Combinatorial small molecule treatment enhances the in vitro maturation of pluripotent stem cell-derived myotubes

Targeted differentiation of pluripotent stem (PS) cells into myotubes enables in vitro disease modeling of skeletal muscle diseases. Although various protocols achieve myogenic differentiation in vitro, resulting myotubes invariably display an embryonic identity. This is a major hurdle for accurately recapitulating disease phenotypes in vitro, as disease typically does not manifest in the embryonic muscle, but at more mature stages. To address this problem, we identified four factors from a small molecule screen whose combinatorial treatment resulted in myotubes with enhanced maturation, as shown by increased expression of fetal, neonatal and adult myosin heavy-chain isoforms. These molecular changes were confirmed by global chromatin accessibility and transcriptome studies. Importantly, we also observed this maturation in three-dimensional muscle bundles, which displayed improved in vitro contractile force generation in response to electrical stimulus. Thus, we established a model for in vitro muscle maturation from PS cells. Overall design: Three replicates each of human iPSC myotubes derived with vehicle (DMSO) or small molecule treatment (SDDF) were analyzed by 1) RNA seq to identify the differentially expressed genes and 2) ATAC-seq to determine changes in chromatin accessibility.