Differentiation of Human Induced Pluripotent Stem Cells (hiPSCs) into Mature and Contractible Skeletal Muscles

Pathophysiological analysis of and drug discovery for human diseases require disease models that suitably recapitulate patient pathology. Disease-specific human induced pluripotent stem cells (hiPSCs) differentiated into affected cell types can potentially recapitulate disease pathology more accurately than existing disease models. Such successful modeling of muscular diseases requires efficient differentiation of hiPSCs into skeletal muscles. hiPSCs transduced with doxycycline-inducible MYOD1 (MYOD1-hiPSCs-hiPSCs) have been widely used; however, they require time- and labor-consuming clonal selection. We previously demonstrated that, without clonal selection, bulk MYOD1-hiPSCs established with puromycin selection rapidly and highly efficiently differentiated into skeletal muscles. This method requires less time and labor than other methods and may minimize clonal variations caused by clonal selection. Moreover, three-dimensional muscle tissues can be fabricated from bulk MYOD1-hiPSCs, which exhibit contractile force upon electrical stimulation, indicating their functionality. In this chapter, we introduce our method for differentiating hiPSCs into two-dimensional skeletal muscles as well as functional three-dimensional muscle tissues.