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

## TL;DR

Researchers developed a serum-free medium using small molecules to efficiently expand bovine muscle stem cells for cultured meat production.

## Contribution

A novel serum-free medium combining forskolin and a p38 inhibitor for efficient muscle stem cell expansion is introduced.

## Key findings

- The 'Beefy-F' medium with forskolin matched serum-controlled levels in bMuSC expansion and maintained cell quality.
- Adding the p38 inhibitor SB202190 (Beefy-F + S) significantly improved bMuSC proliferation and stemness marker expression.
- Transcriptomic analysis showed the medium preserved myogenic identity and modulated cell cycle and ECM pathways.

## Abstract

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.