Summary This project builds upon the Principal Investigator’s extensive experience in studying urothelial differentiation and keratinocyte stem cells, and aims at providing a better understanding of bladder urothelial stem cells. Despite the central importance of urothelial stem cells in the homeostasis, regeneration and tumorigenesis of the bladder, their precise location and properties are still incompletely understood. There are several issues that hinder the progress in this field. For example, since the term “urothelium” is used to describe the uroplakin-expressing epithelium that covers the entire urinary tract including the renal pelvis, ureter, bladder and proximal urethra, it is often assumed that urothelia of all these sites are the same. In addition, studies on bladder urothelial stem cells have been limited to the bladder per se and no attention has been paid to urothelial stem cells outside of the bladder proper. However, we have recently discovered that ureterovesicular junction (UVJ) harbors a large cluster of strongly labeled (label-retaining) stem cells. The presence of a major cluster of stem cells outside of the bladder proper, located near the bladder-ureter orifice, is reminiscent of the corneal epithelium whose stem cells reside outside of the corneal proper in the limbal zone, and the follicular bulge stem cells that, although outside of the epidermis, are involved in epidermal repair. The goals of this project are to better understand the properties and in vivo functions of the UVJ stem cells, and their relationships with the bladder and ureteral urothelial stem cells. These studies can improve our understanding of urothelial homeostasis, regeneration, and diseases including metaplasia and urinary tract infection. To reach these goals, we will pursue three Specific Aims that will study: (i) molecular markers of urothelial lineages and stem cells, (ii) biological functions of the urothelial stem cells, and (iii) the possible roles of the Wnt pathway in regulating the urothelial stem cells.