UCLA

Neuromuscular disorders include over 200 rare, monogenic disorders that collectively exceed an incidence of 1 in 3,000 and have few effective treatments. Amyotrophic Lateral Sclerosis (ALS) is a fatal, late-onset neuromuscular disorder characterized by the progressive loss of both upper and lower motor neurons. Over 30 genes have been implicated in ALS, with the largest proportion of cases attributed to a G4C2 hexanucleotide repeat expansion mutation in the non-coding region of the gene C9orf72. In order to gain further understanding into pathogenic mechanisms involved in neuromuscular disorders, we aimed to recapitulate the physiology of the neuromuscular junction (NMJ) via creation of a patient-derived ‘disease in a dish’ model system using human induced pluripotent stem cells (hiPSCs) from patients carrying C9orf72 G4C2 expansions. We first developed a novel morphogen-directed differentiation protocol to efficiently yield skeletal myotubes from hiPSCs and examined potential C9orf72 G4C2 expansion-related phenotypes manifesting in skeletal muscle and genetically-corrected, isogenic iPSC lines. We then derived both skeletal muscle and spinal motor neurons from hiPSCs originating from healthy controls, patients with G4C2 expansions, or isogenic iPSC lines before combining these two cell types in co-culture in vitro. We show that co-cultures can spontaneously produce functional NMJs as shown by immunocytochemistry, myotube contraction, patch-clamp electrophysiology, and live-cell calcium imaging. Co-cultures consisting of motor neurons derived from G4C2 carriers displayed less total NMJ area versus those from isogenic motor neurons, suggesting that early phenotypes may be detectable in immature iPSC co-cultures. Use of genetically encoded ChannelRhodopsin-2 allowed for optogenetic control of NMJ physiology, which could be paired with multi-electrode arrays for live-cell pharmacological interrogation of neuromuscular physiology. Together, these experiments provide evidence for the establishment of a scalable, tunable, human patient-specific model of the NMJ, adaptable to a broad range of neuromuscular disorders. This new model system can be utilized for investigation into developmental aspects of NMJ formation, maturation, and repair phenotypes, as well as novel therapeutic discovery efforts for neuromuscular disorders.