Fig 2: Comparison of the NPC induction efficiency between HC- and HD-iPSC lines. (a) The size of spheres derived from HD-2 increased over time. Scale bars: 500 µm. (b) qRT-PCR profiling of T, OSR1, PAX2, WT1 and SIX2 of the spheres derived from HC-1 and HD-2 at 5 points during induction from iPSCs to NPSs. (c) RT-PCR for NPC marker gene expression, WT1, PAX2 and SIX2, of HC- and HD-iPSC-derived NPSs. (d) qRT-PCR relative to the expression of WT1, PAX2 and SIX2, between the HC- and HD-iPSC-derived NPSs (n = 12 in each group). (e) Immunostaining of WT1, PAX2 and SIX2 in dissociated HC-1 and HD-2-derived NPSs. Scale bars, 20 µm. (f) Comparison of the percentage of the WT1, PAX2 and SIX2 positive cells between the HC- and HD-iPSC-derived NPSs (n = 12 in each group). (g) Forward and side scatter plots of dissociated NPSs. Blue: DAPI positive dead cells. (h) Flow cytometric analysis of the HC-1 and HD-2-derived NPSs in the gate. (i) Comparison of the percentage of the ITGA8+/PDGFRA− population between the HC- and HD-iPSC-derived NPSs (n = 4 in each group). Full-length gel is presented in Supplementary Figure S5. NPCs, nephron progenitor cells; NPSs, nephron progenitor spheres; HC, healthy controls; HD, haemodialysis. Data are the mean ± SEM (two-tailed, unpaired t-test).

Fig 3: Nephron progenitors are generated from PAX2-null iPS cells. (A) FACS analysis of tissues induced from wild-type (+/+) and PAX2-null (−/−) iPS cells. The percentages of the ITGA8+/PDGFRA− nephron progenitor fraction from three independent induction experiments are determined (mean ± standard error). (B) Nephron progenitor markers are more abundantly expressed in the ITGA8+/PDGFRA− fraction (open bars) than in the ITGA8−/PDGFRA− fraction (closed bars) induced from the wild-type clone. Data represent mean ± standard error of three independent induction experiments. *p < 0.05, **p < 0.01. (C) Tubulogenesis is observed when the ITGA8+/PDGFRA− fraction is co-cultured with spinal cord for 9 days. SP: spinal cord. Scale bars: 200 µm. (D) Expression levels of nephron progenitor markers are unaltered, except for PAX2. qPCR analyses of sorted ITGA8+/PDGFRA− fractions from two wild-type clones (open bars) and two PAX2-null clones (closed bars) are shown (mean ± standard error). **p < 0.01.

Fig 4: 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.