Urothelial organoids originating from Cd49fhigh mouse stem cells display Notch-dependent differentiation capacity

Abstract

Understanding urothelial stem cell biology and differentiation has been limited by the lack of methods for their unlimited propagation. Here, we establish mouse urothelial organoids 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 cells expressing basal markers. Upon differentiation, multilayered organoids undergo reduced proliferation, decreased cell layer number, urothelial program activation, and acquisition of barrier function. Pharmacological modulation of PPARγ and EGFR promotes differentiation. RNA sequencing highlighted genesets enriched in proliferative organoids (i.e. ribosome) and transcriptional networks involved in differentiation, including expression of Wnt ligands and Notch components. Single-cell RNA sequencing (scRNA-Seq) analysis of the organoids revealed five clusters with distinct gene expression profiles. Together with the use of γ-secretase inhibitors, scRNA-Seq confirms that Notch signaling is required for differentiation. Urothelial organoids provide a powerful tool to study cell regeneration and differentiation.

A Cellular Reference Resource for the Mouse Urinary Bladder

“…Intermediate genes had more variable pseudotime profiles, however, each had some increase in expression early in pseudotime with a relative decrease at t=1. The Notch signaling transcriptional effector Hes1 identified as intermediate in our analysis has previously been shown to transiently increase during urothelial differentiation 15 lending credence to these genes constituting a true intermediate set. Transcription factors both with known (e.g.…”

STAG2 loss-of-function affects short-range genomic contacts and modulates urothelial differentiation in bladder cancer cells

“…Most UBC lines are derived from muscle-invasive UBC and, therefore, few of them harbour STAG2 mutations. Furthermore, until recently it has not been possible to permanently maintain normal urothelial cells in culture (Santos et al 2019). Therefore, we aimed at assessing the effects of STAG2 knockdown on one of the most commonly used luminal-type UBC line: RT112.…”

Application of Adult and Pluripotent Stem Cells in Interstitial Cystitis/Bladder Pain Syndrome Therapy: Methods and Perspectives

“…The research data using IC/BPS animal models should be meticulously scrutinized before further clinical trials since animal models have limitations in emulating human diseases. Alternatively, the generation of bladder organoids from human stem cells should be considered in the future for studying the pathogenicity of IC/BPS and drug screening [222,223].…”