|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
In vitro culture and induced differentiation of sheep skeletal muscle satellite cells
Haiqing Wu, Yu Ren, Shuo Li, Wei Wang, Jianlong Yuan, Xudong Guo, Dongjun Liu and Ming Cang1
Key Laboratory of Mammalian Reproductive Biology and Biotechnology Ministry of Education, Inner Mongolia University, Inner Mongolia, Hohhot 010021, Peoples Republic of China
Skeletal muscle satellite cells are adult muscle-derived stem cells receiving increasing attention. Sheep satellite cells have a greater similarity to human satellite cells with regard to metabolism, life span, proliferation and differentiation, than satellite cells of the rat and mouse. We have used 2-step enzymatic digestion and differential adhesion methods to isolate and purify sheep skeletal muscle satellite cells, identified the cells and induced differentiation to examine their pluripotency. The most efficient method for the isolation of sheep skeletal muscle satellite cells was the type I collagenase and trypsin 2-step digestion method, with the best conditions for in vitro culture being in medium containing 20% FBS+10% horse serum. Immunofluorescence staining showed that satellite cells expressed Desmin, α-Sarcomeric Actinin, MyoD1, Myf5 and PAX7. After myogenic induction, multinucleated myotubes formed, as indicated by the expression of MyoG and fast muscle myosin. After osteogenic induction, cells expressed Osteocalcin, with Alizarin Red and ALP (alkaline phosphatase) staining results both being positive. After adipogenic induction, cells expressed PPARγ2 (peroxisome-proliferator-activated receptor γ2) and clear lipid droplets were present around the cells, with Oil Red-O staining giving a positive result. In summary, a successful system has been established for the isolation, purification and identification of sheep skeletal muscle satellite cells.
Key words: adipogenesis, identification, myoblast, osteogenic, sheep, skeletal muscle satellite cell
Abbreviations: ALP, alkaline phosphatase, CEE, chick embryo extract, DMEM/F12, Dulbecco's modified Eagle's medium/Ham's nutrient mixture F-12, ECM, extracellular matrix, FBS, fetal bovine serum, GAPDH, glyceraldehyde-3-phosphate dehydrogenase, MDSC, muscle-derived satellite cell, MRF, myogenic regulatory factor, PPARγ2, peroxisome-proliferator-activated receptor γ2, RT–PCR, reverse transcription–PCR
1To whom correspondence should be addressed (email email@example.com).
Received 11 September 2011/14 November 2011; accepted 11 January 2012
Published as Cell Biology International Immediate Publication 11 January 2012, doi:10.1042/CBI20110487
© The Author(s) Journal compilation © 2012 International Federation for Cell Biology