Fig. 2: PI3K inhibitor-treated kidney organoid nephrons exhibit a shift toward early tubular nephron cell fates.

a UMAP of single-cell RNA-sequencing from day 10 (pre-treatment), day 12 control, and day 12 PI3K inhibitor (LY294002)-treated kidney organoid nephrons. Cells are colored by sample with marker gene annotations. b Heatmap of Z-scores for hierarchically clustered differentially expressed genes across samples. Representative genes are listed, with a Z-score legend. c Feature plots of genes enriched in each sample group from the differentially expressed gene list. d Feature plots of selected genes for each group based on sample characteristics. For c, d human nephron single-cell detection (Supplementary Fig. 1a) is shown on the left, organoid plot on the right. e UMAP of single-cell RNA-sequencing from day 12 control and day 12 LY294002-treated kidney organoid nephrons. Cells match a but exclude day 10 sample. Cells are colored by sample with marker gene annotations. f Heatmap of log₂ fold change (log₂FC) for differentially expressed genes, calculated as the ratio of LY294002-treated to control aggregate expression. Representative genes are listed with a log₂FC legend. g Feature plots of genes enriched in each sample from the differentially expressed gene list. Human nephron single-cell (Supplementary Fig. 1a) detection is on the left, organoid plot on the right. h Split violin plots of genes from the differentially expressed gene list. Top two rows show genes enriched in day 12 control cells; bottom two rows show genes enriched in day 12 LY294002-treated cells. Plot colors match sample colors from (e). i Gene ontology terms for the top 50 differentially expressed genes per sample. Top graph: day 12 control nephron cells; bottom graph: LY294002-treated nephron cells. j–l Whole-mount immunofluorescent stains of day 12 control and LY294002-treated kidney organoids. Boxed regions are magnified. Inset in j shows absence of detectable HNF4A protein (orange) in day 12 samples. Scale bars: 10 microns. m Whole-mount immunofluorescent stain of day 12 control and LY294002-treated kidney organoids. Insets highlight JAG1 and HNF1B protein detection. Scale bars: 500 microns. n Quantification of JAG1⁺ and HNF1B⁺ nephron size (µm²) and HNF1B⁺ intensity (RFU) for n = 3 day 12 organoids each, across all positive segments. SEM error bars shown. Statistical significance determined by two-sided Student’s t-test.