Dissecting cell diversity and connectivity in skeletal muscle for myogenesis.

Characterized by their slow adhering property, skeletal muscle myogenic progenitor cells (MPCs) have been widely utilized in skeletal muscle tissue engineering for muscle regeneration, but with limited efficacy. Skeletal muscle regeneration is regulated by various cell types, including a large number of rapidly adhering cells (RACs) where their functions and mechanisms are still unclear. In this study, we explored the function of RACs by co-culturing them with MPCs in a biomimetic skeletal muscle organoid system. Results showed that RACs promoted the myogenic potential of MPCs in the organoid. Single-cell RNA-Seq was also performed, classifying RACs into 7 cell subtypes, including one newly described cell subtype: teno-muscular cells (TMCs). Connectivity map of RACs and MPCs subpopulations revealed potential growth factors (VEGFA and HBEGF) and extracellular matrix (ECM) proteins involvement in the promotion of myogenesis of MPCs during muscle organoid formation. Finally, trans-well experiments and small molecular inhibitors blocking experiments confirmed the role of RACs in the promotion of myogenic differentiation of MPCs. The RACs reported here revealed complex cell diversity and connectivity with MPCs in the biomimetic skeletal muscle organoid system, which not only offers an attractive alternative for disease modeling and in vitro drug screening but also provides clues for in vivo muscle regeneration.