Simple and efficient differentiation of human iPSCs into contractible skeletal muscles for muscular disease modeling

Abstract

ABSTRACTPathophysiological analysis and drug discovery targeting human diseases require disease models that suitably recapitulate patients’ pathology. Disease-specific human induced pluripotent stem cells (hiPSCs) can potentially recapitulate disease pathology more accurately than existing disease models when differentiated into affected cell types. Thus, successful modeling of muscular diseases requires efficient differentiation of hiPSCs into skeletal muscles. hiPSCs transduced with doxycycline-inducible MYOD1 (MYOD1-hiPSCs) have been widely used; however, they require time- and labor-consuming clonal selection procedures, and clonal variations must be overcome. Moreover, their functionality to exhibit muscular contraction has never been reported. Here, we demonstrated that bulk MYOD1- hiPSCs established with puromycin selection, but not with G418 selection, showed high differentiation efficiency, generating more than 80% Myogenin (MyoG)+ and Myosin heavy chain (MHC)+ muscle cells within seven days. Interestingly, bulk MYOD1-hiPSCs exhibited average differentiation properties compared with those of clonally established MYOD1- hiPSCs, suggesting that the bulk method may minimize the effects of clonal variations. Finally, three-dimensional muscle tissues were fabricated from bulk MYOD1-hiPSCs, which exhibited contractile force upon electrical pulse stimulation, indicating their functionality. Together, the findings indicate that our bulk differentiation requires less time and labor than existing methods, efficiently generates contractible skeletal muscles, and facilitates the generation of muscular disease models.Graphical Abstract