Fig. 3: Kidney organoid nephrons shift toward proximal-biased cell fates following early PI3K inhibitor treatment.

a Single-cell RNA-seq UMAP from day 14 control and PI3K inhibitor-treated (LY294002 from days 10–12, proximal-biased, PB) kidney organoid nephrons. Cells colored by sample with marker gene annotations. b Log₂ fold change (log₂FC) heatmap for differentially expressed genes, calculated as the ratio of proximal-biased to control aggregate expression. Representative genes listed with a legend. c Feature plots of genes enriched in each sample from the differentially expressed gene list. d Split violin plots of differentially expressed genes. Top two rows: genes enriched in day 14 control cells; bottom two rows: genes enriched in day 14 proximal-biased cells. Plot colors match sample colors from (a). e Feature plots of selected genes for each group based on sample characteristics. For c, e human nephron (Supplementary Fig. 1a) detection is on the left, organoid plot on the right. f Immunofluorescent stains of day 14 control and proximal-biased kidney organoids. Boxed regions split and magnified. White arrowheads mark early HNF4A⁺ nephron regions, yellow arrowheads indicate autofluorescence. Scale bars: 10 microns. g Bulk RNA-sequencing (TPM) of select genes on days 10, 12, 14, and 18 from n = 2 whole organoids. SEM error bars shown. h, i FACS of day 18 kidney organoids from an HNF4A-YFP iPSC reporter line³⁵ for control (h) and proximal-biased (i) conditions. Brightfield and YFP channels are shown as merged and split panels. FACS plots in duplicates with YFP⁺ percentages in bottom right. Scale bars: 500 microns. j Immunofluorescent stains of week 16.1 human kidneys during HNF4A⁺, HNF4G⁺ proximal tubule elongation. White arrowheads mark endothelial autofluorescence. Scale bars: 10 microns. k Immunofluorescent stain of control and proximal-biased day 18 organoids. Boxed regions magnify HNF4A detection. Scale bars: 200 microns. l Total HNF4A⁺ nephrons in n = 4 whole day 18 organoids. SEM error bars shown. Statistical significance by two-sided Student’s t-test. m Average area (µm²) of HNF4A⁺ nephrons from n = 4 whole day 18 organoids, across all positive segments. SEM error bars shown. Statistical significance by two-sided Student’s t-test. n Sum total area (µm²) of HNF4A⁺ nephron segments from n = 3 whole day 18 organoids.