Inhibition of GSK3β Promotes Proliferation and Suppresses Apoptosis of Porcine Muscle Satellite Cells

SIMPLE SUMMARY: This study was conducted to investigate the effect of the inhibitor and activator of the Wnt signaling pathway on the proliferation and maintenance of satellite cells in vitro. In this study, we isolated porcine muscle satellite cells (PMSCs) from a 1-day-old piglet and cultured PMSCs by treating the inhibitor (XAV939, Tankyrase inhibitor) and activator (CHIR99021, GSK3β inhibitor) of Wnt signaling. The results revealed that CHIR99021 promoted the proliferation of PMSCs by inhibiting MyoD expression while maintaining the expression of Pax7. CHIR99021 also suppressed apoptosis of PMSCs by regulating the expression of apoptosis-related proteins and genes. In essence, our findings indicate that the inhibition of GSK3β could promote the self-renewal of PMSCs and inhibit apoptosis. ABSTRACT: As the global population increases, interest in cultured meat (a new research field) is gradually increasing. The main raw material for the production of cultured meat is muscle stem cells called satellite cells isolated from livestock. However, how to mass proliferate and maintain satellite cells in vitro without genetic manipulation remains unclear. In the present study, we isolated and purified porcine muscle satellite cells (PMSCs) from the femur of a 1-day-old piglet and cultured PMSCs by treating them with an inhibitor (XAV939, Tankyrase (TNKS) inhibitor) or an activator (CHIR99021, glycogen synthase kinase 3 beta (GSK3β) inhibitor) of Wnt signaling. The CHIR group treated with 3 μM CHIR99021 showed a significantly increased proliferation rate of PMSCs compared to the SC group (control), whereas the XAV group treated with 1 μM XAV939 showed a significantly decreased proliferation rate of PMSCs. CHIR99021 also inhibited the differentiation of PMSCs by reducing the expression of MyoD while maintaining the expression of Pax7 and suppressed apoptosis by regulating the expression of apoptosis-related proteins and genes. RNA sequencing was performed to obtain gene expression profiles following inhibition or activation of the Wnt signaling pathway and various signaling mechanisms related to the maintenance of satellite cells were identified. Our results suggest that inhibition of GSK3β could dramatically improve the maintenance and mass proliferation ability of PMSCs in vitro by regulating the expression of myogenic markers and the cell cycle.