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Satellite Cells

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

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Satellite Cells

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  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 SC Function • Quiescent: withdrawn from cell cycle • Regeneration • SCs “activated” by damage • Re-enter cell cycle • Fully differentiate • Fusemyotubemuscle fiber • Hypertrophy/growth •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 contentmay limitRNA synthesisand proteincontent 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 Lox Lox Lox Start eGFP Stop DTA Stop Start 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 • 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 BLfacilitates 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 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 • 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 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 viaactive ABC transporters • Subset of many stem-likecell 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 (1% @ 18 hr) • Endocrine supplement • eg, insulin Dib & al., 2005

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