Optimized simple culture protocol for inducing mature myotubes from MYOD1-overexpressed human iPS cells

The forced expression system of MYOD1, a master gene for myogenic differentiation, can efficiently and rapidly reproduce muscle differentiation of human induced pluripotent stem cells (hiPSCs). Despite these advantages of the MYOD1 overexpression system, developed myotubes are relatively immature and do not recapitulate several aspects of striated muscle fibers. Here, we developed a simple optimized protocol using an alternative culture medium for maximizing the advantages of the MYOD1 overexpression system, and successfully improved the formation of multinucleated mature myotubes within 10 days. In this study, we generated hiPSCs derived from healthy donors and an individual with congenial muscular dystrophy caused by LMNA mutation (laminopathy), and compared disease-associated phenotypes in differentiated myotubes generated by the conventional method and by our new optimized culture method. Using our optimized method, abnormal myonuclear shape was pronounced in the patient-derived iPSCs. In addition, abnormal accumulation of the nuclear membrane protein emerin was observed in LMNA-mutant hiPSCs. Our new culture method is expected to be widely applicable as a MYOD1 overexpression model of hiPSC-derived skeletal muscle cells for the analysis of a variety of muscle diseases.