In Vitro Culture And Induced Differentiation Of Sheep Skeletal Muscle Satellite Cells

Skeletal muscle satellite cells are adult muscle-derived stem cells receiving increasing attention. Although skeletal muscle satellite cells successfully isolated from several species, such as mouse rat and human, the study for livestock (cattle and sheep) is rarely. In livestock transgenic research and production process, before the transgenic animal production, the expression of the target gene in the donator cells should be identified and verified. Therefore, the establishment of the skeletal muscle satellite cells model of livestock in vitro appears to be particularly important and urgent.In this study, we investigated the in vitro culture and differentiation of sheep skeletal muscle satellite cells in order to establish a sheep skeletal muscle satellite cells line. Our findings provide an experimental basis for the research and application of skeletal muscle satellite cells in other fields such as livestock breeding and regenerative medicine.We have used2-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 the0.1%type Ⅰ collagenase and0.25%trypsin2-step digestion method, with the best conditions for in vitro culture being in medium containing20%FBS+10%horse serum.Immunofluorescence staining and Flow cytometry showed that the isolated cells expressed Desmin, a-Sarcomeric Actinin, MyoDl, Myf5and PAX7, and the cells is skeletal muscle satellite cells.In order to verify the pluripotency of sheep skeletal muscle satellite cells, the cells would be induced to myoblast, osteoblast and adipocyte. After myogenic induction, multinucleated myotubes formed, as indicated by the expression of MyoGand 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 PPARy2(peroxisome-proliferator-activated receptor y2) and clear lipid droplets were present around the cells, with Oil Red-O staining giving a positive result. The results showed that the isolated cells have pluripotency.In summary, a successful system has been established for the isolation, purification and identification of sheep skeletal muscle satellite cells.