Geometrically engineered human motor assembloids-on-a-chip for neuromuscular interaction readout and hypoxia-driven disease modeling

Precision medicine leverages stem cell-derived neuromuscular models to dissect complex interactions underlying neuromuscular damage from severe diseases. However, such reductionist models form stochastic structures without external guidance, while available engineering solutions remain intricate. Here, simplified surface modification engineering was used to implement geometric confinement to render spatially patterned human motor assembloids-on-a-chip. The anisotropic architecture of skeletal muscle organoids (hSkM) can be conferred solely by localized mechanobiological cues within this predefined device without aligned scaffolds or adjuncts. The hSkM-orchestrated coupling of motor neuron spheroids (hMNS) promoted synergistic neuromuscular development. Furthermore, the integration of optogenetic activation with microelectrode array mapping enabled visualization of functional patterning in assembloids. Applied to the oxygen deprivation model, SkM exhibited structural anomalies, fatigable muscle remodeling and weakened contraction, recapitulating the muscle pathologies found in respiratory disorders characterized by intermittent hypoxia (IH). Notably, electrical activity mapping revealed the heterogeneity in neuromuscular responses to IH, elucidating the neuroregulatory etiology of muscle dysfunction. Finally, we identified mitochondrial bioenergetic imbalance as a key target of IH-induced pathology, proposing the evaluation of agents that target NAD+ salvage pathway. Our findings provide a unique platform with translational potential for neuromuscular physiopathology research.