Abstract: In this study, four different initial densities (2.7 × 103 as a control, 5.4 × 103, 1.1 × 104, and 2.2 × 104 cells/cm2) of porcine muscle stem cells were cultured for three days to explore the effect of high-density culture conditions on the fate of porcine muscle stem cells. The counting results showed that the suitable culture density of porcine muscle stem cells was 2 × 104–3 × 104 cells/cm2, and the proliferation-related protein phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) was down-regulated when the initial cell density was outside this range, causing a proliferation inhibitory effect. The transcriptome sequencing results showed that 2 125 differentially expressed genes were identified between the highest density and control groups, mainly enriched in some signaling pathways such as those related to cell senescence and cell cycle and the PI3K-Akt signaling pathway. Furthermore, the expression of the stemness genes PAX7 and PAX3 and the quiescence-related gene SPRY1 was upregulated under high-density conditions. The expression of the differentiation genes MYOD, MYOG and MYH3 and the senescence genes P21, P53 and P15 was upregulated. The results of real-time PCR and Western blot assay showed that the expression of PAX7, SPRY1, MYOG, and P21 were upregulated as the cell density increased. The above results showed that porcine muscle stem cells had multiple fats such as returning to a quiescent state, differentiation and senescence under high-density conditions. The results of this study can help in understanding the mechanism of cell fate determination and provide a theoretical basis for studying the regulation of cell proliferation and differentiation under high-density condition.

Key words: muscle stem cell; high-density culture; senescence; proliferation; stemness; RNA sequencing