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HEMATOPOIETIC STEM CELLS
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Fights infection
Transports oxygen
Critical for clotting
Function of the hematopoietic system
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Function of hematopoietic system
T cellB cell
Macrophage
Granulocyte
Erythrocyte
Megakaryocytes
Cell Types Function
Antigen specific cell killingAntigen specific antibody production
Phagocytosis/antigen presentation
Innate immunity
Oxygen transport
Blood Clotting
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HEMATOPOIETIC ORGANS
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HEMANGIOBLAST
Evidence for common precursors from chick embryos (dye labelling)
From ES cell derived embryoid bodies
Mouse data is more controversial
Embryonic beginnings of HSCs
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Embryonic beginnings of HSCs
Where does the long term intrembryonic hematopoiesis come from?
Initial data for intraembryonic site of hematopoiesis from chick quail chimeras
YS and AGM contribute to Fetal liver hematopoiesis
AGM HSC may arise independently of YS
AGM has more HSC than YS, but both can contribute to hematopoiesis
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TIMELINE OF HEMATOPOIETIC DEVELOPMENT
YS
AGM
FL
NS
BM
e6 e8
BIRTH
e14e10 e12 e16 e18 1 weee20
Thymus
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CMP
MkP
ErP Red Cells
Macrophages
GMP
MEP
HematopoieticStem Cell
LongTerm
ShortTerm
Multipotentprogenitor
CLP
B Cells
T Cells
NK Cells
Dendritic Cells
Pro-B
Pro-T
Pro-NK
Granulocytes
Platelets
Hematopoietic Development
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Pluripotent
Stem Cell
TdT
CD19 HLA-DR
TdT
CD19HLA-DR
Early
Pre-B Cell
Ig
CD10
TdT
CD19HLA-DR
Ig
CD10CD20
Immature
B Cell
sIg DHJH
RearrangedVHDHJH
Rearranged
B cell development
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Myeloid Cell Development
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HISTORICAL PERSPECTIVE
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August 6, 1945
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Table 1. Estimated population size and number of acute (within
two to four months) deaths in Hiroshima and Nagasaki after the
atomic bombings
____________________________________________________________________________
Name Estimated city population Estimated number of
ofCity at the time of the bombings acute deaths
____________________________________________________________________________
Hiroshima 310,000 persons 90,000-140,000
Nagasaki 250,000 persons 60,000-80,000
____________________________________________________________________________
Radiation induced Bone Marrow Failure
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Bone Marrow Transplantation
Early Observations in Mouse Models
Protected from lethal dose if spleen shielded Jacobsen et al, 1951
Protected if received infusion of bone marrow Lorenz et al, 1951
~1000 Rads
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BM mediated protection from
lethal irradiation
(Eldredge/Shelton, 1951)
LONG TERM SURVIVAL AND ENGRAFTMENT
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BOVINE FRATERNAL TWINS ARE
CHIMERIC FOR EACH OTHERS
BLOOD CELLS FOR LIFE
(Ray Owen, 1945)
S O S O O
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FIRST DEMONSTRATION OF
STEM CELL CONCEPT
(Till, McCulloch and Siminovitch 1961)
Transferred donated BM
spleen in irradiated recipients
Colonies were clonal origin
Stem cell concept demonstrated
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ATTEMPTS AT ISOLATION OF HSC
Size and density
cell cycle active drugs
Supravital stains Hoechst33342 (side population)
Rhodamine 123
FACS
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ISOLATION OF MOUSE HEMATOPOIETIC STEM CELLS
MONOCLONAL ANTIBODIES
FACS
SORTER
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ASSAYS FOR HEMATOPOIETIC STEM CELL ACTIVITY
In vitro Assays
No test of self-renewal
Good for differentiation
Not always
Correlated to in vivo activity
methylcellulose
LTC-IC
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BM Fractionated Cells
Long Term Reconstitution AssayCFU-S
BM Fractionated Cells
Analysis of peripheral blood
D8-12 spleen colony formation
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HSC assays in vivo
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ISOLATION OF HSC
1 CFU/10 HSCs (lin, Thy1.1, Sca-1)
1CFU/7200 BM cells
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ISOLATION OF HSC
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ISOLATION OF HSC
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Therapeutic uses of Hematopoietic Stem cells
Regeneration of aged tissue
Regeneration of injured tissue
Transplantation following cancer therapy
Transplantation to repair genetic disorders
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ISOLATION OF HSC
Lin-Thy1.1+Sca-1+ cells were 1000 fold enriched in
HSC activity
Later addition of c-kit
Lin-Thy1.1+Sca-1+c-kit+ enriched HSCs to 2000 fold
Use of Thy1.1 may be replaced by Rhodamine expressionor Flk2 expression.
New isolation protocols should meet long term reconstitution,
Radioprotection, and limiting dilution data.
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Characteristics of HSCs
Short Term, Long Term, Mulitpotent Precursor
with differential self-renewal activity
Frequency of cells: LT:0.007, ST:0.01, MPP:0.03
Quiescent: 4% in cycle in young mice
30% in cycle in old mice
High levels of telomerase activity correlating
with self-renewal
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SIGNALS THAT REGULATE HEMATOPOIETIC STEM
CELL FUNCTION
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Self Renewal
Commitment
Differentiation
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Bone Marrow as a site for adult hematopoiesis
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DIFFERENTIATION
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PERMISSIVE VS. INSTRUCTIVE MODELOF COMMITMENT
PRIMED BY INTRINSIC FACTORSCYTOKINES ALLOW PRECOMMITED CELLS
TO PROLIFERATE
UNCOMMITTED CELLS ARE INDUCEDTO COMMIT
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Megakaryocyte
MYELOID/ERYTHROID DIFFERENTIATION
G-CSF
M-CSF
Erythropoietin
Tpo
Granulocytes
Macrophages
RBCs
*Require other factors
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B cells
LYMPHOID DIFFERENTIATION
IL-7+Stroma
IL-7+Notch
+StromaT cells
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SELF-RENEWAL
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EARLY ATTEMPTS
USING CLASSICAL CYTOKINES
SLF/Tpo: Expands a few cell divisions
Robust proliferation always associated with differentiation
RECENTLY IDENTIFIED SIGNALS
Notch
Shh
Wnt
Bmi
HoxB4
p21
p27
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STEM CELLS AND CANCER
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Signals mediating self renewal
Stem cells as a target cell for mutation
Cancer stem cells
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Signals that stimulate self-renewal
Shared signals between stem cells and cancer cellsShared characteristics of the stem cell and cancer cell niche
Shared patterns of symmetric and asymmetric division
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Shared Signaling
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Notch signaling
Activation leads to extensive
proliferation with reduceddifferentiation:in vitroand in vivoassays in HSCs, germline and neuralstem cells
Mutations in T cell leukemias andmammary tumors
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Sonic Hedgehog Signaling
Enhanced self-renewal in HSCs
in xenograft model,proliferation of neuralprecursors
Mutations in medulloblastoma,basal cell carcinoma, activationin lung cancer
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Wnt signaling
Wnt
b-cat
b
-cat
Fzd LRP
LEF1/
TCF
GSK3b
cyclin D1
Implicated in proliferation and self-
renewal of HSCs, skin stem cells, gutStem cells, Neural stem cells
Mutations in colon cancer, Prostate
cancer, skin cancers, activation inleukemias
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Shared Characteristics of the nicheRichard Gilbertson
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Self Renewal
Commitment
Transformation
AsymmetricSymmetricRenewal
SymmetricCommitment
Contribution of symmetric and asymmetric division
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ASYMMETRIC AND SYMMETRIC DIVISION
HOW CAN A STEM CELL MAINTAIN ITSELF?
(A) (B)
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Stem cells as the cell of origin
Defining the Cell of originIdentifying the Cell of origin
Consequences of knowing the cell of origin
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Stem cells renew for long periods
Stem cells are long lived
Greater propensity to accrue mutations
Phenotypic similarities in AML cells with HSCs
Translocations often found in HSCs, leukemia exhibits later
Progenitors cannot be transformed by some oncogenes
Evidence that cancers can derive from stem cells
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Mouse models with mutations at progenitor stage can recapitulate
cancers
Progenitor cell must reacquire stem cells properties
Evidence that cancers can derive from progenitor cells
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Why does the cell of origin matter?
To compare the tumor cell to the normal cell from which itarose so that critical differences can be identifiedand used for targeting
Different cells of origin give rise to different cancersarising from the same oncogene. These differences may
also lead to differential dependence on signals.
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Cancers propagated by cancer stem cells
How are they defined?Do they exist?
What are the caveats of the assays?What are the therapeutic implications of the concept?
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Small subpopulation within a tumor
High self-renewal capacityResponsible for propagating the tumor in vivo
Maybe quiescent and resistant to chemotherapy
Definition:cancer stem cells
Th id f C t ll
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The idea of Cancer stem cells
Rudolf Virchow/Julius Connheim: Embryonal Rest hypothesisTumors arise from residual embryonic tissues
Van Potter/Barry PierceTumors arise from maturational arrest in tissue specific stem cells
Mouse myeloma cells: 1:100 to 1:10000 form colonies
When transplanted 1-4% forms colonies in the spleen
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The identification ofleukemia initiating cells
Work done by John Dick and Colleagues
Human AML is heterogenous by cell surface markers
Can be sorted into different subfractions
CD34+38- subfraction makes up 0.2% of the leukemia
They were the only cells capable of transplanting the leukemia
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Hunting for cancer stem cells
Dissociate into single cell suspension
Surface markers identified to see if multiple subfractions
Cells isolated by FACS or magnetic sorting
Fractionated and unfractionated cells transplanted into NOD-SCIDs
The fraction that recapitulates tumor formation at the
lowest number is most enrichedfor cancer stem cell activity
C t R h C St C ll i lid
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Current Research: Cancer Stem Cells in solid cancers
Breast Cancer:Mike
Clarke/Kornelia Polyak
CD44+ (more progenitor)
CD24(more differentiated)
Brain Tumors: Peter Dirks
(CD133)
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105 cells 103 cells 102 cells 10 cells
E-myc B lymphoma
Case 1 3/3 (25) 3/3 (25) 3/3 (32) 2/2 (35)
Case 2 3/3 (21) 3/3 (23) 3/3 (24) 3/3 (24)
Case 3Sca-1+
AA4.1hi 3/3 (21) 3/3 (21) ND 3/3 (17)
Sca-1+
AA4.1lo 2/2 (17) 2/2 (28) 2/2 (28) 2/2 (40)
E-N-RAS T lymphoma
Case 1 3/3 (28) 3/3 (42) 3/3 (28) 3/3 (28)
PU.1-/- AML
Case 1 1/1 (54) 2/2 (168) 1/2 (192) 0/2
Case 2 2/2 (84) 2/2 (85) 2/2 (224) 1/2 (114)
Case 3 1/1 (85) 2/2 (62) 2/2 (69) 2/2 (90)
Tumor Growth Need Not Be Driven by Rare Cancer Stem CellsPriscilla N. Kelly,1,2 Aleksandar Dakic,1,2 Jerry M. Adams,1* Stephen L. Nutt,1* Andreas Strasser1*
Recipients that developed tumors (days to kill)
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Xenotransplants may underestimate the number ofcells that can transplant the tumor
The mouse may have a different microenvironmentDifferent cells may be able to migrate or not but can
contribute in the endogenous tumor
Certain tumor models may have less heterogeneity
Possible Caveats
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Implications for therapy
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Targeting therapies to eradicate cancer stem cells:
A paradigm shift in cancer therapy
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