Single cell RNA-sequencing of murine dermal cells and hindlimb skeletal muscle stromal cells.

Traumatic injury often results in muscle loss and impairment, which is worsened under aged and diseased conditions. Activation of resident stem cells or transplantation of myogenic stem cells can promote muscle regeneration. However, major challenges remain in harvesting sufficient autologous myogenic stem cells and expanding such cells efficiently for muscle regeneration therapies. Here, we identified a chemical cocktail that selectively induced a robust expansion of myogenic stem cells from readily-obtainable dermal cells and from muscle stromal cells. By differential plating and lineage tracing, we showed that Pax7+ cells were the major source for chemical-induced myogenic stem cells (CiMCs). We further performed single-cell RNA sequencing (scRNA-seq) analysis to characterize the transcriptomic profile of CiMCs and demonstrate a specific expansion of myogenic cells from heterogeneous dermal cell population. Upon transplantation into the injured muscle, CiMCs were efficiently engrafted and improved functional muscle regeneration in both adult and aged mice. In addition, CiMC transplantation rescued muscle function in mice with Duchenne muscular dystrophy (DMD). Furthermore, an in situ therapeutic modality using this cocktail was developed by loading the chemical cocktail into injectable nanoparticles, which enabled a sustained release of the cocktail in injured muscle and a local expansion of resident satellite cells for muscle regeneration in adult and aged mice. These findings will lead to the development of novel in vitro and in situ stem cell therapies for effective skeletal muscle repair.

Freshly isolated neonatal dermal cells, neonatal dermal cells treated with FR medium for 3 days, adult dermal cells treated with FR medium for 3 days and freshly isolated adult hindlimb skeletal muscle stromal cells were subjected to single cell RNA sequencing. FR medium is 20 μM F, 20 μM R, 50 μg/ml AA, and 50 ng/ml bFGF in high-glucose DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.