Fig. 1: Directed differentiation of kidney organoids for a proximal-to-distal fate switch.

a (left) Schematic of modified Takasato protocol for iPSC-derived kidney organoids. (right) Brightfield images of DD10, DD12, and DD14 organoids. Scale bar: 500 microns. b Human kidney immunofluorescence stains for proximal, medial, and distal domain markers at the PTA, RV, and SSB stages. Corresponding stage-matched kidney organoids at DD10, DD12, and DD14 are stained for the same markers. Yellow arrows denote polarized nephrons. Biological replicates (n > 4) showed similar results. Scale bar: 50 microns. c Bar graph showing percentage of total nephrons per organoid symmetrical vs asymmetrical WT1 expression on DD10, DD12, and DD14 (n = 2). d Schematic of kidney organoid differentiation trajectory from DD10-DD28. e Immunofluorescence stains of human kidneys at the RV, CSB, SSB, and CLSN stages showing transcription factors marking the distal nephron domains. The SSB includes labeled positional relationships of precursors of the CNT (pCNT), DCT (pDT), loop of Henle (pLOH), proximal tubule (pPT), and podocyte (pPod). Scale bar: 50 microns. f Immunofluorescence stains of DD12 organoids showing distal transcription factor expression in polarized vs unpolarized nephrons. Biological replicates (n = 3) showed similar results. Scale bar: 50 microns. g Heatmaps of TPM value z-scores of nephron cell type markers at the SSB stage, expressed across 10 differentiation conditions in DD14 organoids (n = 2). Blue arrow denotes condition 8. h, i Immunofluorescence stains of DD14 control (condition 1) vs DE (condition 8) organoids showing proximal-distal marker expression. Biological replicates (n > 4) showed similar results. Scale bar: 50 microns. j Volcano plot of DESeq analysis comparing DD14 DMSO control and DE organoid total mRNA sequencing data (Wald test p-value, adjusted by FDR correction). Dashed lines show 0.05 p-value cutoff (horizontal) and ±1.5-fold change cutoff (vertical).