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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 thymidinelabeled 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– Myf5 (transcription factor) – Caveolin-1 Laminin M-cadherin Nucelus Irintchev & al 1994 SC Function • Quiescent: withdrawn from cell cycle • Regeneration – SCs “activated” by damage – Re-enter cell cycle – Fully differentiate – Fusemyotubemuscle fiber • Hypertrophy/growth –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 content may limit RNA synthesis and protein content 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 Start Lox eGFP Start Lox DTA Stop Lox Stop DTA Stop Pax7/DTA disrupts SC-function • BaCl2 kills myofibersregeneration • SC-mediated repair is disrupted in pax7/DTA Healthy muscle Injured & 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 chunks Kill fiber with marcaine Count SC (Bischoff, 1990) Satellite cell Basal Lamina shell Killed fiber clot 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-b family (TGF-b, 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-rafMEK-ERK Starvation Rb GSK3 E2F Cyclin-A DNA-pol’ase CDK1 DNA synthesis 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 Wnt receptor – Class A/general • TCF – Class B/Tissue specific M-cadherin GSK3b • MyoD • b-Catenin: bHLH-HDAC deactivator • Inhibitor of differentiation (Id) – HLH, no b – No b = no DNA binding Myostatin Smad2/3 Dishevelled Id3 B-catenin MyoD TCF/LEF Myogenin/MHC DNA synthesis Control of fusion • Scar/SNS/kette mediated actin foci 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 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