isolation and manipulation of skeletal muscle stem...
TRANSCRIPT
Amy J. WagersJoslin Diabetes Center, Dept of Stem Cell and Regenerative Biology, Harvard
University and Harvard Stem Cell Institute
Isolation and Manipulation of SkeletalMuscle Stem Cells
Andreas Vesalius, De fabrica, 1543
Pax7/!1-integrin
• A little less than
half the body’s mass
is skeletal muscle.
Skeletal muscleSkeletal muscle
Andreas Vesalius, De fabrica, 1543
Muscle
Muscle fibers
Muscle fiber
MyofibrilSarcomere
Modified from McMahon, Muscles, Reflexes and Locomotion
Princeton University Press, 1984.
• Generates force/movement
• Voluntary
• Involuntary
• Regulates metabolism
• Glycogen storage
• Insulin uptake
• Catabolism (amino acid
supply)
Skeletal muscle: Skeletal muscle: FunctionsFunctions
Skeletal muscle regeneration
Uninjured Day 3 Day 7 Day 14
Injury
Pax7+
satellite
cell
Amy J. WagersJoslin Diabetes Center, Dept of Stem Cell and Regenerative Biology, Harvard
University and Harvard Stem Cell Institute
Muscle satellite cells
Pax7/!1-integrin
Hawke, T. J. et al. J Appl Physiol 91: 534-551 2001
Skeletal muscle regeneration
Uninjured Day 3 Day 7 Day 14
Injury
Pax7+
satellite
cell
Conboy and Rando, Dev Cell 3, 397 (2002)
Conboy et al., Science 302, 1575 (2003)
Sherwood et al., Cell (2004)
Isolation of skeletal muscle precursor(SMP) cells
Collagenase digestion and tituration
Interstitial cells
+
Single myofibersCollagenase/dispase digestion and tituration
Collagenase digestion and tituration
Interstitial cells
+
Single myofibersCollagenase/dispase digestion and tituration
My
ofi
ber
-ass
oci
ate
d c
ells
•Stain for cell surface markers
•Test myogenic activity of
sorted populations (in vitro)
Skeletal Muscle Precursor (SMP) cell surface markers
CXCR4 !1-integrin
Do not express: CD45
Mac-1
Sca-1
CD31
VE-cadherin
CD45-Sca-1-Mac-1-CXCR4+!1-integrin+ = CSM4B
or Skeletal Muscle Precursor = SMP
Sherwood et al., Cell, (2004)
hematopoietic
mesenchymal
vascular
(also CD34+
Syndecan4+
M-cadherin+)
Conboy and Rando, Dev Cell 3, 397 (2002)
Conboy et al., Science 302, 1575 (2003)
Sherwood et al., Cell (2004) Fibroblast-like colonies Myogenic colonies
Heterogeneity of myofiber-associated cells
Collagenase digestion and tituration
Interstitial cells
+
Single myofibersCollagenase/dispase digestion and tituration
Collagenase digestion and tituration
Interstitial cells
+
Single myofibersCollagenase/dispase digestion and tituration
My
ofi
ber
-ass
oci
ate
d c
ells
100
101
102
103
104
100
101
102
103
104
24.1
100
101
102
103
104
100
101
102
103
100
101
102
103
104
100
101
102
103
104
24.1
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
16.3
CXCR4
!1int.
CD45/Mac1
Sca-1
Non-myogenic
CD45-Mac-1- Sca-1+
Hematopoietic
CD45+
Skeletal muscle
Precursors (CSM4B)
Richard Sherwood, Massimiliano Cerletti
SMPs are the
only
autonomously
myogenic
cells
associated
with
myofibers.
SMPs are a unique myogenicpopulation highly enriched among
myofiber-associated cells.
Are SMPs the
stem cell subset
of adult skeletal
muscle satellite
cells?
SMPs??
SMPs express satellite cell markers, butnot differentiation markers
Massimiliano Cerletti
60x
Pax7 Dapi Merge
activation/differentiation markers
In vitro differentiation of SMPs
Clonal myogenesis in vitro
Sort CSMB4 stem cells
1 cell/well Desmin+Myogenic colony
Up to 80% of sorted cells
form myogenic colonies
day 5
Multinucleated myotubes
MyHC+
Massimiliano Cerletti
!SMPs are committed myogenic cells.
SMPs are committed myogenic cells
Richard Sherwood
Sca1+ cells are fibrogenic and adipogenic.
SMPs never form fibroblasts or adipocytes.
SMPs
Sca1+
Myogenic cultureAdipogenic
culture
•originally identified as a spontaneous mutant.
•nonsense mutation in dystrophin exon 23 truncates the protein.
•dystrophin protein absent at the muscle cell membrane.
•Sicinski et al. Science (1989)
Muscular Dystrophy in mdx mice
mdx WT
WTmdx
H&E
Dystrophin
staining
Engraftment of GFP+ donor SMPcells into recipient mdx mice
MFA
isolation
GFP+ donor
SMP cells
IM transplant
Recipient
immunocompetent
mdx mice
CDTX
SMPs restore dystrophinexpression in mdx mice
MFAisolation
GFP+ donorCSMB4 cells
IM transplant
Recipient
mdx mice
WT
mdx+GFP+ SMPs
mdx
Average regenerative index:
1 myofiber for every 28 SMPs injected
Grafts maintained at least 4 mo.
SMPs initiate a lineage of myogenicdifferentiation
DN
DN
DN
SMPs
SMPs DN
DN
DN
DN
DN
DN
SMPs
SMPs
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
CXCR4
ß1
SMPs
DN
SMPsDN
40X
i.m.
transplant
Transplant function
!Only SMPs show robust in vivo engraftment.
Massimiliano Cerletti
Merged image GFP (donor) HcRed (host)
Transplanted GFP+ SMPs both fuse with existingmyofibers and undergo de novo myogenesis
MFA
isolationGFP+ donor
CSMB4 cells
IM transplant
Recipient
HcRed+ mice
1.8% of donor-derived fibers express GFP only.
10x
60x
SMP transplantation
• SMPs engraft robustly in dystrophic
muscle
– Form both hybrid and de novo fibers
– Restore dystrophin expression
– Improve muscle histology
Does SMP-engrafted muscle
show improved function?
Restoration of function inSMP-transplanted mdx muscle
!Force measured at optimal muscle length and
normalized to muscle cross-sectional area.
!SMP transplanted muscle compared to mock-
transplanted muscle from contralateral leg.
MFA
isolationGFP+ donor
CSMB4 cells
IM transplant
Recipient
mdx mice
Carol Witczak, Mike Hirshman, Massimiliano Cerletti, Laurie Goodyear
SMPs restore function in transplanteddystrophic (mdx) mice
Uninjected (0%GFP+)
y = 4.86x + 0.27
R2 = 0.68
P<0.001
0
1
2
3
4
5
6
7
8
0% 20% 40% 60% 80% 100%
% GFP+ myofibe rs
Fo
ld c
han
ge in
avera
ge
inte
gra
ted
are
a u
nd
er
the c
urv
e
y = 4.33x + 0.36
R2 = 0.71
P<0.001
0
1
2
3
4
5
6
0% 20% 40% 60% 80% 100%
% GFP+ myofibers
Fo
ld c
ha
ng
e i
n a
ve
rag
e
sp
ec
ific
pe
ak
fo
rce
High (94%GFP+)Medium (60.6%GFP+)
A) B)
C)
40x
Low (7.4%GFP+)
SMP transplanted
Stem Cells - definitionStem Cells - definition
!Are unspecialized cells that candifferentiate to generate specialized cells.
!Self-renew and thus maintain cellreplacement potential for long periods oftime.
100
101
102
103
104
PE_CY7-A
100
101
102
103
104
FITC-A
19.4
100
101
102
103
104
PE_CY7-A
100
101
102
103
104
FITC-A
9.26
100
101
102
103
104
PE_CY7-A
100
101
102
103
104
FITC-A
0.94
100
101
102
103
104
PE_CY7-A
100
101
102
103
104
FITC-A
20.4
Untreated Day 3 Day 7 1 Month
Renewal of SMP Populationafter CDTX Injury
SMPs
Injury
SMPs
Sara Jurga
0
20
40
60
80
100
% GFP+ % GFP-
% G
FP
+ o
r G
FP
- cells a
mo
ng
Pax7+
(per
GF
P+
myo
fib
er)
MFA
isolationGFP+ donor
CSMB4 cells
IM transplant
Recipient
mdx mice
Transplanted SMPs re-engraft asstem cells in the host muscle
Single myofiber staining
MergePax7 Dapi GFP+
100x
Transplanted SMPs re-engraft asstem cells in the host muscle
MFA
isolationGFP+ donor
CSMB4 cells
IM transplant
Recipient
mdx mice
0
200
400
600
800
uninjured injured
Avera
ge #
of
GF
P+
Myo
fib
ers
per
clu
ste
r
*P <0.05
*
Re-injured muscle
Massimiliano Cerletti
Stem Cells - definitionStem Cells - definition
!Are unspecialized cells that candifferentiate to generate specialized cells.
!Self-renew and thus maintain cellreplacement potential for long periods oftime.
SMPs are muscle stem cells• SMPs are prospectively isolatable stem
cells for the skeletal muscle.
– Present in satellite cell compartment
– Express early but not late myogenic markers
– Exhibit robust, lineage-selective myogenicdifferentiation in vitro and in vivo
– Self-renew within the satellite cell niche
• SMPs are relevant targets for cell-basedtherapy in muscle, either by directtransplantation….
– Can achieve high level muscle chimerism
– Can enhance muscle function
or by manipulation of endogenous function.
Performance Declines with Aging
--despite maintenance of physical activity
Age (years)
10 20 30 40 50 60
Pe
rfo
rma
nce
(%
of
pe
ak)
0
20
40
60
80
100
Shotput/Discus
Marathon
Basketball (rebounds/game)
D.H. Moore (1975) Nature 253:264-265.
NBA Register, 1992-1993 Edition
Performance Declines with Aging
--despite maintenance of physical activity
Young SMPs
Aged SMPs
regeneration
ageing
Young
Aged
What causes age-dependentdefects in stem cell function and
are they REVERSIBLE?
regenerationX
Heterochronic parabiosis
• Cross-circulation is established 2-3 days after joining.
• Blood chimerism reaches ~50% by 7-10 days.
• Rapid exchange (~1%/min.) of cells and factors across
the vascular junction.Wright et al., 2001
Young
Old
Y YO OY O YO
Restoration of muscle regeneration in agedmice exposed to a young circulatory system
Conboy et al., Nature, 2005
Young SMPs
Aged SMPs
regeneration
ageing
Young
Aged
regeneration
Systemic
factors
X
What causes age-dependentdefects in stem cell function and
are they REVERSIBLE?
Implications
• Identification of the relevant age-
related factors could restore or
maintain healthy tissue function in
aging individuals.
• Alterations in an aged (or diseased)
environment may limit the function
of young, healthy cells transplanted
into aged tissue.
Massimiliano Cerletti Sara Jurga Jennifer Shadrach
Other Wagers lab members: Shane Mayack, Kah Yong Tan, Simone Hettmer,
Michael Lin, Claudia DallOsso, Arthur Young
Flow Core: Joyce LaVecchio and Girijesh Buruzula
Collaborators: Carol Witczak, Mike Hirshman, Laurie Goodyear, Lizi Wu,
Richard Sherwood, Irina Conboy, Tom Rando, Irv Weissman
Funding: HSCI, Jain Foundation, Burroughs-Wellcome Fund, Keck Foundation