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Isolation and Grafting of Single Muscle Fibres

  • Protocol
Stem Cells in Regenerative Medicine

Part of the book series: Methods in Molecular Biology ((MIMB,volume 482))

Abstract

Satellite cells are mononucleate muscle precursor cells resident beneath the basal lamina, which surrounds each skeletal muscle fibre. Normally quiescent in adult muscle, in response to muscle damage satellite cells are activated and proliferate to generate a pool of muscle precursor cells, which subsequently differentiate and fuse together to repair and replace terminally differentiated muscle fibre syncytia. Cells prepared by enzymatic digestion of whole muscle tissue are likely to contain myogenic cells derived both from the satellite cell niche and from other populations in the muscle interstitium and vasculature. Single muscle fibre preparations, in which satellite cells retain their normal anatomical position beneath the basal lamina, are free of interstitial and vascular tissue and can therefore be used to investigate satellite cell behaviour in the absence of other myogenic cell types. Here, we describe methods for the isolation of viable muscle fibres and for grafting of muscle fibres and their associated satellite cells into mouse muscles to assess the contribution of satellite cells to muscle regeneration.

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References

  1. Studitsky, A.N. (1964) Free auto- and homografts of muscle tissue in experiments on animals. Ann N Y Acad Sci 120, 789–801.

    CAS  PubMed  Google Scholar 

  2. Chargé S.B., Rudnicki, M.A. (2004) Cellular and molecular regulation of muscle regeneration. Physiol Rev 84, 209–238.

    Article  PubMed  Google Scholar 

  3. Zammit, P.S., Partridge, T.A., Yablonka-Reuveni, Z. (2006) The skeletal muscle satellite cell: the stem cell that came in from the cold. J Histochem Cytochem 54, 1177–1191.

    Article  CAS  PubMed  Google Scholar 

  4. Mauro, A. (1961) Satellite cells of skeletal muscle fibres. J Biophys Biochem Cytol 9, 493–496.

    Article  CAS  PubMed  Google Scholar 

  5. Moss, F.P., Leblond, C.P. (1971) Satellite cells as a source of myonuclei in the muscles of growing rats. Anat Rec 170, 421–436.

    Article  CAS  PubMed  Google Scholar 

  6. Snow, M.H. (1978) An autoradiographic study of satellite cell differentiation into regenerating myotubes following transplantation of muscles in young rats. Cell Tissue Res 186, 535–540.

    Article  CAS  PubMed  Google Scholar 

  7. Beauchamp, J.R., Heslop, L., Yu, D.S., Tajbakhsh, S., Kelly, R.G., Wernig, A., Buckingham, M.E., Partridge, T.A. and Zammit, P.S. (2000) Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite cells. J Cell Biol 151, 1221–1234.

    Article  CAS  PubMed  Google Scholar 

  8. Seale, P., Sabourin, L.A., Girgis-Gabardo, A., Mansouri, A., Gruss, P. and Rudnicki, M.A. (2000) Pax7 is required for the specification of myogenic satellite cells. Cell 102, 777–786.

    Article  CAS  PubMed  Google Scholar 

  9. Zammit, P.S., Golding, J.P., Nagata, Y., Hudon, V., Partridge, T.A. and Beauchamp, J.R. (2004) Muscle satellite cells adopt divergent fates: a mechanism for self-renewal? J Cell Biol 166, 347–357.

    Article  CAS  PubMed  Google Scholar 

  10. Collins, C.A., Olsen, I., Zammit, P.S., Heslop, L., Petrie, A., Partridge, T.A. and Morgan, J.E. (2005) Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell 122, 289–301.

    Article  CAS  PubMed  Google Scholar 

  11. Shinin, V., Gayraud-Morel. B., Gomes, D., and Tajbakhsh, S. (2006) Asymmetric division and cosegregation of template DNA strands in adult muscle satellite cells. Nat Cell Biol 8, 677–687.

    Article  CAS  PubMed  Google Scholar 

  12. Asakura, A., Seale, P., Girgis-Gabardo, A., Rudnicki, M.A. (2002) Myogenic specification of side population cells in skeletal muscle. J Cell Biol 159, 123–134.

    Article  CAS  PubMed  Google Scholar 

  13. Sampaolesi, M., Torrente, Y., Innocenzi, A., Tonlorenzi, R., D'Antona, G., Pellegrino, M.A., Barresi, R., Bresolin, N., De Angelis, M.G., Campbell, K.P., Bottinelli, R. and Cossu, G. (2003) Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts. Science 301, 487–492.

    Article  CAS  PubMed  Google Scholar 

  14. Kuang, S., Chargé, S.B., Seale, P., Huh, M. and Rudnicki, M. (2006) Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis. J Cell Biol 172, 103–113.

    Article  CAS  PubMed  Google Scholar 

  15. Montarras, D., Morgan, J., Collins, C., Relaix, F., Zaffran, S., Cumano, A., Partridge, T. and Buckingham, M. (2005) Direct isolation of satellite cells for skeletal muscle regeneration. Science 309, 2064–2067.

    Article  CAS  PubMed  Google Scholar 

  16. Bischoff, R. (1975) Regeneration of single skeletal muscle fibers in vitro. Anat Rec 18, 215–235.

    Article  CAS  PubMed  Google Scholar 

  17. Konigsberg, U.R., Lipton, B.H. and Konigsberg I.R. (1975) The regenerative response of single mature muscle fibers isolated in vitro. Dev Biol 45, 260–275.

    Google Scholar 

  18. Rosenblatt, J.D., Lunt, A.I., Parry, D.J. and Partridge, T.A. (1995) Culturing satellite cells from living single muscle fibre explants. In Vitro Cell Dev Biol 31A, 773–779.

    Article  Google Scholar 

  19. Nagata, Y., Partridge, T.A., Matsuda, R. and Zammit, P.S. (2006) Entry of muscle satellite cells into the cell cycle requires sphingolipid signaling. J Cell Biol 174, 245–253.

    Article  CAS  PubMed  Google Scholar 

  20. Cornelison, D.D., Wold, B.J. (1997) Single-cell analysis of regulatory gene expression in quiescent and activated mouse skeletal muscle satellite cells. Dev Biol 191, 270–283.

    Article  CAS  PubMed  Google Scholar 

  21. Collins, C.A., Zammit, P.S., Ruiz, A.P., Morgan, J.E., Partridge, T.A. (2007) A population of myogenic stem cells that survives skeletal muscle aging. Stem Cells 25, 885–894.

    Article  CAS  PubMed  Google Scholar 

  22. Partridge, T.A., Morgan, J.E., Coulton, G.R., Hoffman, E.P. and Kunkel, L.M. (1989) Conversion of mdx myofibres from dystrophin-negative to -positive by injection of normal myoblasts. Nature 337, 176–179.

    Article  CAS  PubMed  Google Scholar 

  23. Kelly, R., Alonso, S., Tajbakhsh, S., Cossu, G. and Buckingham, M. (1995) Myosin light chain 3F regulatory sequences confer regionalized cardiac and skeletal muscle expression in transgenic mice. J Cell Biol 129, 383–396.

    Article  CAS  PubMed  Google Scholar 

  24. Tajbakhsh, S., Rocancourt, D., Buckingham, M. (1996) Muscle progenitor cells failing to respond to positional cues adopt non-myogenic fates in myf-5 null mice. Nature 384, 266–270.

    Article  CAS  PubMed  Google Scholar 

  25. Gross, J.G., Bou-Gharios, G., Morgan, J.E. (1999) Potentiation of myoblast transplantation by host muscle irradiation is dependent on the rate of radiation delivery. Cell Tissue Res 298, 371–375.

    Article  CAS  PubMed  Google Scholar 

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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Collins, C.A., Zammit, P.S. (2009). Isolation and Grafting of Single Muscle Fibres. In: Audet, J., Stanford, W.L. (eds) Stem Cells in Regenerative Medicine. Methods in Molecular Biology, vol 482. Humana Press. https://doi.org/10.1007/978-1-59745-060-7_20

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  • DOI: https://doi.org/10.1007/978-1-59745-060-7_20

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-797-6

  • Online ISBN: 978-1-59745-060-7

  • eBook Packages: Springer Protocols

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