Electro-mechanical stimulation during differentiation of neural stem cells derived from human induced pluripotent stem cells enhances neuronal network organization and activity.

Neural stem cells derived from human-induced pluripotent stem cells (hiPSC-NSCs) offer a powerful model for neurodevelopment. While electrical stimulation is known to enhance the differentiation of hiPSC-NSCs into neuronal cells, the effects of co-stimulation remain unexplored. We applied an electro-mechanical co-stimulation system featuring a stretchable microelectrode array (S-MEA) to investigate its effects on neuronal differentiation and network organization. Co-stimulation during the transition from hiPSC-NSCs to neurons yielded mixed neuronal populations, as confirmed by lineage-specific marker expression. Artificial intelligence-assisted image analysis revealed stimulation-dependent differences in neurite outgrowth and network architecture. In particular, the Co3 condition, combining 20% mechanical strain with an electric field of 1 V cm-1 at 1 Hz, showed prominent directional neurite organization and features of emerging neural microcircuit organization, together with relatively high activity-associated fluorescence responses among the tested conditions. Dynamic calcium-associated and membrane-voltage fluorescence responses differed across the stimulation conditions compared with the unstimulated control. These findings indicate that electro-mechanical stimulation is associated with condition-dependent differences in neuronal structural organization and activity-associated fluorescence responses during differentiation. The observed directional neurite organization and fluorescence responses are consistent with a potential contribution of mechanotransduction-related processes, although the underlying molecular mechanisms were not directly assessed. Overall, electro-mechanical co-stimulation provides a platform for investigating how combined biophysical cues influence neuronal differentiation and network organization in vitro.