Skeletal muscle mediates the beneficial effects of exercise, thereby improving insulin sensitivity and reducing the risk for type 2 diabetes. Current human skeletal-muscle-models in vitro are incapable of fully recapitulating its physiological functions, especially contractility. By supplementation of insulin-like growth factor 1 (IGF1), secreted by myofibers in vivo, we overcome these limitations. We monitored the differentiation process starting from primary human CD56-positive myoblasts in the presence/absence of IGF1 in serum-free medium daily over 10 days. IGF1-supported differentiation formed thicker multinucleated myotubes showing physiological contraction upon electrical-pulse-stimulation following day 6. Myotubes without IGF1 were almost incapable of contraction. IGF1-treatment shifted the proteome toward skeletal muscle-specific proteins that contribute to myofibril and sarcomere assembly, striated muscle contraction, and ATP production. Elevated PPARGC1A, MYH7 and reduced MYH1/2 suggest a more oxidative phenotype further demonstrated by higher abundance of respiratory chain proteins and elevated mitochondrial respiration. IGF1-treatment also upregulated GLUT4 and increased insulin-dependent glucose uptake compared to myotubes differentiated without IGF1. To conclude, utilizing IGF1, we engineered human myotubes that recapitulate the physiological traits of skeletal muscle in vivo vastly superior to established protocols. This novel model enables investigation of exercise on a molecular level, is suitable for drug screening and studies on interorgan-crosstalk.

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