1. Satellite Cells Define the myonuclear domain
Describe the satellite cell niche
Describe the signaling networks controlling SC proliferation and fusion
HGF, FGF, myostatin
MRFs, pax

2. Satellite cell Anatomical definition Mononucleated cells in muscle
Oustide the fiber plasma membrane
Inside the basal lamina
EM of SC on muscle fiber; Dark thymidine-labeled new DNA. Moss & Leblond, 1971

3. Satellite Cell: Molecular definition
Unique
c-Met (HGF receptor)
M-cadherin (adhesion molecule)
Pax3/7 (transcription factor)
Overlapping
CD34 (hematopoetic)
Integrin-a7 (adhesion molecule)
Myf5 (transcription factor)
Caveolin-1
Laminin
M-cadherin
Nucelus
Irintchev & al 1994

4. SC Function Quiescent: withdrawn from cell cycle Regeneration
SCs “activated” by damage
Re-enter cell cycle
Fully differentiate
Fusemyotubemuscle fiber
Hypertrophy/growth
Re-enter cell cycle(?)
Fusemuscle fiber

5. Myonuclear domain Volume of cytoplasm supported by one nucleus
Conserved during growth
Consistent across body size
DNA content may limit RNA synthesis and protein content
Liu & al., 2008

6. SC contribution to hypertrophy
Adams & al., 2002
Irradiation to silence SC
Synergist ablation
Irradiation blocks substantial growth
MN domain conserved

7. Signaling during Ir-hypertrophy
Early (0-3 days)
Muscle IGF-1
Myogenin
Persistent
MGF/myogenin
mTOR effectors
Both groups start the same, but Ir deviates after a few days. Fails to maintain trajectory

8. Another model of SC ablation
McCarthy & al 2011
SC-specific, Tamoxifen-inducible diptheria toxin
“Cre-Lox” recombination
Cre recombinase targets Lox sequences for recombination
Tamoxifen-dependent
Rosa26/DTA: diptheria toxin preceded by floxed transcription stop sequence
Pax7/Cre: satellite-cell specific Cre
Start
Lox
eGFP
Stop
Lox
DTA
Stop
Start
Lox
DTA
Stop

9. Pax7/DTA disrupts SC-function
BaCl2 kills myofibersregeneration
SC-mediated repair is disrupted in pax7/DTA
Healthy muscle
Injured & repaired muscle
(centrally-located nuclei)
Injured & not repaired muscle
(unstructured fibrosis)

10. Pax7/DTA synergist ablation
DTA fails to block substantial growth
MN domain increases

11. Irradiation vs DTA questions
Irradiation kills both SC and bone marrow
Is the initial FO response to the injury or to the overload?
Most clear DTA results at 2 weeks, when edema dominates protein accretion

12. Satellite cell niche Niche: physical environment Basal Lamina/ECM
Circulating growth factors are only part
Physical contacts: myofiber and ECM
Paracrine factors: fiber, inflammatory cells
Mechanical factors
Basal Lamina/ECM
Collagen/laminin
Heparin sulfate proteoglycans
Adhesive substrates
Growth factor chelators
Receptor cofactors

13. Contact control of SC Contact with fiber blocks proliferation
Contact with BL facilitates proliferation
Ground-up muscle helps
Dissociate muscle into Fiber-BL chunks
Kill fiber with marcaine
Count SC (Bischoff, 1990)
Satellite cell
Basal Lamina shell
Killed fiber clot

14. SC Fate
Fiber repair
Self renewal
Seale & Rudnicki, 2000

15. Regulatory control of fate
Proliferation
Cell cycle progression
HGF/FGF
MyoD/Myf5
Differentiation
IGF-1/PGF2a
Myogenin/MRF4
Loss of Pax3/7
Charge & Rudnicki, 2004

16. Activation of quiescent SCs
Some signal is released from damaged muscle
bFGF? HGF?
Cell cycle re-entry (SCadult myoblast)
PI3K-mTORgrow the cell
ERKcell cycle progression
MyoD/myf5be a muscle
Negative controls
Inhibit differentiation
TGF-b family (TGF-b, BMP)
Myostatin

17. Proliferation Mitogens DNA synthesis Cell size integration
GF-R
(c-Met, FGFR)
Mitogens
FGF, EGF, HGF
DNA synthesis
CyD/CDK4 accumulation
Degradation of Rb
Activation of E2F
Cell size integration
GSK3 inhibits CDK4
GSK3 inhibits b-Catenin
Ras-raf-MEK-ERK
Starvation Rb
GSK3
E2F
Cyclin-A
DNA-pol’ase
CDK1
DNA synthesis

18. Differentiation Cell cycle withdrawal Fusion or quiescence
M-cadherinb-catenin/TCF
MYf5/myogeninterminal differentiation
Fusion or quiescence
Pax3/7MRF suppressor
Cytoskeletal rearrangement
Contact recognition of fusion partner

19. Differentiation bHLH transcription factors
Wnt receptor
bHLH transcription factors
Class A/general
TCF
Class B/Tissue specific
MyoD
b-Catenin: bHLH-HDAC deactivator
Inhibitor of differentiation (Id)
HLH, no b
No b = no DNA binding
Dishevelled
M-cadherin
GSK3b
B-catenin
TCF/LEF
MyoD
DNA synthesis
Myogenin/MHC
Myostatin
Smad2/3
Id3

20. Control of fusion
Scar/SNS/kette mediated actin foci

21. Muscle and other stem cells
Pluripotency
The Side Population
Exclude Hoechst dyes via active ABC transporters
Subset of many stem-like cell populations
Muscle SP cells
CD45/Sca1 positive
Pluripotent
Muscle MP cells
Unipotent
Asakura & al, 2002

22. Muscle and other stem cells
Hematopoetic stem cells
Bone marrow derived
Mostly WBC
0.2% SP, pluripotent
May contribute to regen in many tissues
eg: chimeric neurons after BMT
Are muscle SP cells really satellite cells?
Do MP and SP cells contribute equally to regeneration? Hypertrophy?

23. Satellite cell therapies
Muscle normally incorporates new genetic material from proliferating cells
Grow (and engineer) myoblast population
Inject & allow fusion
None of them work, yet.
Some progress in animal models
Skuk & Tremblay, 2003)

24. Myoblast transplantation (DMD)
Correction of genetic defects (Dystrophin)
1e7 donor SC in 100 injections to one muscle
CsA as immunosuppressant
6 month follow-up
Force gain (CsA)
No dystrophin
Miller & al., 1997

25. Satellite cell therapies
Cardiovascular support
Autograft during LVAD implant
1e7-1e8 cells in 3-30 injections into infarct
Some apparent survival > 6 mo
Low efficiency 18 hr)
Endocrine supplement
eg, insulin
Dib & al., 2005