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 typically display an embryonic identity. This is a major hurdle for accurately recapitulating disease phenotypes in vitro, as disease commonly manifests at later stages of development. 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 the expression profile of myosin heavy chain isoforms, as well as the upregulation of genes related with muscle contractile function. These molecular changes were confirmed by global chromatin accessibility and transcriptome studies. Importantly, we also observed this maturation in three-dimensional muscle constructs, 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.
Bibliographical noteFunding Information:
This project was supported by funds from the NIH, grants R01 AR055299 and AR071439 (RCRP) and the NSF CAREER DMR-1151529 (WS). RM-G was funded by CONACyT-Mexico (#394378). We also thank the generous support from ADVault, Inc and MyDirectives.com (RCRP). The cytogenetic analyses were performed in the Cytogenomics Shared Resource at the University of Minnesota with support from the comprehensive Masonic Cancer Center NIH Grant #P30 CA077598-09. The monoclonal antibodies to MyHC, Titin, MyHC-neo and MYH1/2 were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa. We thank Prof. Robert T Tranquillo, Dr. Jeremy Schaefer, and Dr. Lauren Black for the use of the contractile force measurement apparatus and for facilitating data analysis.
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