Neuromuscular junction (NMJ) dysfunction underlies many devastating neuromuscular diseases, highlighting the need for clinically relevant human NMJ models. While embedding motor neurons within 3D skeletal muscle (SkM) improves biochemical and contractile properties in primary cell-derived models, comparable benefits in induced pluripotent stem cell (iPSC) systems remain unexplored. Here, we present an iPSC-derived 3D NMJ model showing an advanced contractile phenotype absent from SkM-only tissues.
3D NMJs and SkM-only control tissues were generated from iPSC-derived myoblasts and motor neurons on Curi Bio’s Mantarray platform in two independent experiments as previously described. Briefly, SkM tissues were combined with iPSC-derived neurospheres via a collagen/matrigel hydrogel to generate NMJs or treated with the hydrogel alone to create SkM-only controls. Contractility was assessed every 2-3 days from Day 17 through Day 31 using electrically stimulated twitch and force–vs-frequency (FVF) protocols.
While twitch and tetanus forces were comparable between groups, NMJ tissues exhibited a distinct FVF plateau above 50 Hz, a phenotype absent from SkM-only controls. By Day 31, NMJs showed a 72% reduction in Frequency50 (the frequency required to elicit 50% of maximal tetanic force) and a 42% decrease in tetanus-to-twitch ratio. Both changes were statistically significant (p<.001). This study demonstrates a robust, previously unreported divergence in the FVF phenotype between SkM-only tissues and NMJs. We hypothesize that this differential FVF response may be due to a shift in myosin isoform and ion channel expression, and our ongoing work will characterize the transcriptomic profiles of SkM-only and NMJ tissues through RNA-seq analysis. This work advances our in vitro model of the NMJ for high‐throughput therapeutic screening, neuromuscular disease modeling, and potency testing for neurotoxins.