Fig. 4: Single-cell transcriptomic and morphological analyses of nephron segment development in proximal-biased kidney organoids across differentiation stages.

a Schematic of single-cell RNA-sequencing timepoints with UMAP reduction of kidney organoid nephrogenic cells, colored by samples. b UMAP reduction of nephrogenic cell clusters in kidney organoids, colored by clusters, with identifying marker gene annotations. UMAPs split by sample type of origin (untreated day 10; control days 12, 14, and 18; or proximal-biased (PB) days 12, 14, and 18) are shown at the top. c Feature plots for select nephron marker genes with insets separating control from proximal-biased cells, and scaled percent quantification of total cells expressing the gene. Whole-mount immunofluorescent stains of control (d) and proximal-biased (e) kidney organoid nephrons from differentiation days 11, 12, 14, 15, and 18, with the bottom row highlighting HNF4A protein detection. Scale bars: 10 microns. Differentiation model for control kidney organoids (f) based on (d), and for proximal-biased kidney organoids (g) derived from (e). h Time-lapse video captures of kidney organoids differentiated from a human HNF4A-YFP iPSC reporter line³⁵ from differentiation days 15–18, showing the emergence and progression of HNF4A-YFP detection in control and proximal-biased conditions. Brightfield and YFP channels are merged. Scale bars: 200 microns. Movies are included as supplementary files. i Whole-mount immunofluorescent stains of control and proximal-biased kidney organoids from differentiation days 14 and 21. Boxed regions are magnified. Scale bars: 10 microns. j Whole-mount immunofluorescent stains of control and proximal-biased kidney organoids from differentiation days 14, 18, and 21. Boxed regions are magnified. Scale bars: 10 microns. k Quantification of nephron segments positive for indicated markers per 15X field of view at differentiation days 14 and 21. Data are from n = 3 independent organoids, including those shown in (i, j). SEM error bars shown. Statistical significance is determined using two-sided Student’s t-test. l Quantification of the average segment length (µm) for indicated proteins in day 18 control and proximal-biased kidney organoids. Data are from n = 4 independent organoids (15X), including those shown in Supplementary Fig. 7c, d. SEM error bars are shown. Statistical significance is determined using two-sided Student’s t-test.