PROJECT SUMMARY Reconstruction of the urinary tract in urologic surgery oftentimes involves utilizing the small intestine to replace a segment of the urinary system, and as a result, patients may suffer from the side effects of connecting the urinary and gastrointestinal tracts. Patients that undergo urinary diversion surgery, for example, are at risk of infection, electrolyte abnormalities, and ileus as a result of this practice. If a better substitute for the urinary tract were available, outcomes from urinary diversion or reconstructive surgery involving the small intestine would be drastically improved. The main goal of this proposal is to develop a source of autologous urothelial stem cells that can potentially be used towards the development of alternative bladder or urothelial substitutes. In this proposal we hypothesize that urothelial stem cells can be generated via direct conversion of fibroblasts and can reconstitute the bladder urothelium in the mouse. First, we will aim to generate urothelial stem cells via direct conversion, or transdifferentiation (Aim 1), and we will achieve this by overexpressing transcription factors associated with bladder development and screening for suprabasal and basal urothelial markers. We will validate our screening results with functional assays with organoids as well as multilayered assembloids. Second, we will map the epigenetic changes that take place during urothelial stem cell differentiation to suprabasal cells (Aim 2), and we will accomplish this by performing Omni ATAC-seq on control bladder organoids and urothelial stem cell organoids in basal and differentiation media conditions. By identifying differences in areas of chromatin hyperaccessibility, we will be able to identify transcription factor binding motifs enriched in stem cell and differentiated cell states. Finally, we will develop a bladder urothelial stem cell transplant protocol using mouse models of urothelial ablation and injury (Aim 3). We will determine if urothelial stem cell transplantation can reconstitute all cell types within the urothelium utilizing urothelial stem cells obtained from mouse bladders as well as those obtained from transdifferentiation, and we will test for functional outcomes. If we are successful in these aims, we will demonstrate that autologous urothelial stem cells can be generated via direct conversion of fibroblasts, and we will establish a source of cells for bladder substitute tissue engineering as well as a basis for cell-based therapy for disorders of the urothelium that are typically treated with surgical reconstruction using the gastrointestinal tract, such as severe radiation cystitis or severe interstitial cystitis. The impact of this work on human health will be significant as this work will potentially make urinary diversion and reconstruction surgery a much less morbid surgical option for patients with severe urothelial disorders.