Epithelial Carcinogenesis Group, Spanish National Cancer Research Centre-CNIO, Madrid, Spain.
CIBERONC, Madrid, Spain.
Nat Commun. 2019 Sep 27;10(1):4407. doi: 10.1038/s41467-019-12307-1.
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 and scRNA-Seq, confirms that Notch signaling is required for differentiation. Urothelial organoids provide a powerful tool to study cell regeneration and differentiation.
了解尿路上皮干细胞的生物学和分化受到缺乏无限增殖方法的限制。在这里，我们建立了可以不间断维持超过 1 年的小鼠尿路上皮类器官。类器官的生长依赖于 EGF 和 Wnt 激活剂。高表达 CD49f/ITGA6 的特征是表达基底标记物的类器官形成细胞的亚群。分化后，多层类器官增殖减少，细胞层数减少，尿路上皮程序激活，并获得屏障功能。PPARγ 和 EGFR 的药理学调节促进分化。RNA 测序突出了增殖类器官中富集的基因集（即核糖体）和参与分化的转录网络，包括 Wnt 配体和 Notch 成分的表达。类器官的单细胞 RNA 测序 (scRNA-Seq) 分析显示了五个具有不同基因表达谱的簇。结合 γ-分泌酶抑制剂和 scRNA-Seq，证实 Notch 信号通路对于分化是必需的。尿路上皮类器官为研究细胞再生和分化提供了强大的工具。
