Methods to Isolate Enteric Glia from hPSC-Derived Enteric Ganglioids.

Enteric glia play pivotal roles in maintaining the enteric nervous system (ENS) homeostasis, supporting neuronal functions, and initiating and developing enteric neuropathies. However, understanding their diversity and functional roles has been formidably challenging due to difficulties in isolating these cells from human tissue. Here, we describe a detailed method for generating enteric glia from human pluripotent stem cells (hPSCs). This approach involves differentiating hPSCs into enteric glial cells through a vagal and enteric neural crest intermediates, replicating in vivo developmental processes. The hPSC-derived enteric glia express canonical markers (SOX10, GFAP, PLP1, S100B) and display the cellular diversity of primary human enteric glia, confirmed by single-nucleus RNA sequencing (snRNA-seq). These cultures are scalable, enabling high-throughput screening and molecular analysis, and they facilitate the study of glial interactions with other cell types, including immune cells, epithelial cells, and the gut microbiome. hPSC-derived enteric glia represent a significant advancement in enteric neurobiology, providing a versatile platform for disease modeling and therapeutic discovery, thereby improving our understanding of enteric glial cell functions in both health and disease.