The human kidney contains up to 2 million epithelial nephrons responsible for blood filtration. Regenerating the kidney requires the induction of the more than 20 distinct cell types required for excretion and the regulation of pH, and electrolyte and fluid balance. We have previously described the simultaneous induction of progenitors for both collecting duct and nephrons via the directed differentiation of human pluripotent stem cells1. Paradoxically, although both are of intermediate mesoderm in origin, collecting duct and nephrons have distinct temporospatial origins. Here we identify the developmental mechanism regulating the preferential induction of collecting duct versus kidney mesenchyme progenitors. Using this knowledge, we have generated kidney organoids that contain nephrons associated with a collecting duct network surrounded by renal interstitium and endothelial cells. Within these organoids, individual nephrons segment into distal and proximal tubules, early loops of Henle, and glomeruli containing podocytes elaborating foot processes and undergoing vascularization. When transcription profiles of kidney organoids were compared to human fetal tissues, they showed highest congruence with first trimester human kidney. Furthermore, the proximal tubules endocytose dextran and differentially apoptose in response to cisplatin, a nephrotoxicant. Such kidney organoids represent powerful models of the human organ for future applications, including nephrotoxicity screening, disease modelling and as a source of cells for therapy.
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This research was supported by National Health and Medical Research Council (NHMRC) of Australia (APP1041277, APP1037320), Australian Research Council (ARC) (SRI110001002, CE140100036), Bontius Stiching and Organovo Inc. M.H.L. and R.G.P. are NHMRC Senior Principal Research Fellows. B.M. is a Rosamond Siemon Postgraduate Scholar. We thank A. Christ and T. Bruxner at the IMB Sequencing Facility for providing NGS service. We also acknowledge the IMB ACRF Imaging Facility and the Australian Microscopy & Microanalysis Research Facility at the Center for Microscopy and Microanalysis at The University of Queensland.
M.T. and M.H.L. are named inventors on a patent relating to this methodology. Some research funding was provided by Organovo Inc.
Extended data figures and tables
Extended Data Figure 1 Antero-posterior intermediate mesoderm specification is regulated by the timing of FGF9 exposure and the presence of RA signalling.
a, Immunofluorescence at day 18 of monolayer differentiation from cultures exposed to different timing of FGF9 addition (after 2, 3, 4 and 5 days of CHIR99021). The ureteric epithelium is represented by GATA3+PAX2+ECAD+ cells. The metanephric mesenchyme and its derivatives are marked by PAX2+GATA3−ECAD− (metanephric mesenchyme) and PAX2+GATA3−ECAD+ (nephrons), respectively. Scale bars, 100 μm. b, Immunofluorescence at day 7 and 18 of monolayer differentiation using 5 days of CHIR99021 followed by RA or AGN193109 (AGN) on top of FGF9. RA reduced the specification of posterior intermediate mesoderm, as indicated by the reduction of HOXD11 at day 7 (top panel). This resulted in less metanephric mesenchyme but some ureteric epithelium by RA at day 18 (bottom panel). Scale bars, 100 μm.
a, b, Immunofluorescence at day 18 of the monolayer differentiation using the 4 days CHIR99021 before FGF9 protocol. The metanephric mesenchyme is marked by SIX2+SIX1+HOXD11+ cells (a). GATA3+PAX2+ECAD+KRT8+ cells representing the ureteric epithelium were also induced (b). Scale bars, 50 μm.
a, Stimulating organoids with 5 μM CHIR99021 for 1 h immediately after aggregation promoted nephrogenesis (CHIR pulse), whereas only limited numbers of nephrogenesis events happened without CHIR99021 (no pulse). Scale bars, 1 mm. b, Without the addition of FGF9 after this CHIR99021 pulse, organoids did not initiate nephrogenesis (−FGF9). Scale bars, 200 μm.
Extended Data Figure 4 The timing of FGF9 exposure affects the ratio of collecting duct to nephron in the kidney organoid.
a, b, Immunofluorescence of kidney organoids at day 18 after-aggregation after exposure to different timings of initial FGF9 exposure (2, 3, 4 and 5 days of CHIR99021 pre-FGF9), demonstrating the regulation of collecting duct/nephron ratio by varying this timing. GATA3+ECAD+ cells represent the collecting duct (a), whereas WT1+NPHS1+ cells mark podocytes of the glomerulus (b). Scale bars, 200 μm.
a–c, Graphs showing expression changes of selected marker genes at 4 time points (day 0, 3, 11 and 18) of the kidney organoid culture. y axis represents the count of detection for each gene in an RNA sequencing analysis. Markers of the nephron progenitor (cap mesenchyme) and collecting duct progenitor (ureteric tip) were peaked by day 3 then dropped (a). Markers of early nephron increased by day 3, while those of mature nephron components (Proximal and distal tubule and Podocytes) started after day 3. Illustrations show expression regions (blue coloured) of each selected gene in the developing kidney (b). Markers of endothelial and renal interstitial cells were also increased by day 11 (c).
Dendrogram showing the hierarchical clustering of day 0, 3, 11 and 18 differentiation experiments and 21 human fetal organs from first and second trimester (Gene Expression Omnibus accession number GSE66302)15. Sample name is composed of individual ID followed by an organ name and gestation week. For instance, ‘DJ1 kidney_9’ represents a kidney at ninth week gestation from individual ID: DJ1. Day 0 and 3 kidney organoids cluster with gonad, in agreement with the common origin of both gonad and kidney from the intermediate mesoderm. Day 11 and 18 kidney organoids show strongest similarity to trimester 1 human kidney. The classifier genes used for this analysis are detailed in Supplementary Table 3.
a, Immunofluorescence of day 11 kidney organoids showing the presence of CD31+KDR+ endothelial cells surrounding NPHS1+ glomeruli. Scale bar, 100 μm. b, Two representative images demonstrating the expression of another endothelium marker SOX17 in CD31+ endothelial cells. Scale bars, 100 μm. c, Immunofluorescence of day 18 kidney organoids displaying endothelia with lumen formation, as indicated by asterisks. This image also shows the endothelial invasion into a glomerulus. Scale bar, 100 μm.
All images were taken from day 18 kidney organoids. a, PDGFRA+ pericytic cells attaching on KDR+ vessels. Scale bar, 50 μm. b, Some glomeruli contained PDGFRA+ cells likely to represent early mesangial cells19. Scale bar, 50 μm. c, Laminin staining (LAM) demonstrates the presence of basement membrane in glomerulus structures (white arrowheads). Scale bar, 100 μm. d, TEM images of avascular glomeruli showing early podocytes surrounding a basement membrane (yellow arrowheads) and exhibiting foot processes on the basement membrane. e, Immunofluorescence showing FOXD1 expression in podocytes (WT1+FOXD1+)18 and a subpopulation of MEIS1+ interstitium (white arrowheads). This is suggestive of the presence of both cortical stroma (FOXD1+MEIS1+) and medullary stroma (FOXD1−MEIS1+). Scale bar, 100 μm.
a, Fluorescent microscopy showing the dextran uptake in both the kidney organoids and E14 mouse embryonic kidneys organ culture after 24 h presence of dextran–Alexa488 (10 μg ml−1) in the culture medium (24 h dextran–Alexa488). 1 h incubation was insufficient for either organoids or mouse kidney explants to uptake dextran from the culture media (1 h dextran–Alexa488). No background signals were detected in a control without dextran (no dextran). Dashed line circles the organoids and kidneys. Scale bars, 1 mm. b, Endocytosis mediator cubilin (CUBN) was present on apical surface of the proximal tubules in kidney organoids (left panel). The same staining without detergent during the process showed the complete absence of CUBN staining on apical surface (right panel), demonstrating that the tubules within the organoids are intact. This explains the requirement for a 24 h incubation with dextran before evidence of apical uptake. Dashed line circles LTL+ proximal tubules. Scale bars, 50 μm. c, Low power immunofluorescence microscopy of day 18 kidney organoids after being treated by cisplatin for 24 h. No apoptosis was observed in proximal tubules in the absence of cisplatin (0 μM, left panel). LTL+ECAD+ proximal tubular cell-specific apoptosis was observed only in response to either 5 μM (not shown) or 20 μM cisplatin (arrowheads in middle panel). Global cell death was observed after culture in 100 μM cisplatin (right panel). Scale bars, 100 μm.
This table contains a list of primer sequences used for qPCR. (XLSX 41 kb)
This table shows RNA-seq profiling of organoid differentiation timecourse (day 0, 3, 11, 18). Data presented is normalized read counts including average counts for each timepoint. (XLSX 5655 kb)
This file contains a table of 85 Classifer genes15 used for the unbiased comparative analysis of RNAseq profiling data generated from trimester 1 (1T) and trimester 2 (2T) human fetal tissues compared to kidney organoids day 0, 3, 11 and 18 after commencement of 3D culture (Figure 2g, Extended Data Figure 6). (XLSX 13 kb)
Confocal microscopic Z-stack images from the bottom to the top of kidney organoids after 11 days in 3D culture. GATA3+ECAD+ CD, yellow with green nucleus; ECAD+ DT, yellow; LTL+ PT, red; NPHS1+ glomerulus, green membrane; DAPI, blue. Fields of view: 354.25 μm x 354.25 μm. (MOV 12982 kb)
Confocal microscopic Z-stack images from the bottom to the top of kidney organoids after 11 days in 3D culture. GATA3+ECAD+ CD, yellow with green nucleus; ECAD+ DT, yellow; LTL+ PT, red; NPHS1+ glomerulus, green membrane; DAPI, blue. Fields of view: 354.25 μm x 354.25 μm. (MOV 14698 kb)
Both videos represent confocal microscopic Z-stack images within a day 18 organoid scanning through vascularizing glomeruli. NPHS1+ podocytes of the glomerulus, green; CD31+ endothelium, pink; DAPI, blue. Fields of view: 212.55 μm x 212.55 μm. (MP4 3231 kb)
Both videos represent confocal microscopic Z-stack images within a day 18 organoid scanning through vascularizing glomeruli. NPHS1+ podocytes of the glomerulus, green; CD31+ endothelium, pink; DAPI, blue. Fields of view: 212.55 μm x 212.55 μm. (MP4 2669 kb)
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Takasato, M., Er, P., Chiu, H. et al. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature 526, 564–568 (2015). https://doi.org/10.1038/nature15695
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