Stem cell therapies for nervous system disorders are hindered by a lack of accessible autologous sources of neural stem cells (NSCs). In this study, neural crest–derived Schwann cells are found to populate nerve fiber bundles (NFBs) residing in mouse and human subcutaneous adipose tissue (SAT). NFBs containing Schwann cells were harvested from mouse and human SAT and cultured in vitro. During in vitro culture, SAT-derived Schwann cells remodeled NFBs to form neurospheres and exhibited neurogenic differentiation potential. Transcriptional profiling determined that the acquisition of these NSC properties can be attributed to dedifferentiation processes in cultured Schwann cells. The emerging population of cells were termed SAT-NSCs because of their considerably distinct gene expression profile, cell markers, and differentiation potential compared to endogenous Schwann cells existing in vivo. SAT-NSCs successfully engrafted to the gastrointestinal tract of mice, migrated longitudinally and circumferentially within the muscularis, differentiated into neurons and glia, and exhibited neurochemical coding and calcium signaling properties consistent with an enteric neuronal phenotype. These cells rescued functional deficits associated with colonic aganglionosis and gastroparesis, indicating their therapeutic potential as a cell therapy for gastrointestinal dysmotility. SAT can be harvested easily and offers unprecedented accessibility for the derivation of autologous NSCs from adult tissues. Evidence from this study indicates that SAT-NSCs are not derived from mesenchymal stem cells and instead originate from Schwann cells within NFBs. Our data describe efficient isolation procedures for mouse and human SAT-NSCs and suggest that these cells have potential for therapeutic applications in gastrointestinal motility disorders.

Hirschsprung disease (HSCR) is characterized by absence of the enteric nervous system (ENS) in the distal bowel. Despite removal of the aganglionic segment, gastrointestinal (GI) problems persist. Cell therapy offers potential treatment but use of genetic models is limited by their poor survival. We have developed a novel model of aganglionosis in which enteric neural crest-derived cells (ENCDCs) express diphtheria toxin (DT) receptor. Local DT injection into the colon wall results in focal, specific, and sustained ENS ablation without altering GI transit or colonic contractility, allowing improved survival over other aganglionosis models. Focal ENS ablation leads to increased smooth muscle and mucosal thickness, and localized inflammation. Transplantation of ENCDCs into this region leads to engraftment, migration, and differentiation of enteric neurons and glial cells, with restoration of normal architecture of the colonic epithelium and muscle, reduction in inflammation, and improved survival.

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.