Functional maturation of enteric neurons derived from human induced pluripotent stem cells

The enteric nervous system (ENS) plays a crucial role in regulating gastrointestinal function, including motility, secretion, and absorption. Enteric neurons and glia derived from human induced pluripotent stem cells (iPSCs) have vast applications in disease modeling, drug screening, and cell replacement therapy, yet our understanding of their functional maturation remains limited. Using a defined protocol for the generation of iPSC-derived enteric neurons, we examined neuronal differentiation with immunohistochemistry, gene expression analysis, and cellular-resolution calcium (Ca²⁺) imaging from day (D)21 to D57 of differentiation. By D21, cells expressing pan-neuronal markers were present, and at D22 the cultures were responsive to K⁺-induced depolarization as well as neurotransmitter receptor agonists, with maturation of responses to high K⁺ depolarization during the differentiation period. In addition, the development of spontaneous neuronal network activity was observed at 2 months post-differentiation. Together these findings provide a timeline of the functional maturation of iPSC-derived ENS cells.