Neurointestinal diseases comprise a group of serious gastrointestinal disorders characterized by congenital or acquired abnormalities of the enteric nervous system (ENS). These diseases, including Hirschsprung disease, intestinal pseudo-obstruction, esophageal achalasia, diabetic gastropathy, and irritable bowel syndrome, affect many individuals, are associated with significant morbidity, and currently have limited treatment options. Transplantation of enteric neuronal stem/progenitor cells (ENSCs) offers a promising approach to replacing missing or abnormal neurons in the intestine. A number of recent studies, including from our laboratory, demonstrate that ENSCs can be isolated from the postnatal intestine, propagated in culture, and transplanted into the gut wall. However, the numbers of neurons generated have been limited and their ability to integrate into neuroglial networks capable of restoring gut function has yet to be demonstrated. Applying our expertise in ENS development, ENSC research, and clinical neurogastroenterology, we propose an innovative developmentally-informed approach to the isolation, expansion, and transplantation of gut-derived ENSCs for colonic neuropathies. We hypothesize that culturing and transplanting ENSCs in a microenvironmental milieu that mimics their environment during embryonic development will optimize their expansion in vitro and enhance their engraftment, survival, migration, and differentiation in vivo. This novel hypothesis will be tested with the following specific aims: (i) To determine the effects of GDNF, ET3, and serotonin in modulating survival, proliferation, and differentiation of postnatal gut-derived ENSCs, (ii) To characterize the abilityof transplanted ENSCs to engraft, differentiate, migrate, and improve gut function in mice with colorectal aganglionosis (Ednrb-/-), hypoganglionosis (Gdnf+/-), or neurotransmitter deficiency (nNOS-/-), and (iii) To isolate, expand, and transplant human gut-derived ENSCs for treatment of colorectal neuropathies in mice. In vitro techniques will include isolation and expansion of gut derived neuronal stem/progenitor cells, lentiviral transduction and nanoparticle fabrication to modify their environment, and quantitative analysis of their survival, proliferation, and differentiation. Cell transplantation into the mouse colorectum will be performed using an innovative microcolonoscopy approach, with thorough immunohistochemical and functional characterization of the transplanted mice, including isometric colonic muscle recordings and in vivo anorectal manometry. Finally, methods for isolating and expanding human-derived ENSCs will be established, and transplantation of these cells into mouse models of neurointestinal disease will lay the groundwork for pilot clinical studies. The results obtained will yield new knowledge about the effects of molecular environmental factors on ENSC survival, proliferation, differentiation, and migration, establish an optimized approach to ENSC transplantation, and demonstrate the potential of cell-based therapy to restore GI function in neurointestinal diseases.