PROJECT SUMMARY The overarching objective of our research is to develop a stem cell therapy from subcutaneous fat tissue (SAT) to replace the congenitally absent enteric nervous system (ENS) in Hirschsprung disease (HSCR). Surgical resection of the affected colon is currently the only viable treatment for HSCR. This is a necessary life-saving procedure; albeit, more than 50% of patients still suffer from postoperative complications including constipation, fecal incontinence, and enterocolitis. To overcome these morbidities, investigations into treatments that can preserve the rectum and its functions are warranted. Replacement of the absent ENS via stem cell therapies is touted as the most promising treatment strategy to achieve this goal. Our group has demonstrated the feasibility of stem cell treatments by harvesting neural stem cells (NSCs) from the gut which engraft, migrate and differentiate into neuronal networks when transplanted into mice with HSCR. For clinical application this would require surgical resection of a piece of intestine. To prevent unnecessary resection surgery, other sources of NSCs are of interest. Human fat (adipose) tissue contains a reservoir of stem cells that are readily accessible. These cells have been examined in over 270 clinical trials for numerous diseases that support favourable patient safety profiles. In our preliminary data we have also identified that nerve fiber bundles from murine fat deposits – subcutaneous adipose tissue (SAT) - harbor an endogenous source of NSCs that are unexplored for the treatment of neuropathies. We predict that the SAT could provide a useful source of NSCs to treat colonic aganglionosis in HSCR; however, it remains undetermined if SAT-NSCs can undergo neurogenesis in the aganglionic (absent ENS) environment of the gut and there are currently no methods to purify and expand human SAT-NSCs. In the first aim of this study, we will determine if purified SAT-NSCs from mice are capable of neurogenesis in aganglionic intestine. The ganglionated ENS is supplemented postnatally by NSCs that migrate into the gut from extrinsic nerve fiber bundles and differentiate into enteric neurons in response to environmental cues from the gut. We will address whether SAT-NSCs can also undergo enteric neurogenesis when provided signalling cues from the ganglionic and aganglionic gut in in vitro coculture systems and via microsurgical SAT-NSC implantation in vivo. To determine how to isolate and expand human SAT-NSCs we will address the paucity of knowledge on the origin of these cells. To accomplish this, cells isolated from human SAT nerve fiber bundles will be unbiasedly characterised by single nuclei RNA-Seq before and after stem cell culture procedures. Cells expressing NSC markers will be purified by fluorescence activated cell sorting and their differentiation potential will be assessed in in vitro culture and in ex vivo transplants to the smooth muscle of the gut. The results of these studies wi...