In vitro culture and induced differentiation of sheep skeletal muscle satellite cells

Abstract

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.

Comparative study of the effects of fetal bovine serum versus horse serum on growth and differentiation of primary equine bronchial fibroblasts

“…Cells in HS were smaller, chain-forming and more compact in shape with dark cytoplasmic granules which can be related to the occurrence of abnormal protein accumulation than those in FBS, and they failed to reach confluence. In concordance, sera types affect other various cell types in a similar way: bovine adipocytes and sheep skeletal muscle satellite cells proliferated rapidly, when FBS was supplemented to growth medium instead of HS; thus, FBS-containing medium is often used as growth medium in these cultures [31,32]. On the contrary, equine chondrocytes could equally proliferate under both conditions [33].…”

Myostatin knockout using zinc-finger nucleases promotes proliferation of ovine primary satellite cells in vitro

“…Isolated satellite cells were seeded on 2% matrigel (BD Biosciences, Bedford, MA) coated 6-well plates and cultured in growth medium (GM) containing DMEM supplemented with 20% fetal bovine serum and antibiotics at 37 • C and 5% carbon dioxide (CO 2 ). Identification of sheep PSCs was performed by immune-fluorescence as described by Wu et al (2012).…”

“…Cao). cells that have strong proliferation and differentiation capacities (Wu et al, 2012). Satellite cells are considered mitotically quiescent until stimulated by growth factors to proliferate, differentiate, and fuse to existing muscle fibers (Dodson et al, 1996).…”

Comparison and analysis of Wuding and avian chicken skeletal muscle satellite cells

“…Pax7 (Seale et al, 2000), M-Cadherin, Desmin, c-Met, MNF, MyoD (Musarò andBarberi, 2010;Wu et al, 2012), MyoG, and Myf5 are specific markers of skeletal muscle satellite cells. Pax7, M-Cadherin, and c-Met can be used to identify quiescent satellite cells and the molecular markers Myf5, MyoD, and Desmin are used to identify proliferating satellite cells.…”