Urothelial organoids originate from Cd49f-High stem cells and display Notch-dependent differentiation capacity

Abstract

The urothelium is a specialized stratified epithelium with unique structural and functional features. Understanding the mechanisms involved in urothelial stem cell biology and differentiation has been limited by the lack of methods for unlimited propagation. Here, we establish normal mouse urothelial organoid (NMU-o) cultures that can be maintained uninterruptedly for >1 year. Organoid growth is dependent on EGF and Wnt activators. High CD49f/ITGA6 expression features a subpopulation of organoid-forming urothelial stem cells expressing basal markers. On induction of differentiation, multilayered organoids show reduced layer number, acquire barrier function, and activate the urothelial program, including expression of uroplakins and tight junction components. Combined pharmacological modulation of PPAR{gamma} and EGFR was most potent driving cell differentiation. Transcriptome analysis of organoids widely validates the system, highlights the transcriptional networks involved, and reveals NOTCH signaling as a novel pathway required for normal urothelial organoid differentiation.