Hematopoietic reconstitution

1
Hematopoietic reconstitution
2
9 Gy
BM 1 x 106
time
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3
9 Gy
BM 1 x 104
time
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4
9 Gy
BM 1 x 104
time
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5
9 Gy
BM 1 x 104
time
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6
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7
Limiting dilution into W41
8
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Patterns of reconstitution at limiting dilution

• local, undetected in blood or tissues
• systemic, transient
• systemic, sustained

10
The stem cell basis of tissue permanence
11
Maintenance of proliferative tissues
growth
differentiation
proliferative potential
12
Maintenance of proliferative tissues
growth
differentiation
proliferative potential
13
Self-renewal
S
S
D

• requires cell division

14
The relationship between self-renewal and clonal
fate
15
Patterns of reconstitution at limiting dilution

• local, undetected in blood or tissues
• systemic, transient
• systemic, sustained

16
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
17
Permanent reconstitution by HSC at limiting
dilution
In 1 x 106 marrow cells 50 endure - LTRC
1
0
0
donor
1
0
1
20
40
100
0
weeks
18
Transient reconstitution by HSC at limiting
dilution
In 1 x 106 marrow cells 200 fade - STRC

• detection of a graft from a single HSC requires
  gt 109 progeny

19
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
20
Detection of "progenitors" in colony assays
BM cells
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Progenitors may self-renew
950 r
22
Regulation of self-renewal in HSC
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HSC numbers increase in regenerating marrow
Reconstitution of irradiated mice
1
0
0
cells
CFC

1
0
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o
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a
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1
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1
0
1
5
0
3
6
0
d
a
y
s
24
Expanded HSC can expand again
normal /animal
days
950 r
25
Stem cell expansion during serial transfer
26
Expansion versus maintenance
27
Self-renewal of HSC in vivo

• HSC number constant in the normal
  steady-state
• HSC number expands during marrow regeneration
• expansion can be extensive
• maintenance versus expansion mode governed by
  extrinsic signals

28
Stem cell extinction in culture
29
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
30
Self-renewal of HSC is regulated
culture with cytokines
steady-state in vivo
regeneration in vivo
31
Hierarchical organization of hematopoiesis
Nk
Macrophage
B
Dendritic
Osteoclast
Microglia
Kupfer
N
Langerhans
Eo
Baso/Mast
Pl
E
32
Hierarchical organization of hematopoiesis
Nk
Macrophage
B
Dendritic
Osteoclast
Microglia
Kupfer
N
Langerhans
Eo
Baso/Mast
Pl
E
33
Phenotypic anatomy of hematopoiesis
Nk
lo
Rho
ABCT
Macrophage
B
Dendritic
Osteoclast
Microglia
Kupfer
N
Langerhans
Eo
Baso/Mast
Pl
E
34
Phenotypic anatomy of hematopoiesis
CD34
B220
c-kit
CD3
Sca1
Eng
Thy1
Ly6A
lo
Nk
lo
Rho
Rho
ABCT
Macrophage
B
Dendritic
Osteoclast
Microglia
Kupfer
N
Langerhans
Flt3
CD49b
Eo
Baso/Mast
Pl
E
35
Gene systems specifying self-renewal in HSC
36
marrow
culture
96 hr
GFP
Ly5.2
37
HOXB4-expressing cells eventually dominate in
culture
100
50
10
38
HOXB4-expressing cells eventually dominate in vivo
39
HoxB4
HOXB4-expressing cells differentiate normally in
vivo
Control
40
HOXB4-expressing HSC regenerate fully in vivo
41
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?

42
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
43
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
44
LTRC and STRC 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
45
LTRC and STRC 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
46
A cDNA sample matrix - HSC
M
G1
Go
Gapdh
LTRC
STRC
47
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
48
Hox expression is linked to cell cycling
HoxA4
M
G1
Go
40 cycles
LTRC
STRC
Pentapotent
Tetrapotent
pE
/Meg
pNeut
/Mac
BFUE
pMeg
pMac
pNeut
CFUE
E
Meg
Mac
Neut
Mast
B
T
49
Implications of Hox expression pattern

• Hox induction may be central to execution of SR
• How do LTRC sustain Hox inducibility
• How do STRC lose it?

50
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

51
Pc required for segment-specific homeotic gene
repression in the fly
Pc
wt
Ubx protein
52
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
53
Why multiple homologs
A mechanism for broadening the repertoire of
cellular identities that can be specified?
54
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
55
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
56
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
57
Differential gene expression in LT and STRC
S
Q
S
S
D
Q
1
S
S
D
1
Q
D
D
D
3
1
1
Q
D
D
2
2
D
3
Hox
LTRC
PcG
STRC
Time
58
Differential gene expression in LT and STRC
S
Q
S
S
D
Q
1
S
S
D
1
Q
D
D
D
3
1
1
Q
D
D
2
2
D
3
Hox
LTRC
PcG
STRC
Time
PcG LTRC
Hox
PcG STRC
Hox