Enhancing Maturation of Human Neuromuscular Organoids via Electrical Stimulation

ABSTRACT Organoids derived from human pluripotent stem cells (hPSCs) are emerging as powerful models for studying development and disease. Despite their physiological relevance, the predictive power of organoids remains limited by the immature state of the constituent cells, posing a major challenge for mechanistic studies of adult physiology and late‐onset disorders. Here, we establish a strategy for enhancing the maturation status of human neuromuscular organoids (NMOs) through chronic Electrical Pulse Stimulation (EPS). We demonstrate that low‐frequency EPS, applied during the early stages of NMO development and maintained over several weeks, enhances neuromuscular maturation and functional output. Independent of stimulation waveform dynamics, EPS‐trained NMOs (EPS‐NMOs) display stronger and more frequent spontaneous contractions that persist long after stimulation has ceased. Quantitative imaging and transcriptomic analyses reveal a robust improvement in EPS‐NMO skeletal muscle and neural tissue morphology, coordinated regulation of lineage‐specific biomarkers, and upregulation of gene programs associated with neuromuscular maturation. Mechanobiological measurements further demonstrate increased tissue stiffness and faster relaxation dynamics in EPS‐NMOs, consistent with enhanced excitation‐contraction coupling (ECC) and force generation. Collectively, these findings establish EPS as a powerful, non‐invasive, and on‐demand modality for promoting the morphological and functional maturation of complex organoid systems.