Interplay between embryonic enteric neural stem cells (ENSCs) and enteric mesenchymal cells (EMCs) in the embryonic gut is essential for normal development of the enteric nervous system. Disruption of these interactions underlies the pathogenesis of intestinal aganglionosis in Hirschsprung disease (HSCR). ENSC therapy has been proposed as a possible treatment for HSCR, but whether the survival and development of postnatal-derived ENSCs similarly rely on signals from the mesenchymal environment is unknown and has important implications for developing protocols to expand ENSCs for cell transplantation therapy. Enteric neural crest-derived cells (ENCDCs) and EMCs were cultured from the small intestine of Wnt1-Rosa26-tdTomato mice. EMCs promoted the expansion of ENCDCs 9.5-fold by inducing ENSC properties, including expression of Nes, Sox10, Sox2, and Ngfr. EMCs enhanced the neurosphere-forming ability of ENCDCs, and this persisted after withdrawal of the EMCs. These effects were mediated by paracrine factors and several ligands known to support neural stem cells were identified in EMCs. Using the optimized expansion procedures, neurospheres were generated from small intestine of the Ednrb −/− mouse model of HSCR. These ENSCs had similar proliferative and migratory capacity to Ednrb +/+ ENSCs, albeit neurospheres contained fewer neurons. ENSCs derived from Ednrb −/− mice generated functional neurons with similar calcium responses to Ednrb +/+ ENSCs and survived after transplantation into the aganglionic colon of Ednrb −/− recipients. EMCs act as supporting cells to ENSCs postnatally via an array of synergistically acting paracrine signaling factors. These properties can be leveraged to expand autologous ENSCs from patients with HSCR mutations for therapeutic application.

Background Enteric nervous system (ENS) abnormalities have been implicated in delayed gastric emptying but studies exploring potential treatment options are limited by the lack of an experimental animal model. We examined the ENS abnormalities in the mouse stomach associated with aging, developed a novel model of gastroparesis, and established a new approach to measure gastric emptying. Methods A modified gastric emptying assay was developed, validated in nNOS −/− mice, and tested in mice at multiple ages. Age‐related changes in ENS structure were analyzed by immunohistochemistry. Gastric aganglionosis was generated in Wnt1‐iDTR mice using focal administration of diphtheria toxin (DT) into the anterior antral wall. Key Results Older mice (>5 months) exhibit hypoganglionosis in the gastric antrum and a decreased proportion of nNOS neurons as compared to younger mice (age 5‐7 weeks). This was associated with a significant age‐dependent decrease in liquid and solid gastric emptying. A novel model of gastric antrum hypoganglionosis was established using neural crest‐specific expression of diphtheria toxin receptor. In this model, a significant reduction in liquid and solid gastric emptying is observed. Conclusions & Inferences Older mice exhibit delayed gastric emptying associated with hypoganglionosis and a reduction in nNOS‐expressing neurons in the antrum. The causal relationship between antral hypoganglionosis and delayed gastric emptying was verified using a novel experimental model of ENS ablation. This study provides new information regarding the pathogenesis of delayed gastric emptying and provides a robust model system to study this disease and develop novel treatments.

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