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Hematopoietic stem cells:insights into bone marrow biology
ESH-EBMTLatimer 2009
Aleksandra Wodnar-FilipowiczDepartment of BiomedicineUniversity Hospital Basel
Switzerland
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Stem Cells - Definition:
Cells capable • to divide for indefinite period,• to self-renew, and • to give rise to specialized cells
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Hematopoietic
LYMPHOIDSTEM CELL
T-CELL
B-CELL PLASMA CELL
ERYTHROCYTE
NEUTROPHIL
MONOCYTE
EOSINOPHIL
BASOPHIL
MEGAKARYOCYTE
MYELOIDSTEM CELL
BFU-E
CFU-GM
CFU-Eo
CFU-Baso
CFU-Meg
MACROPHAGE
PLATELETS
Totipotent Stem CellZygote
Pluripotent Stem CellEmbryonic Stem (ES) Cells
Multipotent Tissue Stem Cells
Neural Muscle EpidermisLiver
Embryonic und adult tissuestem cells
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Retina
CancerCancer Stem CellsStem Cells
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Adult tissuestem cells
Developmental Potential of Stem Cells
Developmental potential
Specialization
Embryonicstem cells
Zygote
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“Trans-differentiation”
“De-differentiation”
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CancerCancerStem CellsStem Cells
Embryonicstem cells
Adult tissuestem cells
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1. Characterisation of bone marrow - resident hematopoietic stem cells
Phenotype Functional assays Stem cell niche Leukemia stem cells
2. Stem cell research and challenges of regenerative medicine
ES cells iPS cells Plasticity of bone marrow - resident stem cells Therapeutic outlook - 2009
Presentation outline:
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Hematopoietic stem cells
Mouse
PhenotypeLin-
sca1+
ckit+
tie2+
flk2-
CXCR4+
Abcg2+
SLAM-Rs:CD150+CD244-CD48-
TLR4+
LSK cells0.007%of BM
c-ki
t
BL6
%
LSK cells:
100 101 102 103 104SCA1-Height
R3
sca-1
c-ki
t 0.007%
Human
PhenotypeLin-
CD34+ (CD34- ?)CD38-
ckit+
flk2- (flk2+ ?)CD133+ (?)CXCR4+
ABCG2+
?
CD34+CD38- cells0.1%of BM
Hoe
chst
Blu
e
Hoechst Far Red
Side population (SP)(ABCG2+transporter)
Hoechst far red
Hoe
chst
blu
e
0.2%SP
The phenotype of human HSC remains poorly defined
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Function
• In vitro CFU assays• In vivo transplantation:
xenografts in immunologically permissive mouse strains (e.g. ..NOD/SCID; NOD/SCID-c-/-; Rag2-/-c-/-)
Human hematopoietic stem cells
Clinical transplantation
1º 2º 3º
BMT BMT
humanCD34+cells(i.v. or i.f.)
SRC
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CD45ISO FLCD45
CFSE/ DAPI/ OPN
BONE
BM
endosteum
os
HSC
Wilson et al.2004
Labeled HSC in the bone marrow niche Cytokine expressionin the bone marrow niche
In situ analysis of bone marrow “niche”
Tie2+CD45+ cells(yellow)adhere to the endosteal surface
Arai et al. 2004
Cytokine receptor expression in the bone marrow niche
CD45/Flt3 ligand
Kenins & Wodnar-Filipowicz 2005
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Bone marrow “niche”2009
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Endosteal Endosteal nicheniche
Precursor cells
Self-renewal
MigrationProliferation
Differentiation
Cellular components of stem cell niches in the adult bone marrow
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Trabecular bone
HSC
Sinusoid endothelial cells
MSC
MSC
MSC
HSC
Osteoblasts
Osteoblasts - osteogenic cells lining the inner surface of the bone
CAR cells - (CXCL12 abundant reticular) cells close tothe bone surface and endothelium
CARcells
Stroma fibroblasts
Vascular Vascular nicheniche
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Molecular interactions in the bone marrow nicheExtrinsic mechanisms:• cytokines• chemokines• adhesion molecules• negative regulators (osteopontin)• proteases (MMP-9, cathepsin K)• hormones (PTH, PGE2)• sympathetic nerves• oxygen status• calcium concentration• circadian rhytm
HSC
cell-celladhesionmigration
quiescenceself-renewalexpansion
Ang-1 - tie2SCF - c-kitFlt3-L - Flt3TPO - mplWnt - Frizzled Jagged/Delta - Notch
Cadherins VCAM - VLAICAM - LFACXCL12 - CXCR4HA - CD44
Gene expression
OSTEOBLASTPTH, PGE2PTH, PGE2 nervesnerves
Intrinsic mechanisms:• signalling molecules
• cytoplasmic• nuclear, incl.
transcription factors• epigenetic mechanisms
• DNA/histone modif.• microRNAs
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Leukemia Stem Cells
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Normal stem cells and cancer cells share the ability to self-renew and many signaling pathways involvedin the regulaton of normal stem cell development are mutated or epigenetically activated in cancer.
Leukemia stem cells:Transformed hematopoietic stem or commited progenitor cellsthat have amplified or acquired the capacity for self-renewal, albeit in a poorly regulated fashion.
Cancer stem cells
HSC
Prog.HSC
LSC
LSC
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Human AMLperipheral blood
Leukemia stem cells: 1st identified cancer stem cells
SL-ICCD34+CD38-
CD34- ?CD33+ ?CD44+ ?CD71+ ?CD96+ ?CD117- ?CD123+ ?CD133+ ?CLL-1+ ?
NOD/SCID
Phenotype Function
CD34+CD38-
not CD34+CD38+
(?)
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Functional analysis of LSCs
AMLcells
BM BM
1o 2o 3o
FACS:humanCD45+
cells
iv
if
Non-injected bones Injected femur Peripheral blood
2 weeks 1o CFU 2o CFU 3o CFU
AMLcells
CD45
CD
33
Langenkamp &Wodnar-Filipowicz2008
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• resistant to conventional treatment• lead to relapse and fatal outcome
Conventional treatment
Elimination of LSC
Relapse
Leukemic cell growth
Degeneration
Terminal differentiation
Remission
Remission
Leukemic stem cells
Challenge in stem cell biology/stem cell-based therapies:understanding the molecular and the functional programmes of leukemic vs normal stem cells
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CrossCross--talk in the AML niche in the bone marrow ?talk in the AML niche in the bone marrow ?
Bone marrow stromal cells are important for the maintenance of normal HSCs (cell cycle, differentiation, proliferation)and of AML-LSCs(CD44-dependent interactions) Van Etten et al. Nat Med 2006
CFSE/ DAPI/ OPN
Wilson et al. Genes & Dev 2004
boneBM
endosteum
os
HSC
HSCBM stromaBM stroma
LSCor
Transition of SDF-1- to SCF - expressing environment, which disrupts the behaviour of normal HSC/progenitorsColmone et al. Science 2008
Bone marrow stromal cells can in turn be influenced by leukemic cells
Is the stem cell niche in leukemia normal or not ?“Seed or soil” or both ?
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Self-renewal mechanism of stem cells
Normal stem cell Cancer stem cell
Mathematical model:Skipping assymetric division every 4-6 divisions issufficient for the tumor to grow.
80%asymmetric
80%symmetric
stem cell number:
stable
increased
s. as.
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1. Characterisation of bone marrow - resident hematopoietic stem cells
Phenotype Functional assays Stem cell niche Leukemia stem cells
2. Stem cell research and challenges of regenerative medicine
ES cells iPS cells Plasticity of bone marrow - resident stem cells Therapeutic outlook - 2009
Presentation outline:
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Generation of ES cell lines from human blastocysts
Donated IVFblastocyst
Inner cell mass „immortal“cell lines
Thomson et al. Science 282:1145 (1998)
Somatic differentiation in vitro into various tissue types
CFU’s
Kaufman et al. PNAS 98:10716 (2001)
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„Therapeutic Cloning“ (SCNT)
Immunologically-compatible tissue for transplantation
Human ES cells with patient’sgenetic information
Enucleated donor oocyte
Nuclear-transfer From adult cell
Fusion
ES cell lines
Blastocyst
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Human iPS cells (induced pluripotent stem cells) from adult dermal fibroblasts
Takahashi et al. Cell 131:1 (2007)Yu et al. Science 318:1917 (2007)
Zhou et al. Cell Stem Cell 4:381 (2009)
Oct3/4Sox2c-mycKlf4
iPS (piPS)ES-like cells
e.g.neural cells,
heart muscle cells
EctodermMesodermEntoderm
Differentiation into cells of all germ layers:
Oct4Sox2NanogLin28
± 20 days
Human ES morphology Normal karyotype Telomerase activity Cell surface markers of ES cells Gene expression profile of ES cells
fibroblasts
Fusion-proteins
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Adult bone marrow - derived stem cells
for organ regeneration and repair
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Cell type
Hematopoietic stem cells (HSC)
Mesenchymal stem cells (MSC)
Hemangioblasts (EPC)
[Multipotent adult progenitor cells (MAPC)]
Bone marrow - resident stem cells
Differentiation potential
blood cellsother tissues = plasticity (?)
bone, cartilageadipose tissue, muscleother tissues = plasticity (?)
endotheliumblood cells, muscle
multilineage
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Adult stem cell plasticity by “trans-differentiation”
Ability of tissue-specific stem cells to acquire,under defined microenvironmental conditions,the fate of cell types different from the tissue of originand belonging to all three germ layers,i.e. similar to the differentiation ability of ES cells.
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HSC
Muscle (Heart) contamination
Hepatocyte (Liver) cell fusion
Neural (CNS) not reproducible
Adipocytes not reproducible
= BM-HSC remained true to their roots
Criteria to prove adult stem cell plasticity by “trans-differentiation”:
- initiated by single self-renewing stem cell- generation of all functional cell types of tissue of originand unrelated tissue
- robust repopulation of tissue of origin and unrelated tissue in vivo
have not been fulfilled in the existing studies.
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BM
CardiacVascularLungBrain stroke, Paraplegia, ALSBreast cancerRetina degenerationCartillage degenerationOsteogenesis imperfecta GvHD
BM tissue regeneration (clinical trials)
G/GM-CSFmobilization
BM (MNCs, CD34+) autologous
Diseases
MSC-infusionsallogeneicautologous
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d.0-d.14 w.2-w.8 mo.3-mo.9 after birth
Pre-embryo Embryo Fetus Adult
All duplicated if starting from diseased cells
8 cell(blastomeres)
32 cell(blastocyst)
d.1-d.2 d.5 d.8-9
Implantationin utero
Stem cells: Key words
Parthenogenesis(chemical treatment)
Oocyte+
Sperm Zygote *
Yolk sac *Placenta *Amniotic fluid *EG cells *
Epiblast *AGM region *
Extraembryonic tissueEmbryo
animal oocyte(chimerae)
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Nuclear transfer(SCNT)
± Genetic modifications
CordBlood *
BoneMarrow *
Fetal Liver *
Adult cells
iPS *
Gene transfer(reprogramming)
Natural plasticity of adult cells ??
Cell fusions (adult/adult; adult/ES)
trophoblastES cells *= ICM
ES cell lines *
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2009: Prospects for stem cell-based therapyof degenerative diseases
Stem cell choice:• adult (multipotent) or embryonic (pluripotent) • “off-the-shelf” from master-stocks (= cell banks) or “customized” for ... ......individual patientsStem cell number:• increase homing and engraftment in tissue of choice• develop drugs that augment the endogenous stem cell poolsStem cell immunogenicity:• generate isogenic (genetically equivalent) cells:
• iPS cells • ES cells after nuclear transfer (“therapeutic cloning”) • parthenogenetic embryos as ES cell source (pES)
• overcome the immune response: engineer cells deficient in class I ...and II HLA genes/NK ligands or antagonizing immune responsesStem cell gene therapy: ES/iPS terato/cancerogenicity:• control insertional mutagenesis ?
ES iPSHSCMSC
NSCSCNT
CB-HSC
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Literature - Reviews:
Stem cells - all aspects:Issue of Cell 132; Feb 22 (2008)
HSC and niches:Kiel & Morrison, Nature 8:290-301 (2008)Adult stem cell (general) niches:Jones & Wagers, Nature 9:11-21 (2008)
Cancer stem cells - targetingTrumpp & Wiestler, Nature Clinical Oncol 5:337 (2008)Leukemia stem cellsChan & Huntly, Seminars in Oncology 35:326 (2008)
Plasticity:Scadden DT, J Clin Invest 117:3653-3655 (2007)
Deconstructing Stem Cell Tumorigenicity:A Roadmap to Safe Regenerative Medicine.Knoepfler PS, Stem Cells 29:1050-1056 (2009)