Santos, Catarina P.Lapi, EleonoraMartínez de Villarreal, JaimeÁlvaro-Espinosa, LauraFernández Barral, AsunciónBarbáchano, AntonioDomínguez, OrlandoLaughney, Ashley M.Megías, DiegoMuñoz, AlbertoReal, Francisco X.2019-11-062019-11-062019Santos CP, Lapi E, Martínez de Villarreal J, Álvaro-Espinosa L, Fernández-Barral A, Barbáchano A et al. Urothelial organoids originating from Cd49fhigh mouse stem cells display Notch-dependent differentiation capacity. Nat Commun. 2019;10(1):4407. DOI: 10.1038/s41467-019-12307-12041-1723http://hdl.handle.net/10230/42751Understanding 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 and scRNA-Seq, confirms that Notch signaling is required for differentiation. Urothelial organoids provide a powerful tool to study cell regeneration and differentiation.application/pdfeng© The Author(s) 2019. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.info:eu-repo/semantics/articlehttp://dx.doi.org/10.1038/s41467-019-12307-1BladderDifferentiationStem cellsinfo:eu-repo/semantics/openAccess