Satellite Cells Define the myonuclear domain Describe the satellite cell niche Describe the signaling networks controlling SC proliferation and fusion.

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

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

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-cadherinNucelusIrintchev & al 1994

SC Function• Quiescent: withdrawn from cell cycle• Regeneration

– SCs “activated” by damage– Re-enter cell cycle– Fully differentiate– Fusemyotubemuscle fiber

• Hypertrophy/growth– Re-enter cell cycle(?)– Fusemuscle fiber

Myonuclear domain• Volume of cytoplasm supported by one nucleus

– Conserved during growth– Consistent across body size

• DNA contentmay limitRNA synthesisand proteincontent

Liu & al., 2008

SC contribution to hypertrophy• Adams & al., 2002• Irradiation to silence SC• Synergist ablation• Irradiation blocks

substantial growth• MN domain conserved

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

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

eGFP DTAStart Stop StopLox Lox

DTAStart StopLox

Pax7/DTA disrupts SC-function• BaCl2 kills myofibersregeneration• SC-mediated repair is disrupted in pax7/DTA

Healthy muscleInjured & repaired muscle (centrally-located nuclei)Injured & not repaired muscle (unstructured fibrosis)

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

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

Satellite cell niche• Niche: physical environment

– 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

Contact control of SC• Contact with fiber blocks proliferation• Contact with BL

facilitates proliferation• Ground-up muscle helps

Dissociate muscle into Fiber-BL chunksKill fiber with marcaineCount SC (Bischoff, 1990)

Killed fiber clotBasal Lamina shell

Satellite cell

SC Fate• Fiber repair• Self renewal

Seale & Rudnicki, 2000

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

Activation of quiescent SCs• Some signal is released from damaged muscle

– bFGF? HGF?

• Cell cycle re-entry (SCadult myoblast)– PI3K-mTORgrow the cell– ERKcell cycle progression– MyoD/myf5be a muscle

• Negative controls– Inhibit differentiation– TGF- family (TGF-, BMP)– Myostatin

Proliferation• 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

GF-R(c-Met, FGFR)

Ras-raf-MEK-ERK

Rb

E2F

Cyclin-ADNA-pol’ase

CDK1

DNA synthesis

Starvation

GSK3

Differentiation• Cell cycle withdrawal

– M-cadherinb-catenin/TCF– MYf5/myogeninterminal differentiation

• Fusion or quiescence– Pax3/7MRF suppressor– Cytoskeletal rearrangement– Contact recognition of fusion partner

Differentiation• 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

Wnt receptor

Dishevelled

GSK3b

B-catenin

TCF/LEF

DNA synthesis

MyoD

Myogenin/MHCMyostatin Smad2/3 Id3

M-cadherin

Control of fusion• Scar/SNS/kette mediated actin foci

Muscle and other stem cells• Pluripotency• The Side Population

– Exclude Hoechst dyes viaactive ABC transporters

– Subset of many stem-likecell populations

• Muscle SP cells– CD45/Sca1 positive– Pluripotent

• Muscle MP cells– Unipotent

Asakura & al, 2002

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?

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)

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

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 (1% @ 18 hr)

• Endocrine supplement– eg, insulin

Dib & al., 2005