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    Hematopoietic stem cells: insights into bone marrow biology

    ESH-EBMT Latimer 2009

    Aleksandra Wodnar-Filipowicz Department of Biomedicine University 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

  • 3

    Hematopoietic

    LYMPHOID STEM CELL

    T-CELL

    B-CELL PLASMA CELL

    ERYTHROCYTE

    NEUTROPHIL

    MONOCYTE

    EOSINOPHIL

    BASOPHIL

    MEGAKARYOCYTE

    MYELOID STEM CELL

    BFU-E

    CFU-GM

    CFU-Eo

    CFU-Baso

    CFU-Meg

    MACROPHAGE

    PLATELETS

    Totipotent Stem Cell Zygote

    Pluripotent Stem Cell Embryonic Stem (ES) Cells

    Multipotent Tissue Stem Cells

    Neural Muscle EpidermisLiver

    Embryonic und adult tissue stem cells

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    Retina

    CancerCancer Stem CellsStem Cells

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    Adult tissue stem cells

    Developmental Potential of Stem Cells

    Developmental potential

    Specialization

    Embryonic stem cells

    Zygote

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    “Trans-differentiation”

    “De-differentiation”

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    CancerCancer Stem CellsStem Cells

    Embryonic stem cells

    Adult tissue stem 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:

  • 7

    Hematopoietic stem cells

    Mouse

    Phenotype Lin- sca1+ ckit+ tie2+ flk2- CXCR4+ Abcg2+ SLAM-Rs: CD150+CD244-CD48-

    TLR4+

    LSK cells 0.007% of BM

    c- ki

    t

    BL6

    %

    LSK cells:

    100 101 102 103 104 SCA1-Height

    R3

    sca-1

    c- ki

    t 0.007%

    Human

    Phenotype Lin- CD34+ (CD34- ?) CD38- ckit+ flk2- (flk2+ ?) CD133+ (?) CXCR4+ ABCG2+ ?

    CD34+CD38- cells 0.1% of BM

    H oe

    ch st

    B lu

    e

    Hoechst Far Red

    Side population (SP) (ABCG2+transporter)

    Hoechst far red

    H oe

    ch st

    b lu

    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

    human CD34+cells (i.v. or i.f.)

    SRC

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    CD45 ISO FL CD45

    CFSE/ DAPI/ OPN

    BONE

    BM

    endosteum

    os

    HSC

    Wilson et al. 2004

    Labeled HSC in the bone marrow niche Cytokine expression in 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

    Migration Proliferation

    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 to the bone surface and endothelium

    CAR cells

    Stroma fibroblasts

    Vascular Vascular nicheniche

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    Molecular interactions in the bone marrow niche Extrinsic 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-cell adhesion migration

    quiescence self-renewal expansion

    Ang-1 - tie2 SCF - c-kit Flt3-L - Flt3 TPO - mpl Wnt - Frizzled Jagged/Delta - Notch

    Cadherins VCAM - VLA ICAM - LFA CXCL12 - CXCR4 HA - CD44

    Gene expression

    OSTEOBLAST PTH, 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 involved in the regulaton of normal stem cell development are mutated or epigenetically activated in cancer.

    Leukemia stem cells: Transformed hematopoietic stem or commited progenitor cells that have amplified or acquired the capacity for self-renewal, albeit in a poorly regulated fashion.

    Cancer stem cells

    HSC

    Prog.HSC

    LSC

    LSC

  • 15

    Human AML peripheral blood

    Leukemia stem cells: 1st identified cancer stem cells

    SL-IC CD34+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

    AML cells

    BM BM

    1o 2o 3o

    FACS: human CD45+ cells

    iv

    if

    Non-injected bones Injected femur Peripheral blood

    2 weeks 1o CFU 2o CFU 3o CFU

    AML cells

    CD45

    C D

    3 3

    Langenkamp & Wodnar-Filipowicz 2008

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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

    bone BM

    endosteum

    os

    HSC

    HSC BM stromaBM stroma

    LSCor

    Transition of SDF-1- to SCF - expressing environment, which disrupts the behaviour of normal HSC/progenitors Colmone 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 ?

  • 19

    Self-renewal mechanism of stem cells

    Normal stem cell Cancer stem cell

    Mathematical model: Skipping assymetric division every 4-6 divisions is sufficient for the tumor to grow.

    80% asymmetric

    80% symmetric

    stem cell number:

    stable

    increased

    s. as.

  • 20

    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:

  • 21

    Generation of ES cell lines from human blastocysts

    Donated IVF blastocyst

    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’s genetic 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

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