Enhanced Media Optimize Bovine Myogenesis in 2D and 3D Models for Cultivated Meat Applications [in vitro scRNA-Seq]

Livestock farming and conventional meat production pose significant environmental, health, and animal welfare challenges. In seeking sustainable alternative solutions, cultivated meat technology typically utilizes differentiation of myogenic progenitor cells (MPCs) into muscle cells for in vitro meat production. However, understanding the molecular determinants governing MPC differentiation into muscle cells, and the potential enhancement of this process through modulation of signaling pathways, remains limited. Herein, we characterized the molecular landscape associated with bovine MPC differentiation in vitro by employing multiomics, and explored its augmentation by small molecules, together leading to identification of media that enhanced myogenic differentiation compared with conventional methods in both 2D cultures and tissue-engineered 3D skeletal muscle constructs. Through bulk and single-cell transcriptomics and proteomics, we compared conventional and enhanced differentiation media, demonstrating that the enhanced media gave rise to unique progenitor-like cell populations, while simultaneously promoting differentiation into myocytes and contractile myotubes expressing a wide array of myogenic markers that more closely resemble bovine muscle cells in vivo. The improved method for promoting myogenic differentiation in 2D and 3D formats, together with the corresponding molecular roadmap, may prove valuable for cultivated meat applications. Overall design: Analysis of myoblast- and differentiated myoblast- cultures using base media (myoblast medium and iMPC medium) and three different differentiation media (conventional differentiation medium, iFR_hi and iFRC) on a transcriptome level. Cultures were analyzed at day 2 or day 10.