Improved long-term proliferation and myogenic differentiation of bovine muscle stem cells through CDKN2A gene editing

The production of cultured meat is constrained by the limited proliferation and differentiation capacity of seed cells. Here, we report that prolonged in vitro culture induces cellular senescence in bovine muscle stem cells (bMuSCs), impairing their proliferative and myogenic potential. We then examined the expression of cyclin-dependent kinase inhibitors (CKIs) and found that CDKN2A expression was markedly upregulated during passaging and its knockout substantially enhanced the expansion of bMuSCs. Subsequently, we established two CDKN2A-knockout (CDKN2A-KO) monoclonal cell lines, which showed significantly enhanced proliferation over long-term culture and exhibited an overall reduction in the expression of CKIs compared to wild-type (WT) controls. Further, CDKN2A-KO bMuSCs displayed elevated expression of myogenic regulatory factors and improved myogenic differentiation under both low-serum and serum-free conditions. Notably, in a serum-free three-dimensional hydrogel system, CDKN2A-KO cells showed enhanced myotube formation in comparison with WT cells. Moreover, CDKN2A knockout did not induce anchorage-independent growth, indicating a non-tumorigenic phenotype in these cells. These results demonstrate that targeting CDKN2A effectively delays cellular senescence and enhances the myogenic potential of bMuSCs without tumorigenic risk, providing a promising strategy to improve large-scale cell expansion for cultured meat.