DESCRIPTION (adapted from the application) Diseases of the bladder that impact normal urothelial homeostasis cause significant morbidity and mortality for millions of Americans. A critical component of urothelial homeostasis is the ordered process of cellular maturation events known as urothelial differentiation, yet our understanding of the details of urothelial differentiation is restricted by the limitations of current in vitro models of human urothelium. The ideal human urothelial model would be an immortalized cell line that is cultured easily and indefinitely and yet retains the capacity to undergo urothelial differentiation in vitro. However, current studies utilize either primary urothelial cultures with a finite life span in culture or bladder carcinoma cell lines that lack the capacity for terminal differentiation. Our hypothesis is that clonal human urothelial cell lines can be generated by immortalization of primary cultures and that the resultant cell lines will retain the capacity for inducible expression of uroplakins, the terminal differentiation markers of urothelium. In preliminary studies, we generated a cell line that expressed uroplakins following either organotypic or semi-solid culture by immortalizing normal primary urothelial cells with papillomavirus E6E7. Therefore, two alternative methods, E6E7 and telomerase, will be compared for immortalizing primary urothelial cultures (Aim 1), since telomerase offers the advantage of conferring genomic stability. To compare the capacity of the resultant urothelial fines to differentiate (Aim 2), E6E7- and telomerase-immortalized cell lines will be induced to differentiate using both semi-solid and organotypic culture models, and induction of uroplakin and cytokeratin differentiation markers will be evaluated. Finally, an indicator cell line will be developed for capturing purified urothelial cell populations at specific stages of the differentiation process (Aim 3) by stably transfecting immortalized cells with a gene for the marker green fluorescent protein (GFP) whose expression will be driven by a differentiation specific promoter. By inducing differentiation of the indicator cell line in semi-solid cultures, cell populations at distinct stages of differentiation will be sorted and harvested by levels of GFP expression using fluorescence-activated cell sorting. Thus, these models of human urothelium will provide tools for future efforts to dissect the molecular and biochemical details of urothelial differentiation.