Fig 2: Human iPSC-derived kidney organoids contain 17 distinct cell clusters, including well-developed podocytes showing filtration slits. (A) Schematic of the iPSC-derived kidney organoid culture protocol. iPSCs were differentiated in 2D either towards anterior intermediate mesoderm (AIM) or posterior intermediate mesoderm (PIM) and aggregates (300,000 cells) were made using a 1:2 AIM/PIM ratio on day 7. Organoids were cultured on the air-liquid interface of Transwell™ filters for an additional 18 days, using a mixture of growth factors as indicated. BMP7, bone morphogenic protein 7; CHIR, CHIR 99021; EGF, epidermal growth factor; FGF9, fibroblast growth factor 9. (B) Uniform manifold approximation and projection (UMAP) integration of 3900 single cells from human iPSC-derived kidney organoids results in 17 different cell clusters. CD, collecting duct precursors; DT, distal tubular cells; ECp, endothelial cell precursors; LH, Loop of Henle cells; NP, neuronal progenitors; PT, proximal tubular cells. (C-C?) Segmented patterning in human kidney organoids shows the presence of podocytes (NPHS1, magenta), proximal tubules [lotus tetragonolobus lectin (LTL), blue], distal-like [E-cadherin (ECAD), green] and collecting duct-like [ECAD (green) and GATA binding protein 3 (GATA3) (red)] cells. (D-E') TEM micrographs of organoid-derived podocytes showing cell bodies and interdigitating foot processes including a filtration slit, indicated by an arrowhead in E'. (F-H) Filtration slit proteins podocin (red) and nephrin (green) are expressed at the podocyte cell membranes. (I-K) The podocyte-specific transcription factor Wilms' Tumor 1 (WT1, green) is expressed in podocyte nuclei (podocin, red; nuclei, blue). (L-N) Phospholipase A2 receptor (PLA2R, red) expression at the podocyte cell membranes (nephrin, green). (O-P') This cluster was subsetted and merged with the subsetted podocytes from the organoids. Super-resolution filtration slit imaging of nephrin in organoid-podocytes (O,O') and in a morphologically healthy appearing glomerulus of a Munich Wistar Frömter rat (P,P').

Fig 3: Single-Cell RNA-Seq Analysis of Kidney Organoids Reveals ACE2 Expression in Proximal Tubule Cells, Related to Figure 4(A) UMAP plot displaying the results after unbiased clustering. Subpopulations of renal endothelial-like, mesenchymal, proliferating, podocyte and tubule cells were identified. (B) Expression of ACE2 projected in the UMAP reduction. (C) Expression of different cellular markers: SLC3A1, SLC27A2 (Proximal Tubule); PODXL, NPHS1, NPHS2 (Podocyte); CLDN4, MAL (Loop of Henle) and CD93 (Renal Endothelial-like cells).

Fig 4: Extended monolayer culture in CDBLY2 increases SIX2+ progenitor contribution to nephrons and proximalisation.a Confocal immunofluorescence of D7 + 14 (standard protocol) and D13 + 14 (extended protocol) organoids derived from the SIX2Cre/Cre:GAPDHdual lineage tracing iPSC line. Images depict merged and separated channels showing lineage-traced SIX2+ cells (mCherry; red), distal tubules (ECAD; green), podocytes (NPHS1; grey) and proximal tubules (LTL; blue). Scale bars represent 100 µm. b Flow cytometry of SIX2Cre/Cre:GAPDHdual lineage tracing organoids derived from extended (13 day + CDBLY2) and standard (7 day + E6 media) differentiations depicting SIX2-derived mCherry + (red) cell contribution to the EPCAM + (nephron) population. Flow plots shown in top panels are representative of the replicates across multiple experiments. Percentage mCherry contributions from flow cytometry are depicted in bar graph (bottom panel), where error bars represent SEM from n = 4 biological replicates across 3 independent experiments. Statistical significance was determined using an unpaired t test. Asterisks represent two-tailed P values adjusted for multiple comparisons using the Holm–Sidak method, alpha = 0.05 (*P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001 [Experiment 1: P = 0.000924; Experiment 2: P = 0.004899; Experiment 3: P = 0.004845]). Source data are provided as a Source data file. c Confocal immunofluorescence of D13 + 14 organoids demonstrating (left panel) aligned nephron morphology with nephron segmentation makers (nephron epithelium [EPCAM; green], distal tubule/Loop of Henle [SLC12A1; red], proximal tubules [LTL; blue], and podocytes [NPHS1; grey]) and (right panel) the presence of few GATA3 + connecting segment/ureteric epithelium structures (red), co-stained for nephron epithelium (EPCAM; green), podocytes (NPHS1; grey), and proximal tubules (LTL; blue). Inset in right panel shows GATA3 and LTL alone. Scale bars in (i) and (ii) represent 200 µm and 100 µm, respectively. d Confocal immunofluorescence of a D7 + 14 (standard) organoid depicting homogenous distribution of podocytes (NPHS1; grey), proximal tubules (LTL; blue), and nephron epithelium (EPCAM; green), as well as the presence of extended segments of centralised GATA3 + (red [nuclear]) connecting segment/ureteric epithelium (also highlighted in insets). Scale bar represents 200 µm.

Fig 5: Extended monolayer culture in CDBLY2 increases SIX2+ progenitor contribution to nephrons and proximalisation.A. Confocal immunofluorescence of D7+14 (standard protocol) and D13+14 (extended protocol) organoids derived from the SIX2Cre/Cre:GAPDHdual lineage tracing iPSC line. Images depict merged and separated channels showing lineage-traced SIX2+ cells (mCherry; red), distal tubules (ECAD; green), podocytes (NPHS1; grey) and proximal tubules (LTL; blue). Scale bars represent 100 µm. B. Flow cytometry of SIX2Cre/Cre:GAPDHdual lineage tracing organoids derived from extended (13 day + CDBLY2) and standard (7 day + E6 media) differentiations depicting mCherry contribution to the EPCAM+ (nephron) population. Flow plots shown in (i) are representative of the replicates across multiple experiments. Percentage mCherry contributions from flow cytometry are depicted in bar graph (ii). Error bars in (ii) represent SEM from n = 4 biological replicates across 3 independent experiments. Statistical significance was determined using an unpaired t test. Asterisks represent P values adjusted for multiple comparisons using the Holm-Sidak method, alpha = 0.05 (*; P = 0.05, **; P = 0.01, ***; P = 0.001, ****; P = 0.0001). C. Confocal immunofluorescence of D13+14 organoids demonstrating (i) aligned nephron morphology with nephron segmentation makers (nephron epithelium [EPCAM; green], distal tubule/Loop of Henle [SLC12A1; red], proximal tubules [LTL; blue], and podocytes [NPHS1; grey]) and (ii) the presence of few GATA3+ connecting segment/ureteric epithelium structures (red), co-stained for nephron epithelium (EPCAM; green), podocytes (NPHS1; grey), and proximal tubules (LTL; blue). Inset in (ii) shows GATA3 and LTL alone. Scale bars in (i) and (ii) represent 200 µm and 100 µm, respectively. C. Confocal immunofluorescence of a D7+14 (standard) organoid depicting homogenous distribution of podocytes (NPHS1; grey), proximal tubules (LTL; red), and nephron epithelium (EPCAM; green), as well as the presence of extended segments of centralised GATA3+ connecting segment/ureteric epithelium (also highlighted in insets). Scale bar represents 200 µm.