Embryonic skeletal muscle microexplant culture and isolation of skeletal muscle stem cells.
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.
Smith
Janet
J
School of Biosciences, University of Birmingham, B15 2TT Birmingham, UK.
Merrick
Deborah
D
D000818:Animals; D017209:Apoptosis; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D002999:Clone Cells / Q000166:cytology / Q000378:metabolism; D015925:Cryopreservation; D004622:Embryo, Mammalian; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D007621:Karyotyping; D008297:Male; D051379:Mice; D042282:Microdissection; D018482:Muscle, Skeletal / Q000166:cytology*; D034622:RNA Interference; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D014162:Transfection; D019076:Transgenes / Q000235:genetics