Transcriptome profiles of bovine muscle stem cells cultured in serum-containing condition and small molecule–supplemented serum-free media by RNA-seq analysis

Cultured meat, as an emerging sustainable food production technology, offers multiple advantages. However, its large-scale application still faces critical technical bottlenecks, most notably the strong reliance on fetal bovine serum (FBS). In recent years, serum-free culture systems for bovine muscle stem cells (bMuSCs) have made certain progress, such as the Beefy-9 system and its subsequent optimized versions; nevertheless, existing serum-free formulations remain insufficient to fully replace traditional serum-containing culture conditions in both cost control and functional performance. Meanwhile, the use of small-molecule compounds to precisely regulate cell proliferation and fate determination has gradually emerged as a potentially effective strategy to reduce culture costs, simplify culture systems, and enhance functional stability. In this study, through a functional screening of proliferation activators in bMuSCs, we identified forskolin and a p38 pathway inhibitor SB202190 to robustly promote cell proliferation and maintain myogenic capacity of bMuSCs under serum-free conditions. Based on this, RNA-sequencing analysis was committed to systematically elucidate the molecular differences between serum-free small-molecule regulatory strategies and conventional serum-containing conditions in bMuSCs. Overall design: BMuSCs with biological replicates were collected simultaneously for RNA extraction and RNA-seq analysis. Specifically, at the fifth passage of proliferation (corresponding to three consecutive passages under different culture conditions), cells cultured in medium containing 15% FBS (the FBS group), in Beefy-9 medium (the Beefy 9 group), and in Beefy-9 medium supplemented with forskolin or/and SB202190 (the Beefy F group, the Beefy S group, and the Beefy FS group) were harvested, with three biological replicates included for RNA-seq analysis.