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Hematopoietic Stem Cells

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Bone marrow is a factory for blood cells. Multiple assembly ... Jagged/Notch. Wnt/Frizzled:LRP. TGF. b / BMP-4. leptin. NGF. Shh/Patched. Angiopoietin/Tie-2 ... – PowerPoint PPT presentation

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Title: Hematopoietic Stem Cells


1
Hematopoietic Stem Cells
N. Iscove
2
The problem of marrow maintenance
3
Multiple kinds of blood cell
4
Bone marrow is a factory for blood cells
5
Multiple assembly lines in marrow
6
The neutrophil assembly line
7
The neutrophil assembly line
8
The neutrophil assembly line
9
The neutrophil assembly line
10
The neutrophil assembly line
11
Multiple assembly lines
12
Where do precursor cells come from?
13
Hematopoietic reconstitution
14
Polyclonal reconstitution
9 Gy
6
BM 1 x 10
time
BM
R
e
c
o
n
s
t
i
t
u
t
i
o
n

o
f

i
r
r
a
d
i
a
t
e
d

m
i
c
e
1
0
0
cells

1
0
precursors
n
o
r
m
a
l
/
a
n
i
m
a
l
1
7
1
4
21
0
3
6
0
d
a
y
s
15
Reconstitution at limiting dilution
9 Gy
3
BM 1 x 10
time
BM
R
e
c
o
n
s
t
i
t
u
t
i
o
n

o
f

i
r
r
a
d
i
a
t
e
d

m
i
c
e
1
0
0
cells

1
0
precursors
n
o
r
m
a
l
/
a
n
i
m
a
l
1
7
14
21
0
3
6
0
d
a
y
s
16
Reconstitution at limiting dilution
9 Gy
3
BM 1 x 10
time
BM
17
Reconstitution at limiting dilution
9 Gy
3
BM 1 x 10
time
BM
18
(No Transcript)
19
Clones generated by reconstituting cells contain
new reconstituting cells
1
0
0

1
0
n
o
r
m
a
l
cells
/
a
n
i
m
a
l
precursors
1
7
14
21
0
3
6
0
d
a
y
s
120000 cells
20
(No Transcript)
21
Patterns of reconstitution at limiting dilution
1
0
0
donor
1
0
1
20
40
100
0
weeks
  • local, undetected in blood or tissues
  • lt 109 progeny
  • systemic, transient
  • gt 109 progeny
  • systemic, sustained

22
The stem cell basis of tissue permanence
23
Multiple assembly lines
24
Where do precursor cells come from?
25
Precursor cells arise continuously from "stem
cells"
26
Stem cells self-renew
Stem cell
Committed precursors
Differentiating precursors
27
Stem cells self-renew
Stem cell
28
Stem cells self-renew
Stem cell
29
Stem cells self-renew
Stem cell
Committed precursor
30
Stem cells self-renew
Stem cell
Committed precursor
Differentiating precursor
31
Maintenance of proliferative tissues
growth
differentiation
proliferative potential
32
The relationship between self-renewal and clonal
fate
33
Patterns of reconstitution at limiting dilution
1
0
0
donor
1
0
1
20
40
100
0
weeks
  • local, undetected in blood or tissues
  • lt 109 progeny
  • systemic, transient
  • gt 109 progeny
  • systemic, sustained

34
Self-renewal
S
S
D
  • requires cell division

35
Transient reconstitution by HSC at limiting
dilution
D
D
D
D
D
D
D
S
1
0
0
D
D
D

D
1
0
n
o
r
m
a
l
D
/
a
n
i
m
a
l
D
D
1
extinction
7
14
21
0
3
6
0
d
a
y
s
  • detection of a graft from a single HSC requires
    gt 109 progeny

36
Transient reconstitution by HSC at limiting
dilution
D
D
D
D
D
D
D
S
1
0
0
D
D
D
S
donor
1
0
D
D
D
1
20
40
100
0
delayed extinction
weeks
  • detection of a graft from a single HSC requires
    gt 109 progeny

37
Permanent reconstitution by HSC at limiting
dilution
1
0
0
donor
1
0
1
20
40
100
0
weeks
38
Self-renewal determines clonal outcomes
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
S
S
S
D
D
D
S
D
D
S
D
D
S
S
S
D
D
D
S
S
D
S
S
D
extinction
expansion
maintenance
39
Detection of "progenitors" in colony assays
9 GY
BM cells
40
Progenitors may self-renew
9 Gy
41
Progenitors may self-renew
9 Gy
42
Self-renewal sustained versus unsustained
  • SR is a general feature of primitive cells in the
    hematopoietic hierarchy
  • Permanently reconstituting stem cells differ from
    later cells by sustaining SR through multiple
    generations of progeny

43
Regulation of self-renewal in HSC
44
HSC numbers increase in regenerating marrow
9 Gy
BM
time
BM
Reconstitution of irradiated mice
1
0
0
cells
CFC

1
0
n
o
r
m
a
l
/
a
n
i
m
a
l
1
5
1
0
1
5
0
3
6
0
d
a
y
s
45
Expanded HSC can expand again
normal /animal
days
9 Gy
46
Stem cell expansion during serial transfer
47
Expansion versus maintenance
D
D
D
D
D
D
D
D
D
D
D
D
D
D
S
S
D
D
S
D
S
D
S
S
D
D
S
S
S
S
expansion
maintenance
48
Self-renewal of HSC in vivo
  • HSC number constant in the normal steady-state
    (asymmetric SR divisions)
  • HSC number expands during marrow regeneration
    (symmetric SR divisions)
  • expansion can be extensive
  • maintenance versus expansion mode governed by
    extrinsic signals

49
Stem cell extinction in culture
50
Hematopoietic cytokines marrow environment
c-kit ligand
Target
Source
Flt3 ligand
IL-7
early cells
fibroblast
TPO
IL-11
IL-6
LIF
  • significant growth stimuli only in
    combination

Oncostatin M
FGF
Jagged/Notch
/ BMP-4
HGF/Met
Wnt/FrizzledLRP
leptin
NGF
Shh/Patched
SDF-1/CXCR4
VEGF/Flk-1
Angiopoietin/Tie-2
Angpt-like
Epo
pE
kidney
M-CSF
Mac
Osteocl
fibroblast
opgl
Osteocl activation
osteoblast
G-CSF
pN
Mac
51
Hematopoietic cytokines local vs inflammatory
marrow environment
immune origin (Th)
GM-CSF IL-3 IL-5 targets progenitors and
leukocytes
52
Self-renewal of HSC is regulated
culture with cytokines
steady-state in vivo
regeneration in vivo
53
Hierarchical organization of HSC
54
Patterns of reconstitution at limiting dilution
1
0
0
1
0
0
1
0
0
donor
donor
donor
1
0
1
0
1
0
1
1
1
20
40
100
0
20
40
100
0
20
40
100
0
weeks
weeks
weeks
  • local, undetected in blood or tissues
  • lt 109 progeny
  • systemic, transient
  • gt 109 progeny
  • systemic, sustained

55
Detection of "progenitors" in colony assays
9 GY
BM cells
56
Hierarchical organization of hematopoiesis
Nk
Macrophage
B
Dendritic
Osteoclast
Microglia
Kupfer
N
Langerhans
Eo
Baso/Mast
Pl
E
LTRC
57
Hierarchical organization of hematopoiesis
Nk
Macrophage
B
Dendritic
Osteoclast
Microglia
Kupfer
N
Langerhans
Eo
Baso/Mast
Pl
E
ITRC
58
Hierarchical organization of hematopoiesis
Nk
Macrophage
B
Dendritic
Osteoclast
Microglia
Kupfer
N
Langerhans
Eo
Baso/Mast
Pl
E
STRC
progenitors
59
Hierarchical organization of hematopoiesis
Nk
Macrophage
B
Dendritic
Osteoclast
Microglia
Kupfer
N
Langerhans
Eo
Baso/Mast
Pl
E
STRC
progenitors
60
Hierarchical organization of hematopoiesis
Nk
Macrophage
B
Dendritic
Osteoclast
Microglia
Kupfer
N
Langerhans
Eo
Baso/Mast
Pl
E
61
Phenotypic anatomy of hematopoiesis
Nk
lo
Rho
ABCT
Macrophage
B
Dendritic
Osteoclast
Microglia
Kupfer
N
Langerhans
Eo
Baso/Mast
Pl
E
62
Phenotypic anatomy of hematopoiesis
c-kit
Sca1
Nk
lo
Rho
ABCT
Macrophage
B
Dendritic
Osteoclast
Microglia
Kupfer
N
Langerhans
Eo
Baso/Mast
Pl
E
63
Phenotypic anatomy of hematopoiesis
c-kit
Sca1
CD34
Flt3
Nk
lo
Rho
ABCT
Macrophage
B
Dendritic
Osteoclast
Microglia
Kupfer
N
Langerhans
Eo
Baso/Mast
Pl
E
64
Phenotypic anatomy of hematopoiesis
c-kit
Sca1
CD34
Flt3
Nk
lo
Rho
ABCT
Macrophage
B
Dendritic
Osteoclast
Microglia
Kupfer
N
Langerhans
Eo
Baso/Mast
KSL
Pl
E
65
Phenotypic anatomy of hematopoiesis
c-kit
Sca1
CD34
Flt3
Nk
lo
Rho
ABCT
Macrophage
B
Dendritic
Osteoclast
Microglia
Kupfer
N
Langerhans
Eo
Baso/Mast
RKSL
Pl
E
66
Heterogeneity of CD49b in RKSL cells
1st sort
256
256
192
192
B220/CD3
SSC
128
128
64
64
0
0
0
256
192
128
64
0
256
192
128
64
FSC
Rho123
2nd sort
Kit
Sca-1
Sca-1
CD49b
67
Only RKSL/CD49b cells sustain long-term
reconstitution
N 6
1 x 106 Gpi1a marrow
0.5
9.5 Gy
100 cells
0.4
C57BL/6
100 Rho49b
Gpi1a/Ly5.1
Reference
0.3
1 x 106 wt
Proportion donor red cells
4 - 40 wk
Blood
0.2
100 Rho49b
RBC
0.1
0
0
4
8
16
24
32
weeks in vivo
68
Phenotypic anatomy of hematopoiesis
c-kit
Sca1
CD34
Flt3
Nk
lo
Rho
ABCT
Macrophage
B
Dendritic
Osteoclast
Microglia
Kupfer
N
Langerhans
Eo
Baso/Mast
Pl
E
69
Phenotypic anatomy of hematopoiesis
c-kit
Sca1
CD34
Flt3
Nk
lo
Rho
ABCT
Macrophage
B
Dendritic
Osteoclast
Microglia
Kupfer
N
Langerhans
Eo
CD49b
Baso/Mast
Pl
E
LTRC
ITRC
70
LTRC and ITRC are initially quiescent
1.0
RKSL
-
Rho
49
0.8
lo
CD49b-
CD49b
Rho
49
-
-
0.6
Rho
49
CFC
Proportion divided
KL/FL/7/11
0.4
0.2
Onset of cell division
0.0
  • functional distinction maintained

0
20
40
60
80
Hours
71
Gene systems specifying self-renewal in HSC
72
Marrow (Ly5.1)
culture
96 hr
GFP
Ly5.2
73
HOXB4-expressing cells eventually dominate in
culture
100
50
10
74
HOXB4-expressing cells eventually dominate in vivo
75
HoxB4
HOXB4-expressing cells differentiate normally in
vivo
Control
76
HOXB4-expressing HSC regenerate fully in vivo
77
Potential role of Hox genes in self-renewal
  • Enforced expression of Hox genes enhances
    self-renewal
  • Do Hox genes have that role normally?
  • Are they expressed in HSC?
  • Do they determine maintenance or execution of
    self-renewal?
  • How are they regulated?

78
LTRC and ITRC are initially quiescent
1.0
RKSL
-
Rho
49
0.8
lo
CD49b-
CD49b
Rho
49
-
-
cDNA
0.6
Rho
49
CFC
Proportion divided
0.4
cDNA
0.2
Onset of cell division
0.0
0
20
40
60
80
Hours
79
A cDNA sample matrix - HSC
M
G1
Go
Gapdh
LTRC
ITRC
80
A cDNA sample matrix - Progenitors
Gapdh
cells
MultiB
MultiB
Penta
PCR
Tetra
fate
fate
fate
pE/Meg
pNeut/Mac
cDNA
BFUE
pMeg
pMac
pNeut
CFUE
E
Meg
Mac
Neut
Mast
B
T
81
Hox expression is linked to cell cycling
HoxA4
M
G1
Go
40 cycles
LTRC
ITRC
Pentapotent
Tetrapotent
pE
/Meg
pNeut
/Mac
BFUE
pMeg
pMac
pNeut
CFUE
E
Meg
Mac
Neut
Mast
B
T
82
Do ITRC and LTRC differ in stability of Hox4
expression?
LTRC
ITRC
83
Do ITRC and LTRC differ in stability of Hox4
expression?
LTRC
ITRC
84
Implications of Hox expression pattern
  • Hox induction may be central to execution of SR
  • Enforced expression of Hox4 converts ITRC to LTRC
  • How do LTRC sustain Hox inducibility
  • How do ITRC lose it?

85
Regulation of Hox expression
  • Trithorax group genes required for sustained Hox
    expression
  • Polycomb group genes required for specific
    repression of Hox expression
  • Both systems work by epigenetic modification of
    chromatin
  • Preserve memory of active or silenced chromatin
    states across multiple cell divisions

86
Pc required for segment-specific homeotic gene
repression in the fly
Pc
wt
Ubx protein
87
Multiple homologs of PcG elements in mammals
Maintenance
Initiation
Su(z)12
Su(z)12
RING
RING
Psc
Psc
Ph
Ph
Esc
Esc
Fly
Pc
Pc
E(z)
GAF/Zeste
Gaf/Zeste
Gaf/Zeste
Me
Me
Me
Ring1A
Phc1/Rae28
Ring1B
Phc2
Bmi
-
1
Mammal
Phc3
Mel
-
18
Suz12
Suz12
Cbx2 Cbx4
Mblr
Eed
Eed
Ezh1
Cbx6 Cbx7
X
X
Ezh2
Cbx8
Me
Me
88
Why multiple homologs
A mechanism for broadening the repertoire of
cellular identities that can be specified?
89
Ezh1 predominates in quiescent and cycling LTRC
M
G1
Go
Ezh1
LTRC
STRC
MultiB
MultiB
Penta
Tetra
pE/Meg
pNeut/Mac
BFUE
pMeg
pMac
pNeut
CFUE
E
Meg
Mac
Neut
Mast
B
T
90
Phc1 predominates in quiescent and cycling LTRC
M
G1
Go
Phc1
LTRC
STRC
MultiB
MultiB
Penta
Tetra
pE/Meg
pNeut/Mac
BFUE
pMeg
pMac
pNeut
CFUE
E
Meg
Mac
Neut
Mast
B
T
91
Cbx7 predominates in quiescent and cycling LTRC
M
G1
Go
Cbx7
LTRC
STRC
MultiB
MultiB
Penta
Tetra
pE/Meg
pNeut/Mac
BFUE
pMeg
pMac
pNeut
CFUE
E
Meg
Mac
Neut
Mast
B
T
92
Self-renewal versus differentiation
Hox genes, their regulation upstream, their
targets downstream
genes guiding lineage specification and divergence
93
Reading
  • Cell fate determination from stem cells
  • AJ Wagers, JL Christensen and IL Weissman
  • Gene Therapy (2002) 9, 606612
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