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. 2019 Feb 4;9:1172. doi: 10.1038/s41598-018-37793-z

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© 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/.

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Vascular development is limited in the reconstituted kidney organoids in vitro (A) Scheme for kidney organoid reconstitution and culture in vitro. Three types of kidney progenitors at E11.5 (NPs, SPs, and Hoxb7-GFP+ UBs) were aggregated overnight with or without ECs. The organoids were subsequently cultured at the air-liquid interface for 2–6 days (days 3–7). (B) Sorting of ECs, NP, and SPs. CD31+ and/or Flk1+ cells were sorted as ECs (left), and the cells in the CD31−/Flk1− fraction (left) were further sorted into Itga8+ NP and Pdgfra+ SP fractions (right). (C–F) Whole-mount staining of kidney organoids aggregated with ECs and cultured for the indicated days. GFP+ UB branching and CD31+ vasculature formation (C,D), as well as Nephrin+ glomerulus formation (E), were observed. No Cx40+ arterioles (F) were observed. (G) Section staining of glomeruli (nephrin). No CD31+ ECs were detected in glomeruli. (H–L) Whole-mount (H–K) or section (L) staining of kidney organoids aggregated without ECs and cultured for the indicated days. No apparent differences between the organoids with (C–G) and without (H–L) ECs were observed. Scale bars: 100 µm (C–F, H–K); 10 µm (G,L). Representative images of three organoids each with and without ECs from three independent experiments are shown.