Isolating and maintaining the appropriate stem cell for large scale cell culture is essential in tissue engineering or food production. For bovine satellite cells an optimized isolation and purification protocol is lacking and there is also no detailed understanding on the factors that maintain stemness of these cells. Here, we set up a fluorescence-activated cell sorting strategy to enrich bovine satellite cells. We found that p38-MAPK signalling is activated and PAX7 expression is gradually lost during satellite cell proliferation. The p38 inhibitor (SB203580) treatment maintained PAX7 expression but inhibited the fusion of satellite cells in a concentration-dependent way in short-term incubation. The mechanism of p38 inhibition was confirmed by inhibiting canonical p38 signalling, i.e. HSP27. Long-term culture with an appropriate concentration of p38i enhanced the proliferation and PAX7 expression, while the differentiation capacity recovered and was enhanced compared to vehicle control. These studies indicate that bovine satellite cells maintenance depends on cell purity and p38 MAPK signalling. Inhibition of p38 MAPK signaling is a promising strategy to facilitate large scale cell expansion of primary cells for tissue engineering and cultured meat purposes.

Pig is an important food source and an excellent system to model human diseases. Careful characterization of the swine skeletal muscle stem cells (satellite cells) will shed lights on generation of swine skeletal muscle disease model and efficient production of porcine meat for the food industry. Paired box protein 7 (Pax7) is a highly conserved transcription factor shared by satellite cells from various species. However, the sequence of Pax7 has not been characterized in pig. The lack of method to isolate highly purified satellite cells hinders the thorough characterization of the swine satellite cells. Here we found molecular markers for swine satellite cells and revealed that the porcine satellite cells were heterogeneous in various pieces of skeletal muscle. We further developed a method to isolate highly purified satellite cells directly from porcine muscles using fluorescence-activated cell sorting. We next characterized the proliferation and differentiation abilities of isolated satellite cells in vitro; and found that long-term culturing of satellite cells in vitro led to stemness loss.