Characterization of Functional Human Skeletal Myotubes and Neuromuscular Junction Derived—From the Same Induced Pluripotent Stem Cell Source

Abstract

In vitro generation of functional neuromuscular junctions (NMJs) utilizing the same induced pluripotent stem cell (iPSC) source for muscle and motoneurons would be of great value for disease modeling and tissue engineering. Although, differentiation and characterization of iPSC-derived motoneurons are well established, and iPSC-derived skeletal muscle (iPSC-SKM) has been reported, there is a general lack of systemic and functional characterization of the iPSC-SKM. This study performed a systematic characterization of iPSC-SKM differentiated using a serum-free, small molecule-directed protocol. Morphologically, the iPSC-SKM demonstrated the expression and appropriate distribution of acetylcholine, ryanodine and dihydropyridine receptors. Fiber type analysis revealed a mixture of human fast (Type IIX, IIA) and slow (Type I) muscle types and the absence of animal Type IIB fibers. Functionally, the iPSC-SKMs contracted synchronously upon electrical stimulation, with the contraction force comparable to myofibers derived from primary myoblasts. Most importantly, when co-cultured with human iPSC-derived motoneurons from the same iPSC source, the myofibers contracted in response to motoneuron stimulation indicating the formation of functional NMJs. By demonstrating comparable structural and functional capacity to primary myoblast-derived myofibers, this defined, iPSC-SKM system, as well as the personal NMJ system, has applications for patient-specific drug testing and investigation of muscle physiology and disease.