
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Human Embryonic Stem CellsHuman Embryonic Stem Cells• 22 NIH-registered human embryonic stem cells
available• No current human trials with embryonic stem
cells
• Primary current enthusiasm is to use as disease models for diseases such as ALS
Blastocyst Trophoblast cells removed
Inner Cell Mass Human ESCs
HF Chen et al. Hum Reprod. 2006 Oct 27
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California Institute for Regenerative Medicine (CIRM)
• Proposition 71 established "California Institute for Regenerative Medicine" to regulate stem cell research and provide funding through grants and loans.
• Established constitutional right to conduct stem cell research; prohibited funding of human reproductive cloning research.
• Authorized issuance of general obligation bonds to finance Institute activities up to $3 billion subject to annual limit of $350 million.
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Problems with Embryonic Stem CellsProblems with Embryonic Stem Cells
• Cells are immunogenic
• Difficult to control and differentiate in vivo, possible tumorgenicity
• NIH approved lines are maintained on murine feeder cells for necessary ex vivo expansion
• Composition of growth media, factors, conditions for culture unclear
• Ethical and religious
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““EmbryonicEmbryonic--likelike”” Stem CellsStem Cells
• Amniotic sac derived stem cells contain oct-4 and nanog like ES cells
• Cells not immortal, but can be differentiated (Stemnion, Inc.)
• Similar claims made for placenta derived mesenchymal cells (Stemcell Pharma, Inc.) and cord blood
• Unknown immunogenicity
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Embryonic Stem Cells
• We know all the genes that are upregulated in embryonic stem cells
• What happens if we turn on these genes in normal (non-stem cells) using gene therapy?
• This approach is called epigenetic reprogramming of somatic cells
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InducedInduced Pluripotent Stem (iPS) Pluripotent Stem (iPS) CellsCells
• 22 stem cell genes added to skin fibroblasts using retroviruses
• Results in embryonic stem cell-like cells
• Teratomas are formed in vivo
• In vitro differentiation into all 3 germ layers
Takahashi and Yamanaka. Cell 126, 2006.
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Confirmation and RefinementTransduction with only four genes: Oct3/4, Klf4, Sox2, c-Myc (Okita, et al. Nature 448(19), 2007) or…Activation of endogenous Oct4 or Nanoggene (Wernig, et al. Nature 448(19), 2007)Results in iPS cells whose DNA methylation pattern, gene expression, and chromatin state are identical to ES cells
.
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Further Characterization of iPS CellsFurther Characterization of iPS Cells
• Proliferation similar to ES cells, with slightly longer doubling times
• Transplantation into nude mice resulted in teratomas with all three germ layers
Okita, et al. Nature 448(19), 2007.
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Reprogrammed Fibroblasts Yield Viable Chimeras
• Can function as pluripotent stem cells
• GFP labeling shows high contribution to pup development
Wernig, et al. Nature 448(19), 2007.
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The Promise• Reprogrammed iPS cells are nearly
indistinguishable from ES cells
• iPS cells develop into a wide variety of tissues, just like embryonic stem cells
• iPS cells contribute to the germ line and yield viable transgenic animals
• Can normal fibroblasts transduced with a handful of genes replace embryonic stem cells?
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The Reality
Okita, et al., report that 20% of iPS cell-derived mice developed cancer, probably from Myc activation
Retroviral vectors have previously caused leukemia in human gene therapy trials
Thus clinical application is a ways off, but at least we are beginning to understand “stem-ness”
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Adult Bone Marrow Stem CellsAdult Bone Marrow Stem Cells
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Adult Bone Marrow Derived Adult Bone Marrow Derived Stem CellsStem Cells
• Hematopoietic Stem Cells (HSCs)
• Bone Marrow Derived Mesenchymal Stem Cells (BM-MSCs)
• Endothelial Progenitor Cells (EPCs)
• Cord Blood Derived Stem Cells
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Hematopoietic stem cellsHematopoietic stem cells
1. Portion of the bone marrow that forms RBCs, WBCs, etc.
2. Unlike other putative stem cells, discrete markers have been identified (SCA1+, lin-, and c-Kit+, Flk2+/Thy1.1lo ) allowing a pure population of these cells to be isolated and studied
3. True stem cells, able to self-renew and differentiate
Reya T, Morrison SJ, Clarke MF, Weissman IL., Nature 2001;414:105-111
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Established Clinical ApplicationsEstablished Clinical Applications
• 40 year track record of success• HSC harvested from bone marrow or
cytokine mobilized peripheral blood• Treatment of leukemia, blood
dyscrasias, mucopolysaccharidoses and cancer
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Other Proposed Applications of Hematopoietic Stem Cells
• HSCs have been purported to differentiate into myocardium, endothelium, small intestine, skeletal muscle, lung, and liver
• Most of these reports are now being attributed to cell fusion• Cell fusion is being explored for therapeutic purposes
PNAS 2006;103;13156-13161
Nature Medicine 2004: 10, 744 - 748
HSC to endothelial cell
HSC to hepatocyte
PNAS 2006;103;6321-6325
HSC to intestinal epithelium
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Bone Marrow Derived Mesenchymal Stem Cells
• No widely accepted Markers
• Exist within the lin-, CD45-, Sca-1+ fraction of the bone marrow
• Ability to mobilize from bone marrow and home to injured tissue remains controversial
http://www.nih.gov/news/stemcell/scireport.htm
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Characterization of Bone MarrowCharacterization of Bone Marrow
HSC:Lin-Sca+Ckit+CD34+ (human)CD45+Thy1.1+
Shizuru, et al, Ann Rev Med, 2005
MSC:Lin-CD14-CD45-CD29+CD90+CD95+CD166+
Wang, et al, Stem Cells, 2005
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Bone Marrow Mesenchymal Stem CellTherapy of Myocardial InfarctionTherapy of Myocardial Infarction
• BM MSC injected intra-arterially increase vasculogenesis in mycardium
• BM MSC increase perfusion and viability of myocardium
Nature Medicine. 2001 Apr;7(4):430-6
Circulation. 2002 Oct 8;106(15):1913-8
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““SideSide”” Population Marrow Cells Population Marrow Cells for Wound Healingfor Wound Healing
• Topical application accelerated wound healing in diabetic mice
• Side population defined as c-kit+, lin-, sca-1+, CD 34-
• Orgill et al, PRS, in press
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Cord Blood Stem CellsCord Blood Stem Cells
• Obtained from blood retrieved from the umbilical cord and placenta at the time of birth
• Isolated by Fluorescence Activated Cell Sorting (FACS)
• Infused via an IV and find their way to the bone marrow, “stem cell homing”
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Established Clinical Applications Cord Blood Stem Cells
Autologous Transplant• To date over 6,000 cord
blood transplants have been performed
• In the US ½ of all pediatric marrow transplants from unrelated donors come from cord blood
• In Japan, this is true for adults as well
Dana-Farber Cancer Institute
Allogenic Transplant
Johns Hopkins
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Other Proposed Uses of Umbilical Cord Stem Cells
Medical Hypothesis 2006
Circulation 114 (Suppl.1) 125-131, 2006
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“Overall, the results of these studies of a combined total of 376 patients do not promote the use of intracoronary infusions of autologous bone marrow to improve ventricular function”
NEJM September 21,2006
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The only established clinical The only established clinical application for hematopoietic application for hematopoietic stem cells or cord blood stem stem cells or cord blood stem
cells is in the treatment of blood cells is in the treatment of blood disorders and cancerand cancer
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Adult Tissue Derived Stem CellsAdult Tissue Derived Stem Cells
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Adult Tissue Specific Stem CellsEctoderm
epithelial stem cellEndoderm
intestinal crypt cell
Spradling A, Drummond-Barbosa D, Kai T. Stem cells find their niche. Nature 414, 98-104, 2001
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Adult Tissue Specific Stem CellsAdult Tissue Specific Stem Cells
• Neural Stem Cells
• Adipose Derived Mesenchymal Stem Cells (AdipoMSCs)
• Skin Stem Cells
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Neural Stem CellsNeural Stem Cells• Subventricular zone along lateral ventricles of
human brain have greatest density of NSCs
Sanai N. N England J Med. 2005 Aug 25;353(8):811-22
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Neural Stem CellsNeural Stem Cells
• Progenitor cells that can differentiate to neurons or glial cells
• Neural stem cells have also been implicated in development of gliomas
• Bone morphogenetic proteins (BMPs), EGF and FGF-2 implicated in neural stem cell differentiation
Sanai N. N England J Med. 2005 Aug 25;353(8):811-22
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NeurogenesisNeurogenesis• Progenitor neural stem cells can potentially
mature into neurons and support axonal growth• Potential application in neurodenerative
disorders ie. Alzheimer’s
Steindler DA. Glia. 2006 Dec;54(8):815-25
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Proposed Clinical Application: Proposed Clinical Application: Spinal Cord InjurySpinal Cord Injury
•The adult central nervous system has a limited capacity to regenerate after injury
•Neurogenesis occurs at sites of injury but is very inefficient
•No human trials yet due to concerns regarding source and safety of neural stem cell transplants Journal of Neuroscience21(23):9334-9344, 2001.
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Adipose Derived Mesenchymal Stem Cells (AdipoMSCs)
Suction-assisted lipectomy(elective cosmetic surgery patients)
Raw lipoaspirate
Extensive washingproteolytic digestion
(collagenase)
Collection ofcells = PLA
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In Vitro Differentiation of AdipoMSCs
OsteogenicMyogenic
AdipogenicNeurogenicChondrogenic
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Proposed Clinical Application:Proposed Clinical Application:Bone RegenerationBone Regeneration
Bioengineered Scaffolds
Regeneration
AdipoMSCs
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AdipoMSCs Form Bone in Modified Standard Media
Von Kossa
at 28 days
Alkaline Phosphatase
at 7 days
Control Media
ODM
D E F
A B C
ODM + RA + BMP-2
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Nature Biotechnology May, 2004
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Case ReportCase ReportAdipose derived stem cells harvested and injected into calvarial defect (Lendeckel S et al. Journal of Cranio-
Maxillofacial Surgery 2004: 32, 370–373.)
Pre Operative
Post Operative
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Proposed Clinical Application for Proposed Clinical Application for AdipoMSCs: Soft Tissue AugmentationAdipoMSCs: Soft Tissue Augmentation
• Historical basis in fat grafting which has been performed in plastic surgery for over a hundred years
• Enormous interest in identifying the ideal soft tissue “filler” for cosmetic and reconstructive applications
• Ideal filler would be integrated, vascularized and non-immunogenic
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QuestionsQuestions1. What are we injecting?
Adipocytes, Fibroblasts, Smooth Muscle Cells, Perivascular Cells, Endothelial Cells, Stem Cells
2. What happens after we inject it?
3. What impact does this have on tumorigenesis in the breast?
?
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Adipose Tissue Contains Multipotent Cells
• Limiting Dilution Assay and Cloning Rings used to isolate 500 cell clones
• 7 of 500 clones (1.4%) demonstrated in vitrodifferentiation along 3 mesodermal lineages• Osteogenic• Adipogenic• Chondrogenic
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PBS WashCollagenase Digest
Culture ofAdherent Cells
ADIPOGENESIS OSTEOGENESIS CHONDROGENESIS
Alcian Blue StainOil Red O Stain Alizarin Red Stain
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Different Processing:Different Processing:LABORATORY ISOLATION: 1. Collagenase digestion2. Red cell lysis3. Centrifugation4. Plating and Culture
1. CLINICAL ISOLATION: 2. Lipoaspirate undergoes
centrifugation 3. Separate into oil, adipose,
and red cell layers
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Fat grafting performed by current techniques is not predominantly stem
cell transfer
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Survival of Implanted CellsSurvival of Implanted CellsAngiogenesis• Diffusion is unable to sustain
cells at a distance of greater then 2mm from a blood vessel
• Recent advances in fat grafting have probably increased cell viability
• Still unclear what the optimal injection volume per pass is to maximize cell viability
• Any non viable cells can result in calcification
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Commercialization of Adipose “Stem Cell Isolation” for Fat Transfer
The Celution™ System makes cells available in about one hour and these cells should be enriched for MSCs
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Duality of Stem CellsDuality of Stem Cells
Tissue Regeneration Tumorigenesis andCancer Stem Cells
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In Animal Models, Mesenchymal Stem Cells Increase Breast Cancer Metastases
The authors show that human breast cancer cell lines become metastatic when in physical contact with mouse mesenchymal stem cells.
Using subcutaneous xenografts of human and mouse cells combined, Karnoub et al. show increased metastases to the lung while control grafts having human or mouse cells alone did not result in metastasis.
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Even without stem cells, tissue damage to leads Even without stem cells, tissue damage to leads to the release of tumorigenic factorsto the release of tumorigenic factors
• Published techniques delivering 0.2 to 0.02 cc per “pass” require up to 13,900 “passes” to deliver the average volume (278cc) over an 4-6 hour period.
• Fat grafting is traumatic and results in the deposition of a disorganized mixture with mechanical damage, inflammation, and necrosis.
• Fibroblasts activated by surgical trauma and infiltrating immune cells secrete cytokines that have been shown to potentiate tumor growth including TGF-β, FGF-2 , and PDGF (Sieweke, Science, 1990; Blobe, N. Engl. J.Med. 2000; Skobe, Proc. Natl Acad. Sci. 1998).
• Specific to breast tissue, Lochter (J. Cell Biol. 2000) demonstrate inflammation specific activation of matrix proteases are involved in breast cancer progression.
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AdipoMSCs for Soft Tissue AdipoMSCs for Soft Tissue AugmentationAugmentation
Plastic and Reconstructive Surgery, Supplement 4, 2006
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Other Proposed Applications of Other Proposed Applications of AdipoMSCsAdipoMSCs
• To increase vascularity in cardiovascular and wound healing applications
• As substitutes for neural progenitor cells • Advantages are ease of harvest and
excellent yields
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Skin Stem Cells and Skin Stem Cells and Wound HealingWound Healing
*modified from Singer & Clark, NEJM, 1997
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Endogenous Sources of Stem/Progenitor Cells During Wound Healing
Blood Borne,(EPC, HSC)
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Not a new ideaNot a new idea……
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Epidermal Multipotent Stem CellsEpidermal Multipotent Stem CellsMultipotent epidermal stem cells reside in the bulge (Bu) zone of hair follicle (HF)
Blanpain C, Lowry WE, Geoghegan A, Polak L, Fuchs E. Cell, Vol. 118, 635–648, September 3, 2004,
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Multipotent epidermal stem cells Multipotent epidermal stem cells can differentiate into:can differentiate into:
1. epidermis2. sebaceous gland 3. hair follicles
Transplanted Bulge Hair Regeneration
Blanpain C, Lowry WE, Geoghegan A, Polak L, Fuchs E. Cell, Vol. 118, 635–648, September 3, 2004,
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Sebaceous Gland Sebaceous Gland RegenerationRegeneration
• Blimp1+ cells responsible for sebaceous gland regeneration and controls the size, activity, and number of sebocytes Blimp1+
SG
Fuchs E et al. Cell. 2006 Aug 11;126(3):597-609
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Epidermal Stem Cells and Epidermal Stem Cells and Wound HealingWound Healing
• Bulge epidermal stem cells migrate to healing wounds and are involved in re-epithelialization after injury
• Are eventually eliminated as wound heals
Cotsarelis G et al. Nature Medicine. 2005 Dec;11(12):1351-4
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Hair Follicle Regeneration During Wound Healing
Ito, et al. Nature 447(17), 2007.
• De novo hair regeneration occurs after wound closure in mice
• Histology shows that regenerating hair follicles mimic stages of normal embryonic hair development
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Regenerated Hair Originates from Interfollicular Stem Cells
Genetic lineage analysis using transgenic mice:
1) Tg(Krtl-15-cre/PGR)22Cot;R26R LacZ + only in bulge cells (arrow)
Only TRANSIENT presence of bulge cells in regenerated hair follicles
2) Krtl-15-CrePR*LacZ + in 70% bulge and 50% non-bulge epidermal cells (red arrowhead)
Hair follicles are chimeric, indicating multiple progenitor cells are involved
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Hair Follicle Development Requires Wnt Protein
KRT17
• Regenerated follicles parallel embryonic follicle development at molecular level:
• KRT17, alkaline phosphatase, Lef1, Wnt10b, Shh Alkaline
phosphatase
• Secreted DKK1, a Wnt inhibitor, blocks follicular neogenesis
• Overexpression of secreted Wnt7aresults in significant follicular neogenesis
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The Bottom Line• New hair follicles can be formed following
wound formation in mice.
• Wnt signaling plays an important role in hair follicle neogenesis.
• It may be possible to regrow hair follicles in humans using local trauma (dermabrasion) and wnt signaling.
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Skin Regeneration Using Autologous Stem Cells
Nature 414, 98-104, 2001
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Unanswered Questions and Unanswered Questions and Obstacles Obstacles
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Do stem cells incorporate into Do stem cells incorporate into tissues or do they function as tissues or do they function as cytokine/growth factor delivery cytokine/growth factor delivery
vehicles?vehicles?
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The answer to this question is critical The answer to this question is critical for the problem of immunogenicityfor the problem of immunogenicity
1. With investigation, most stem cells and their progeny have been found to be immunogenic
2. Thus will need HLA matching and/or immunosuppression if they are permanent cell replacement
3. However if they are not permanent this will be less of an obstacle
www.osiris.com
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Therapeutic Cloning to Avoid Therapeutic Cloning to Avoid ImmunogenicityImmunogenicity
http://stemcells.nih.gov/
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Secondary Questions
1.1. If these are functioning as cytokine/growth If these are functioning as cytokine/growth factor delivery systems, is there an factor delivery systems, is there an advantage for stem cell based therapy over advantage for stem cell based therapy over differentiated cell based therapy?differentiated cell based therapy?
2. If they are incorporating, hohow can we control the differentiation of cells in vivo to produce a physiologic effectwhile preventing tumor creation?
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How can we isolate and purify rare stem cell subsets in the absence of discrete markers?
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In the case of EPCs what we know so far is:– Resident bone marrow cells– Respond to ischemic stimuli– Mobilize into the circulation– Differentiate into mature
endothelial cells in new blood vessels
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What is an What is an ““EPCEPC””??1. Markers and
precise identification currently unclear
2. Nomenclature and identification in the literature inconsistent
3. Are circulating cells identical bone marrow resident cells?
4. Are one or many different cells involved?
Authors Marker Profile Origin Species
Asahara et al. Science (1997) CD34+/ KDR (VEGF-R2) Blood Mouse
Kalka and Asahara PNAS (2000) CD31/VE-Cadherin Blood MouseKDR/LDL-R/UAE-1 R
Kawamoto et al. Circ. (2001) CD31/UAE-1 binding Blood Human
Walter et al. Circ. (2002) Tie2? BM Mouse, RatHuman
Takahashi et al. Nat Med. (1999) CD31/Tie2/Sca1 BM Mouse
Dimmler et al. Circ. Res. (2001) CD34/KDR Blood Human
Rauscher et al. Circ. (2003) CD31/CD-45_ BM Mouse
Asahara and Isner Stem Cells (2004) Flk-1/AC133/CD34 BM? HumanTie2/c-Kit/Sca-1
Tie2/VE-cadherin BM MouseCD34/CD31
Chan and Verma Transl. Physiol. (2005) CD34/VE-cadherin Blood Human
Gurtner et al. Nat Med. (2004) CD34+/Flk1+/CD133+ Blood HumanCD11b-
Blood (2005) CD34+/Flk1+/sca-1+ Blood, BM Mouselin-/CD11b-
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Embryonic Vasculogenesis
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What is the What is the ““EPCEPC”” Lineage?Lineage?
Hemangioblast
HSC
EPC
Hemangioblast
HSC EPC
Hemangioblast
HSC EPC
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Regulatory Issues: FDA Guidelines Regulatory Issues: FDA Guidelines for Stem Cell Therapyfor Stem Cell Therapy
N Engl J Med. 2006 Oct 19;355(16):1730-5
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Can stem cells overcome a Can stem cells overcome a hostile environment to produce a hostile environment to produce a
physiologic effect? physiologic effect?
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Stem Cell Mediated Regeneration: Importance of Seed and Soil
Seeds
Soil Growth
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Seed and Soil in Stem Cell BiologySeed and Soil in Stem Cell Biology
Soil
Seed-stem cell-resident cell
Soil
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Regenerative MedicineRegenerative Medicine
STEM CELLS
TISSUE ENGINEERING
REGENERATIVE MEDICINE
Nanosciences/devices
Developmental biology
Material Science
Bioengineering
Genomics
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Tissue Engineered BladderTissue Engineered Bladder
Lancet 2006Biodegradable matrix (PLGA)
seeded with bladder smooth muscle cells from biopsy
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Integration and Colonization by Integration and Colonization by Native UrotheliumNative Urothelium
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Dermal SubstitutesDermal Substitutes
•Alloderm, MTF, Dermamatrix
•Acellular dermal matrix from cadavers
•Will also revascularize
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Breast Contour AbnormalitiesBreast Contour Abnormalities
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Implant Positioning and Implant Positioning and CoverageCoverage
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Less inflammatory response in Less inflammatory response in implant apsulesimplant apsules
No Matrix Matrix
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Prefabrication of Synthetic Matrix Prefabrication of Synthetic Matrix with a Vascular Pediclewith a Vascular Pedicle
Neumeister, PRS, 2007
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ConclusionsConclusions
• Regenerative Medicine and Tissue Engineering are already here and will play an increasing role in clinical practice
• New products will combine regenerative cells and the proper environmental cues for maximal effect
• New advances in developmental biology may make personalized stem cells a reality in the next 5 years