isolation and manipulation of skeletal muscle stem...

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

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24.1

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24.1

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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

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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

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103

104

PE_CY7-A

100

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104

FITC-A

19.4

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PE_CY7-A

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FITC-A

9.26

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FITC-A

0.94

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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