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, 16 (11), 1112-1119

Organoid Cystogenesis Reveals a Critical Role of Microenvironment in Human Polycystic Kidney Disease


Organoid Cystogenesis Reveals a Critical Role of Microenvironment in Human Polycystic Kidney Disease

Nelly M Cruz et al. Nat Mater.


Polycystic kidney disease (PKD) is a life-threatening disorder, commonly caused by defects in polycystin-1 (PC1) or polycystin-2 (PC2), in which tubular epithelia form fluid-filled cysts. A major barrier to understanding PKD is the absence of human cellular models that accurately and efficiently recapitulate cystogenesis. Previously, we have generated a genetic model of PKD using human pluripotent stem cells and derived kidney organoids. Here we show that systematic substitution of physical components can dramatically increase or decrease cyst formation, unveiling a critical role for microenvironment in PKD. Removal of adherent cues increases cystogenesis 10-fold, producing cysts phenotypically resembling PKD that expand massively to 1-centimetre diameters. Removal of stroma enables outgrowth of PKD cell lines, which exhibit defects in PC1 expression and collagen compaction. Cyclic adenosine monophosphate (cAMP), when added, induces cysts in both PKD organoids and controls. These biomaterials establish a highly efficient model of PKD cystogenesis that directly implicates the microenvironment at the earliest stages of the disease.


Fig. 1
Fig. 1. Removal of adherent cues establishes a highly efficient model of PKD cystogenesis
a, Still images from Movie 1 showing cyst formation from a PKD organoid in adherent culture. b, Schematic of high-efficiency organoid cystogenesis protocol. c, Representative images of kidney organoids and d, quantification of cyst formation after two weeks of suspension culture (CTRL1 vs. PKD1-/-, n=3 separate experiments, ± s.e.m., t(3.663)=21.05, p=5.8949×10-5; CTRL2 vs. PKD2-/-, n=4 separate experiments, ± s.e.m., t(5.458)=10.66, p=7.3731×10-5). e, 6-well (3.5 cm) dishes containing PKD or control organoids after 9 months of culture. Scale bars, 100 μm (a-c) and 1 cm (e).
Fig. 2
Fig. 2. Organoid PKD cysts phenotypically resemble PKD patient cysts
a, Paraffin sections dyed with hematoxylin and eosin from PKD organoids, or human kidney biopsies taken from patients with autosomal dominant PKD (ADPKD), autosomal recessive PKD (ARPKD), and Meckel syndrome. Identifying labels are provided for orientation and emphasis of specific histological features (c, kidney capsule; z, nephrogenic zone; i, inflammatory infiltrate; cy, large cyst; post, postnatal). b, Confocal immunofluorescence showing nephron segment markers in PKD organoid cysts or c, PKD patient kidneys. Zoom shows close-up of dotted boxed region. Arrow represents an area of specific enrichment for LTL. Glomeruli (g) do not appear cystic. Neither LTL nor ECAD is detected in a large ADPKD cyst, whose epithelium has dedifferentiated (*). d, Percentage of PKD organoid cysts labeling positive for LTL, ECAD, or both markers (n=3 separate experiments, ± s.e.m.). e, Confocal optical sections showing LTL affinity in a representative cyst in suspension. Higher magnification (hi mag) image shows LTL in the adjoining organoid remnant portion of this cyst. f, LTL in cyst-lining epithelial cells. g, Cilia (acetylated α-tubulin) and tight junctions (ZO1) in representative cyst-lining epithelial cells. h, Representative confocal images showing stromal markers in PKD organoid cyst and patient cysts. Scale bars, 200 μm or 25 μm (f-g).
Fig. 3
Fig. 3. PKD organoid cysts arise from hyperproliferative KTECs
a, Representative images and b, quantification of pH3 in adherent PKD organoid cysts under adherent conditions. Boxes show 25th and 75th percentiles, whiskers indicate min and max values (n = 115 tubules pooled from 7 separate experiments and 26 cysts pooled from 6 separate experiments, ± s.e.m., t(37.16)=3.491, p=0.0013). c, Three-dimensional confocal reconstruction of a large cyst in suspension. Arrowhead indicates anaphases. See also Movie 3. d, Representative images showing microdissection of large cysts in suspension. e, Cell counts in organoids immediately after placement in suspension (org.) or in microdissected cysts grown for several months (cyst). Dashed lines represent non-linear breaks in the y-axis. f, Heat maps from microarray analysis of cysts and tubule remnants from cultured organoids, showing differentially expressed genes (p-value ≤ 0.05) contributing to activation of E2F targets, mTORC1 signaling, and Myc. Columns represent samples and row represent gene; red indicates greater than the mean (white) and blue, less than the mean values. Scale bars, 100 μm.
Fig. 4
Fig. 4
Outgrowth of PKD cell lines reveals a critical deficiency in PC1 expression. a, Phase contrast image of organoid explants on days 1, 4, and 12 after replating. b, Wide-field fluorescence and c-d, confocal sections showing epithelial and kidney-specific marker expression in representative kidney organoid cell monolayers. e, Representative immunoblots of PC1 and PC2 in kidney organoids and f, undifferentiated hPSCs. g, PC1 protein levels in undifferentiated hPSCs, normalized to b-actin loading control (ctrl, n = 6; PKD1-/- and PKD2-/-, n=3, ± s.e.m., ctrl vs. PKD1-/-, t(6.936)=6.603, p=0.00031 (***); ctrl vs. PKD2-/-, t(4.837)=5.669, p=0.0026 (**). h, PC2 protein levels in undifferentiated hPSCs, normalized to b-actin loading control (ctrl, n=6; PKD1-/- and PKD2-/-, n=3, ± s.e.m., ctrl vs. PKD1-/-, t(6.451)=0.9247, p=0.3884 (ns); ctrl vs. PKD2-/-, t(5)=8.006, p=0.0005 (***)). i, Representative immunoblot and j-k, quantification of PC1 and PC2 levels in hPSCs treated with four different PKD2 siRNAs (pooled or individually) or a scrambled (scr) siRNA control (n=3). j, Unpaired t test with Welch's correction, scr vs. pool, t(2)=11, p=0.0075; #2 vs. scr, t(2)=1.747, p=0.2227; #3 vs. scr, t(2)=22.66, p=0.0019; #4 vs. scr, t(2)=9.467, p=0.0110; #5 vs. scr, t(2)=11.56, p=0.0074. k, Unpaired t test with Welch's correction, scr vs. pool, t(2)= 16.92, p=0.0035; #2 vs. scr, t(2)=2.912, p=0.1005 (ns, not significant); #3 vs. scr, t(2)=31.93, p=0.0010; #4 vs. scr, t(2)=77.64, p=0.0002; #5 vs. scr, t(2)=20.28, p=0.0024. Scale bars, 100 μm (a-b) or 10 μm (c-d). ns, not significant.
Fig. 5
Fig. 5. Organoids remodel their matrix microenvironment in a PKD-dependent manner
a, Photograph of organoids implanted into collagen balls and cultured in suspension for two weeks. b, Diameters of empty (n=17) and organoid-implanted (ctrl, n=19; PKD, n=16) collagen droplets, pooled from three experiments (empty vs. ctrl, t(21.96)=13.53, p=3.9334×10-12; ctrl vs. PKD, t(24.74)=11.33, p=1.7989×10-6). Each droplet is indicated by a single data point. c, Wide-field immunofluorescence image of whole droplet compacted by a PKD organoid, stained for tubule segment markers. Zoom of dashed boxed region is shown for fluorescent channels. d, Confocal immunofluorescence at the edge of a representative droplet after contraction, adjacent to the collagen interior (col). The dashed boxed region is shown for each individual channel at higher magnification. e, Phase contrast images showing the edge of a representative droplet at an early stage of compaction. f, Sirius red staining of collagen droplets. g, TEM 25,000X images of collagen filament structure in the interior of droplets. Zoom of dashed boxed region is shown for each image. h, Schematic of collagen droplet compaction by organoids. KTECs migrate out and surround the scaffold, which contracts towards the outgrowth (curved arrows). Scale bars, 500 μm (b-f) or 500 nm (g).

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