stem cell module.ppt

7/28/2019 stem cell module.ppt

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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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