Download Hematopoietic stem cells: insights into bone marrow biology

Hematopoietic stem cells:
insights into bone marrow biology
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Aleksandra Wodnar-Filipowicz
Department of Biomedicine
University Hospital Basel
Switzerland
ESH-EBMT
1
Latimer 2009
Stem Cells - Definition:
Cells capable
• to divide for indefinite period,
• to self-renew, and
• to give rise to specialized cells
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Totipotent Stem Cell
Zygote
Embryonic und adult tissue
stem cells
Pluripotent Stem Cell
Embryonic Stem (ES) Cells
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T-CELL
LYMPHOID
STEM CELL
B-CELL
Multipotent Tissue Stem Cells
BFU-E
Neural Muscle Liver Epidermis Retina Hematopoietic
PLASMA CELL
ERYTHROCYTE
NEUTROPHIL
CFU-GM
MONOCYTE
MACROPHAGE
Cancer Stem Cells
MYELOID
STEM CELL
CFU-Eo
EOSINOPHIL
CFU-Baso
BASOPHIL
PLATELETS
CFU-Meg
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MEGAKARYOCYTE
Developmental Potential of Stem Cells
Zygote
Embryonic
stem cells
Adult tissue
stem cells
“De-differentiation”
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“Trans-differentiation”
Specialization
Developmental potential
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Embryonic
stem cells
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Adult tissue
stem cells
Cancer
Stem Cells
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Presentation outline:
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
6
Hematopoietic stem cells
Mouse
Human
Phenotype
Phenotype
LSK cells
Lin0.007%
sca1+
of BM
ckit+
tie2+
flk2CXCR4+
Abcg2+
SLAM-Rs:
CD150+CD244-CD48TLR4+
c-kit
0.007%
R3
BL6
c-kit
%
101
102
SCA1-Height
103
sca-1
Hoechst
blue
Hoechst Blue
Side population (SP)
(ABCG2+transporter)
LSK cells:
100
CD34+CD38- cells
Lin0.1%
CD34+ (CD34- ?)
of BM
CD38ckit+
flk2- (flk2+ ?)
CD133+ (?)
CXCR4+
ABCG2+
?
0.2%
SP
104
Hoechst Far Red
Hoechst
far red
The phenotype of human HSC remains poorly defined
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Human hematopoietic stem cells
Function
• In vitro CFU assays
• In vivo transplantation:
xenografts in immunologically permissive mouse
strains
(e.g. ..NOD/SCID; NOD/SCID-c-/-; Rag2-/-c-/-)
SRC
human
CD34+cells
(i.v. or i.f.)
BMT
1º
BMT
2º
3º
Clinical transplantation
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In situ analysis of bone marrow “niche”
Labeled HSC in the bone marrow niche
CFSE/
DAPI/
OPN
Cytokine expression
in the bone marrow niche
CD45/
Flt3 ligand
BONE
HSC
os
BM
Wilson et al.
2004
ISO
CD45
FL
CD45
Kenins & Wodnar-Filipowicz 2005
endosteum
Cytokine
receptor expression
in the bone marrow niche
Tie2+CD45+ cells
(yellow)
adhere to the endosteal surface
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Arai et al. 2004
Bone marrow “niche”
2009
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Cellular components of stem cell niches
in the adult bone marrow
Sinusoid
endothelial cells
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MSC
Trabecular bone
MSC
MSC
Endosteal
niche
HSC
Vascular
niche
Migration
Proliferation
Differentiation
Precursor
cells
HSC
CAR
cells
Osteoblasts
Osteoblasts - osteogenic cells
lining the inner surface of the bone
Stroma
fibroblasts
Self-renewal
CAR cells - (CXCL12 abundant
reticular) cells close to
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the bone surface and endothelium
Molecular interactions in the bone marrow niche
Intrinsic
Extrinsic
mechanisms:
mechanisms:
• signalling molecules
• cytokines
• cytoplasmic
• chemokines
• nuclear, incl.
• adhesion molecules
transcription factors
• negative regulators (osteopontin)
• epigenetic mechanisms
• proteases (MMP-9, cathepsin K)
• DNA/histone modif.
HSC
• hormones (PTH, PGE2)
• microRNAs
• sympathetic nerves
Gene expression
• oxygen status
• calcium concentration
• circadian rhytm
Ang-1 - tie2
quiescence SCF - c-kit
self-renewal Flt3-L - Flt3
expansion TPO - mpl
Wnt - Frizzled
Jagged/Delta - Notch
Cadherins
VCAM - VLA
ICAM - LFA
CXCL12 - CXCR4
HA - CD44
cell-cell
adhesion
migration
OSTEOBLAST
PTH, PGE2
nerves
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Leukemia Stem Cells
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Cancer stem cells
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.
HSC
HSC
LSC
Prog.
LSC
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1st
Leukemia stem cells:
identified cancer stem cells
Phenotype
CD34+CD38CD34- ?
CD33+ ?
CD44+ ?
CD71+ ?
CD96+ ?
CD117- ?
CD123+ ?
CD133+ ?
CLL-1+ ?
Function
SL-IC
Human AML
peripheral blood
NOD/SCID
CD34+CD38not
CD34+CD38+
(?)
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Functional analysis of LSCs
BM
BM
FACS:
human
CD45+
cells
iv
AML
cells
2o
1o
if
Injected femur Peripheral blood
CD33
Non-injected bones
3o
Langenkamp &
Wodnar-Filipowicz
2008
CD45
AML
cells
2 weeks
1o CFU
2o CFU
3o CFU
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Leukemic stem cells
Conventional
treatment
• resistant to conventional treatment
• lead to relapse and fatal outcome
Leukemic
cell growth
Relapse
Degeneration
Remission
Terminal
differentiation
Remission
Elimination
of LSC
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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Cross-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)
CFSE/ DAPI/ OPN
bone
BM
os
HSC
endosteum
Van Etten et al. Nat Med 2006
Wilson et al. Genes & Dev 2004
Bone marrow stromal cells can in turn be influenced by leukemic
cells
BM stroma
HSC
or
LSC
Transition of SDF-1- to SCF - expressing
environment,
which disrupts the behaviour of normal
HSC/progenitors
Colmone et al. Science 2008
Is the stem cell niche in leukemia normal or not ?
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“Seed or soil” or both ?
Self-renewal mechanism of stem cells
Normal stem cell
s.
Cancer stem cell
stem cell number:
as.
stable
80%
asymmetric
increased
80%
symmetric
Mathematical model:
Skipping assymetric division every 4-6 divisions is
sufficient for the tumor to grow.
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Presentation outline:
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
20
Generation of ES cell lines from human blastocysts
Thomson et al. Science 282:1145 (1998)
Donated IVF
blastocyst
Inner cell mass
„immortal“
cell lines
Somatic differentiation in vitro
into various tissue types
CFU’s
Kaufman et al. PNAS 98:10716 (2001)
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„Therapeutic Cloning“ (SCNT)
Enucleated donor oocyte
Fusion
Blastocyst
Nuclear-transfer
From adult cell
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ES cell lines
Human ES cells with patient’s
genetic information
Immunologically-compatible
tissue for transplantation
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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)
Oct3/4
Sox2
c-myc
Klf4
Fusionproteins
iPS (piPS)
ES-like cells
fibroblasts
± 20 days
Oct4
Sox2
Nanog
Lin28





Differentiation into cells
of all germ layers:
Ectoderm
e.g.
Mesoderm
neural cells,
Entoderm heart muscle cells
Human ES morphology
Normal karyotype
Telomerase activity
Cell surface markers of ES cells
Gene expression profile of ES cells
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Adult bone marrow - derived stem cells
for organ regeneration and repair
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Bone marrow - resident stem cells
Cell type
Differentiation potential
Hematopoietic stem cells (HSC)
blood cells
other tissues = plasticity (?)
Mesenchymal stem cells (MSC)
bone, cartilage
adipose tissue, muscle
other tissues = plasticity (?)
Hemangioblasts (EPC)
endothelium
blood cells, muscle
[Multipotent adult progenitor cells
(MAPC)]
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 origin
and belonging to all three germ layers,
i.e. similar to the differentiation ability of ES cells.
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Muscle (Heart)
Neural (CNS)
HSC
Adipocytes
 contamination
 not reproducible
 not reproducible
Hepatocyte (Liver)
 cell fusion
= 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 origin
and 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  tissue regeneration (clinical trials)
Diseases
G/GM-CSF
mobilization
BM
Cardiac
Vascular
Lung
BM (MNCs, CD34+)
Brain stroke, Paraplegia, ALS
autologous
Breast cancer
Retina degeneration
Cartillage degeneration
MSC-infusions
Osteogenesis imperfecta
allogeneic
GvHD
autologous
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Stem cells: Key words
d.0-d.14
w.2-w.8
mo.3-mo.9
Pre-embryo Embryo
after birth
Fetus
Adult
Parthenogenesis
(chemical treatment)
d.1-d.2
Oocyte
+
Sperm
Adult cells
8 cell
(blastomeres)
Zygote *
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d.8-9
32 cell
Implantation
(blastocyst)
in utero
ES cells *
= ICM
animal oocyte
(chimerae)
Nuclear transfer
(SCNT)
trophoblast
Embryo
ES cell lines *
Gene transfer
(reprogramming)
iPS *
d.5
Extraembryonic tissue
Epiblast *
AGM region *
EG cells *
Yolk sac *
Placenta *
Amniotic fluid *
Natural plasticity of adult cells ??
Cell fusions (adult/adult; adult/ES)
All duplicated if starting from diseased cells
Fetal
Cord
Bone
Liver * Blood * Marrow *
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± Genetic modifications
2009: Prospects for stem cell-based therapy
of degenerative diseases
ES
SCNT
iPS
MSC
CBHSC
HSC
NSC
Stem cell choice:
• adult (multipotent) or embryonic (pluripotent)
• “off-the-shelf” from master-stocks (= cell banks) or “customized” for ... ...
...individual patients
Stem cell number:
• increase homing and engraftment in tissue of choice
• develop drugs that augment the endogenous stem cell pools
Stem 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 responses
Stem cell gene therapy:
ES/iPS terato/cancerogenicity:
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• control insertional mutagenesis
?
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 - targeting
Trumpp & Wiestler, Nature Clinical Oncol 5:337 (2008)
Leukemia stem cells
Chan & 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)
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