PROJECT SUMMARY Mucosal healing is the primary goal of all therapies for inflammatory bowel disease (IBD). No currently approved therapy directly promotes wound healing of the bowel epithelium, which forms a single-cell barrier between the host and lumen and regulates the immune response. The cellular and molecular mechanisms that define the healing process remain to be defined. The intestinal crypt maintains the normal turnover of epithelial cells within a vertical lineage hierarchy of basal stem and terminally differentiated luminal cells. During wound healing, this lineage hierarchy is suspended, and epithelial cells can undergo dedifferentiation to mediate re- epithelialization. In recent work, we have identified an alternate source of wound healing in the distal colon of mice. In acute and chronic models of colitis, a skin-like cell population at the anal transition zone (ATZ), bordering the sharp squamocolumnar anorectal junction, migrates into the colon and forms a permanent hybrid epithelial structure called squamous neo-epithelium of colon (SNEC). SNEC represents the end-product of wound healing by cells of the ATZ. The ATZ is an anatomically small region that is composed of a unique population of epithelial stem cells that have a mixed colonic/epidermal phenotype and are capable of wound- healing plasticity. Moreover, these stem cells are partially resistant to the damage of colitis. The study of these cells could highlight new pathways for colonic epithelial regeneration in the context of IBD. However, the lineage diversity and relationships within the myriad tissue types of the human ATZ are not known, and furthermore it remains to be defined whether ATZ stem cells might provide enduring intestinal function, protect from potential oncogenic sequela, or repair ulcers throughout the colon. In the proposed work, we will test whether ATZ-derived stem cells could be suitable reagents to mediate colonic epithelial wound healing and identify key pathways that contribute to their proliferative potential. The Specific Aims of the project are: 1) to define regenerative cell populations in the human ATZ, 2) to define long-term outcomes of endogenous colonic wound healing by ATZ cells, and 3) to identify mechanisms of functional ATZ plasticity in wound healing. A deeper investigation of the mechanisms, implications, and potential translation of this noncanonical form of colonic wound healing could reveal new therapeutic targets to direct mucosal healing in IBD.