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<p>Cultured meat represents a promising sustainable food source, yet the development of efficient serum-free media remains a key bottleneck. Small molecules offer a cost-effective approach to system optimization. Through a functional screening of proliferation activators in bovine muscle stem cells (bMuSCs), we identified forskolin and formulated the serum-free “Beefy-F” medium. Over six passages, Beefy-F yielded 1.9 times more bMuSCs than the basal serum-free control (Beefy-9), matching serum-controlled levels while maintaining bMuSC morphology, myogenic gene expression, and differentiation potential. Subsequent synergistic screening revealed that the p38 inhibitor SB202190 enhanced forskolin’s effects. The optimized “Beefy-F + S” medium significantly outperformed both the basal serum-free control and single-supplemented formulations after three passages, upregulated the stemness marker <i>PAX7</i>, and preserved differentiation capacity. Transcriptomic analysis revealed that the Beefy-F + S medium broadly altered the bMuSC transcriptome to maintain myogenic identity, upregulate cell cycle genes, and reshape extracellular matrix (ECM) pathways, with forskolin sustaining myogenic factors and the p38 inhibitor promoting proliferation and modulating ECM interactions. Overall, this study establishes a cost-effective, small molecule-based strategy for the robust serum-free expansion of bMuSCs.</p>

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A small molecule strategy with forskolin and p38 inhibitor for serum-free muscle stem cell expansion

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年2月2日

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  • Hao Lu,
  • Zheng Liu,
  • Xiaoyu Liu,
  • Yijia Pan,
  • Dan Yan,
  • Xinpei Cheng,
  • Longfei Zhang,
  • Shijie Ding,
  • Chunbao Li,
  • Guanghong Zhou,
  • Renpeng Guo

摘要

Cultured meat represents a promising sustainable food source, yet the development of efficient serum-free media remains a key bottleneck. Small molecules offer a cost-effective approach to system optimization. Through a functional screening of proliferation activators in bovine muscle stem cells (bMuSCs), we identified forskolin and formulated the serum-free “Beefy-F” medium. Over six passages, Beefy-F yielded 1.9 times more bMuSCs than the basal serum-free control (Beefy-9), matching serum-controlled levels while maintaining bMuSC morphology, myogenic gene expression, and differentiation potential. Subsequent synergistic screening revealed that the p38 inhibitor SB202190 enhanced forskolin’s effects. The optimized “Beefy-F + S” medium significantly outperformed both the basal serum-free control and single-supplemented formulations after three passages, upregulated the stemness marker PAX7, and preserved differentiation capacity. Transcriptomic analysis revealed that the Beefy-F + S medium broadly altered the bMuSC transcriptome to maintain myogenic identity, upregulate cell cycle genes, and reshape extracellular matrix (ECM) pathways, with forskolin sustaining myogenic factors and the p38 inhibitor promoting proliferation and modulating ECM interactions. Overall, this study establishes a cost-effective, small molecule-based strategy for the robust serum-free expansion of bMuSCs.