Construction of contractile human iPSC-derived skeletal muscle tissues on 96-well scale microdevices

Tissue-engineered three-dimensional (3D) skeletal muscles can be potentially used in contractile force-based phenotypic screening to elucidate the mechanisms of skeletal muscle dysfunction and develop preventive and therapeutic strategies. Human induced pluripotent stem cells (hiPSCs) expressing tetracycline-inducible myogenic differentiation 1 (MYOD1) are a promising cell source for construction of tissue-engineered skeletal muscles. Although we successfully constructed contractile tissues using these hiPSCs in a previous study, further improvements are required because of their weak contractile force and inefficient screening capabilities. In this study, we aimed to construct iPSC-derived muscle tissues with high contractile force using a 96-well scale microdevice that we had previously developed. To increase the contractile force, we optimized the time of supplementation of the transforming growth factor-β (TGF-β) inhibitor, SB431542 (SB), to identify culture conditions that enhance contractile force. The maximum contractile force with addition of SB was approximately five times greater than that without SB (58.45 ± 20.14 μN with SB compared to 11.64 ± 4.86 μN without SB). Various analyses, including immunostaining, transmission electron microscopy, gene expression analysis, and proteomics, revealed enhanced myotube differentiation and muscle tissue maturation in the presence of SB. Experiments using inhibitors indicated that TGFβ1, not myostatin, is partially involved in these effects. Furthermore, we confirmed that tissues constructed from iPSCs derived from patients with Duchenne muscular dystrophy also showed improved contractility following addition of SB. Therefore, iPSC-derived muscle tissues cultured with SB on the 96-well microdevice provide a promising platform for screening compounds that can ameliorate disease pathology.