Anabolic Factors and Myokines Improve Differentiation of Human Embryonic Stem Cell Derived Skeletal Muscle Cells

Skeletal muscle weakness is linked to many adverse health outcomes. Current research to identify new drugs has often been inconclusive due to lack of adequate cellular models. We have previously developed a scalable monolayer system to differentiate human embryonic stem cell (hESC) into mature skeletal muscle cells (SkMC) within 26 days without cell sorting or genetic manipulation. Here, building on our previous work, we show that differentiation and fusion of myotubes can be further enhanced using the anabolic factors testosterone (T) and follistatin (F) in combination with a cocktail of myokines (C). Importantly, combined TFC treatment significantly enhanced both hESC-SkMC fusion index and expression of various skeletal muscle markers including the motor protein Myosin Heavy Chain (MyHC). Transcriptomic and proteomic analysis revealed oxidative phosphorylation as the most up-regulated pathway and a significantly higher level of ATP and increased mitochondrial mass were also observed in TFC-treated hESC-SkMCs, suggesting enhanced energy metabolism is coupled to improved muscle differentiation. This cellular model will be a powerful tool for studying in vitro myogenesis and for drug discovery to further enhance muscle development or treat muscle diseases. ### Competing Interest Statement The authors have declared no competing interest. * hESC : human embryonic stem cell SkMC : skeletal muscle cells T : testosterone F : follistatin C : cocktail of myokines TFC : testosterone follistatin and cocktail of myokines MyHC : myosin heavy chain hPSC : human pluripotent stem cells hiPSC : human induced pluripotent stem cells MRF : myogenic regulatory factors IL4 : interleukin-4 IL6 : interleukin-6 BDNF : brain derived neurotrophic factor VEGF : vascular endothelial growth factor NTC : non-treated cells