Skip to main content
SpringerLink
Log in

Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

  • Protocol
  • First Online:
Mouse Cell Culture

Part of the book series: Methods in Molecular Biology ((MIMB,volume 633))

Abstract

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The microdissected explant technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used microdissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The microexplant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors. Uniquely, microexplant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the microexplant method.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Yaffe, D., and Saxel, O. (1997) Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270, 725–727.

    Article  Google Scholar 

  2. Askanas, V., and Engel, W. K. (1975) A new program for investigating adult human skeletal muscle grown aneurally in tissue culture. Neurology 25, 58–67.

    CAS  PubMed  Google Scholar 

  3. Smith, J., and Schofield, P. N. (1994) The effects of fibroblast growth factors in long-term primary culture of dystrophic (mdx) mouse muscle myoblasts. Exp. Cell. Res. 210, 86–93.

    Article  CAS  PubMed  Google Scholar 

  4. Fell, H. (1958) The Cell in Culture. J. Clin. Pathol. 11, 489–495.

    Article  CAS  PubMed  Google Scholar 

  5. Stewart, D. C., and Kirk, P. L. (1952) The simultaneous measurement of several parameters of embryo heart explant growth in vitro. J. Cell. Physiol. 40, 183–198.

    Article  CAS  PubMed  Google Scholar 

  6. Harvey, A. L., Robertson, J. G., and Witkowski, J. A. (1979) Maturation of human skeletal muscle fibres in explant tissue culture. J. Neurol. Sci. 41, 115–122.

    Article  CAS  PubMed  Google Scholar 

  7. Zammit, P. S., Heslop, L., Hudon, V., Rosenblatt, J. D., Tajbakhsh, S., Buckingham, M. E., Beauchamp, J. R., and Partridge, T. A. (2002) Kinetics of myoblast proliferation show that resident satellite cells are competent to fully regenerate skeletal muscle fibers. Exp. Cell Res. 281, 39–49.

    Article  CAS  PubMed  Google Scholar 

  8. Konigsberg, I. (1986) The embryonic origin of muscle. McGraw-Hill, New York.

    Google Scholar 

  9. Trainor, P. A., Tan, S. S., and Tam, P. P. (1994) Cranial paraxial mesoderm: regionalisation of cell fate and impact on craniofacial development in mouse embryos. Development 120, 2397–2408.

    CAS  PubMed  Google Scholar 

  10. Tajbakhsh, S., Rocancourt, D., Cossu, G., and Buckingham, M. (1997) Redefining the genetic hierarchies controlling skeletal myogenesis: Pax-3 and Myf-5 act upstream of MyoD. Cell 89, 127–138.

    Article  CAS  PubMed  Google Scholar 

  11. Tremblay, P., Dietrich, S., Mericskay, M., Schubert, F. R., Li, Z., and Paulin, D. (1998) A crucial role for Pax3 in the development of the hypaxial musculature and the long-range migration of muscle precursors. Dev. Biol. 203, 49–61.

    Article  CAS  PubMed  Google Scholar 

  12. Tajbakhsh, S., Bober, E., Babinet, C., Pournin, S., Arnold, H., and Buckingham, M. (1996) Gene targeting the myf-5 locus with nlacZ reveals expression of this myogenic factor in mature skeletal muscle fibres as well as early embryonic muscle. Dev. Dyn. 206, 291–300.

    Article  CAS  PubMed  Google Scholar 

  13. Tam, P. P. (1986) A study of the pattern of prospective somites in the presomitic mesoderm of mouse embryos. J. Embryol. Exp. Morphol. 92, 269–285.

    CAS  PubMed  Google Scholar 

  14. Cossu, G., Kelly, R., Di Donna, S., Vivarelli, E., and Buckingham, M. (1995) Myoblast differentiation during mammalian somitogenesis is dependent upon a community effect. Proc. Natl. Acad. Sci. USA 92, 2254–2258.

    Article  CAS  PubMed  Google Scholar 

  15. Shainberg, A., Yagil, G., and Yaffe, D. (1969) Control of myogenesis in vitro by Ca 2 + concentration in nutritional medium. Exp. Cell Res. 58, 163–167.

    Article  CAS  PubMed  Google Scholar 

  16. Dodson, M. V., Martin, E. L., Brannon, M. A., Mathison, B. A., and McFarland, D. C. (1987) Optimization of bovine satellite cell-derived myotube formation in vitro. Tissue Cell 19, 159–166.

    Article  CAS  PubMed  Google Scholar 

  17. Smith, J. (1996) Muscle Growth factors, ubiquitin and apoptosis in dystrophic muscle: Apoptosis declines with age in the mdx mouse. B. Appl. Myol. 6, 279–284.

    Google Scholar 

  18. Smith, J., Fowke, G., and Schofield, P. (1995) Programmed cell death in dystrophic (mdx) muscle is inhibited by IGF-II. Cell Death Differ. 2, 243–251.

    CAS  PubMed  Google Scholar 

  19. Smith, J., and Schofield, P. N. (1997) Stable integration of an mdx skeletal muscle cell line into dystrophic (mdx) skeletal muscle: evidence for stem cell status. Cell Growth Differ. 8, 927–934.

    CAS  PubMed  Google Scholar 

  20. O’Shea, L., Johnson, C., Rooney, M., Gleeson, R., Woods, K., and Smith, J. (2001) Adipogenesis and skeletal muscle ageing. In: Abstracts ‘Stem cells, stress and senescence: Abstracts of presentations at the Annual Scientific Meeting of the BSRA held in London on 14 July 2000’. Mech. Ageing Dev. 122, 1354–1355.

    Google Scholar 

  21. Merrick, D. (2006) A role for Igf-2 in fast skeletal muscle specification during myogenesis in dystrophic and wild type embryos. PhD Thesis, University of Birmingham.

    Google Scholar 

  22. Stadler, L. K. J., Merrick, D., and Smith, J. (2008) Morphological, stem cell and fast myosin abnormalities in the cav-3 –/– and mdx dystrophic embryo reveal an embryonic basis for muscular dystrophy. Abstr. Genet. Res. 90, 281–289, Cambridge University Press.

    Google Scholar 

  23. Merrick, D., Stadler, L. K. J., Larner, D. P., and Smith, J. (2009) Muscular dystrophy begins early in embryonic development deriving from stem cell loss and disrupted skeletal muscle formation. Dis. Model Mech. 2(7–8), 374–388.

    Google Scholar 

  24. Matschak, T., Stickland, N. C., and Smith, J. (1997) Explants of embryonic Atlantic salmon muscle in culture. Proc. Soc. Exp. Biol. A 1, 23.

    Google Scholar 

  25. Smith, J., and Hooper, M. L. (1989) Dominance and independent segregation of metabolic cooperation – competence and pluripotency in an embryonal carcinoma cell hybrid. Exp. Cell Res. 181, 40–50.

    Article  CAS  PubMed  Google Scholar 

  26. Bulfield, G., Siller, W. G., Wight, P. A., and Moore, K. J. (1984) X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc. Natl. Acad. Sci. USA 81, 1189–1192.

    Article  CAS  PubMed  Google Scholar 

  27. Hagiwara, Y., Sasaoka, T., Araishi, K., Imamura, M., Yorifuji, H., Nonaka, I., Ozawa, E., and Kikuchi, T. (2000) Caveolin-3 deficiency causes muscle degeneration in mice. Hum. Mol. Genet. 9, 3047–3054.

    Article  CAS  PubMed  Google Scholar 

  28. Westbury, J., Watkins, M., Ferguson-Smith, A. C., and Smith, J. (2001) Dynamic temporal and spatial regulation of the cdk inhibitor p57(kip2) during embryo morphogenesis. Mech. Dev. 109, 83–89.

    Article  CAS  PubMed  Google Scholar 

  29. Merrick, D., Ting, T., Stadler, L. K. J., and Smith, J. (2007) A role for Insulin-like growth factor 2 in specification of the fast skeletal muscle fibre. BMC Dev. Biol. 7, 65

    Article  PubMed  Google Scholar 

  30. Paddison, P. J., Caudy, A. A., and Hannon, G. J. (2002) Stable suppression of gene expression by RNAi in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 1443–1448.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Patrick Paddison for his gift of the shRNAi shuttle vector. Angela Sloan generated the GFP RNAi image in Fig. 3.3. We also thank the following funding bodies for their support: Muscular Dystrophy Campaign grant number RA2/592/2; SPARKS grant number 02BHM04, The Royal Society grant number 574006.G503/1948./JE and BBSRC grant number 6/SAG10077.

Author information

Authors and Affiliations

  1. School of Biosciences, University of Birmingham, B15 2TT, Birmingham, UK

    Janet Smith & Deborah Merrick

Authors
  1. Janet Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Deborah Merrick
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Dept. Biology & Biochemistry, University of Bath, Claverton Down, Bath, BA2 7AY, United Kingdom

    Andrew Ward

  2. Dept. Biology & Biochemistry, University of Bath, Claverton Down, Bath, BA2 7AY, United Kingdom

    David Tosh

Rights and permissions

Reprints and permissions

Copyright information

© 2010 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Smith, J., Merrick, D. (2010). Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells. In: Ward, A., Tosh, D. (eds) Mouse Cell Culture. Methods in Molecular Biology, vol 633. Humana Press. https://doi.org/10.1007/978-1-59745-019-5_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-019-5_3

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-772-3

  • Online ISBN: 978-1-59745-019-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation