PROJECT SUMMARY Hirschsprung disease is a potentially lethal congenital disorder characterized by the absence of enteric nervous system (ENS) along variable lengths of distal intestine due to the failure of neural crest-derived cells to colonize the entire intestine during development. The surgical treatment of Hirschsprung disease involves removing the aganglionic segment. While this is life- saving, significant complications commonly occur after surgery, including constipation, fecal incontinence, and enterocolitis. Transplantation of enteric neural crest-derived cells (ENCCs) offers a promising new approach to replacing missing or abnormal enteric neurons in Hirschsprung disease and other neurointestinal diseases. Recent studies, including from our laboratory, demonstrate that ENCCs can be isolated from the postnatal intestine, propagated in culture, and transplanted into the gut wall. However, major challenges remain: (1) the numbers of neurons generated have been limited, (2) their ability to integrate into neuroglial networks capable of restoring gut function has yet to be demonstrated, and (3) mice with Hirschsprung disease die at 4-6 weeks of age, limiting the time window available for analysis after cell transplantation. To overcome these challenges, we propose the following aims: (1) to incorporate an innovative approach to isolating and expanding postnatal gut-derived ENCCs that generates greater numbers of progenitor ENCCs than prior methods and (2) to transplant autologously-derived ENCCs into a novel non-lethal model of colorectal aganglionosis generated by local injection of human diphtheria toxin (DT) into transgenic mice whose neural crest-derived cells express DT receptor. To optimize ENCC expansion, a combinatorial drug screen approach will be used. ENCCs will be cultured with candidate molecules, together with known growth factors, to identify the optimal “cocktail” for progenitor cell expansion. Single cell RNAseq will be performed to characterize the transcriptome profile of cells prior to transplantation. Autologously-derived donor cells will be delivered into the experimentally- generated aganglionic segment of colon. Analyses will include quantitative determination of ENCC survival, proliferation, and neuronal differentiation, and functional characterization of neuronal activity. The results obtained will establish an optimized approach to ENCC isolation, expansion, and transplantation, and demonstrate the potential of cell-based therapy to restore GI function in Hirschsprung disease and other neurointestinal diseases.