Electron microscopic analysis of the influence of iPSC-derived motor neurons on bioengineered human skeletal muscle tissues.

Abstract

3D bioengineered skeletal muscle macrotissues are increasingly important for studies of cell biology and development of therapeutics. Tissues derived from immortalized cells obtained from patient samples, or from pluripotent stem cells, can be co-cultured with motor-neurons to create models of human neuromuscular junctions in culture. In this study, we present foundational work on 3D cultured muscle ultrastructure, with and without motor neurons, which is enabled by the development of a new co-culture platform. Our results show that tissues from Duchenne muscular dystrophy patients are poorly organized compared to tissues grown from healthy donor and that the presence of motor neurons invariably improves sarcomere organization. Electron micrographs show that in the presence of motor neurons, filament directionality, banding patterns, z-disc continuity, and the appearance of presumptive SSR and T-tubule profiles all improve in healthy, DMD-, and iPSC-derived muscle tissue. Further work to identify the underlying defects of DMD tissue disorganization and the mechanisms by which motor neurons support muscle are likely to yield potential new therapeutic approaches for treating patients suffering from Duchenne muscular dystrophy.