hematopoietic stem cells: insights into bone marrow...

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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



Retina

CancerCancer Stem CellsStem Cells

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Adult tissuestem cells

Developmental Potential of Stem Cells

Developmental potential

Specialization

Embryonicstem cells

Zygote



“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



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)

hematopoietic stem cells: insights into bone marrow...

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