Skip to main content
. 2022 Oct 12;11:e80165. doi: 10.7554/eLife.80165

Figure 3. Defective kidney organoid differentiation from CEP83-deficient pluripotent stem cells.

(A, B) Brightfield images of organoids after a total of 25 days of culture (D25) indicate the formation of multiple kidney-like structures in WT organoids (A), whereas CEP83−/− organoids are composed of uniform clusters (B). (C, D) Representative images of hematoxylin-eosin–stained sections of organoids. WT organoids (C) display glomerulus-like (yellow arrows) and tubular (red arrow) components, whereas CEP83−/− organoids (D) are composed of monomorphic mesenchymal-like cells. (E–F) Whole mounting immunostaining of organoids for NPHS1 (podocyte marker), LTL (proximal tubule marker), and CDH1 (distal tubule marker) indicates segmented nephron-like structures in WT organoids (E), and the absence of such structures in CEP83−/− organoids (F). (G) Quantitative analysis of brightfield images indicating the estimated percentage of organoid area composed of nephron-like structures, organoids were collected from three different experiments. (H–L) Gene expression (transcripts per million [TPM]) of NPHP1 (H), LRP2 (I), HNF1B (J), PAX2 (K), and PAX8 (L) in WT and CEP83−/− cells at the indicated time points based on bulk RNA sequencing. n=3 clones per group. Data are mean ± SD. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. ns = not significant. Panels A–F: Bar = 50 μm. See Figure 3—source data 1 and 2. See also Figure 3—figure supplements 1–2.

Figure 3—source data 1. The data shows the quantitative analysis of the percent of nephron formation per organoid in knockout organoids versus WT organoids.
Figure 3—source data 2. The sheet shows the plotted TPM values of mRNA sequencing analysis in Figure 3L-H for the expression of renal epithelial marker genes in KO and WT at days 0, 7, and 25 of the differentiation.

Figure 3.

Figure 3—figure supplement 1. Whole mount immunostaining of the wildtype organoids shows positive staining for NPHS1 (podocyte marker), LTL (proximal tubule marker), and CDH1 (distal tubule marker).

Figure 3—figure supplement 1.

Bar = 50 µm.
Figure 3—figure supplement 2. mRNA analysis of organoids differentiated for 7+ (18) days indicates marked differences in global gene expression in CEP83−/− (KO1–KO3) compared to wildtype (WT1–WT3) organoids.

Figure 3—figure supplement 2.

(A) Heatmap displaying the expression of the top 1000 highly variable genes (see Methods, transcripts per million [TPM] ≥10) using bulk RNA within WT (WT1, WT2, WT3) and CEP83−/− (KO1, KO2, KO3) organoids. Hierarchical clustering of clones indicating that global gene expression is profoundly different in WT and KO organoids. Ontology analysis of the biological processes (BPs) using the top 100 downregulated genes (based on fold change values) in CEP83−/− organoids (TPM >2, fold change >1.5, p-value calculated on log10 TPM <0.05) using DOSE and cluster profile packages in R2. The analysis shows downregulation of many BPs associated with kidney development in CEP83-mutated organoids, as shown in the dot plot (B). Bulk RNA sequencing shows downregulation of specific renal epithelial cells marker genes at day 25, including (C–E) PODXL, WT1, and PTPRO for podocytes, (F– H) EPCAM, EMX2, and MAL2 marker genes for the distal nephron precursor cells. RT-PCR confirms that the expression of some nephron markers, including (I) NPHS1 (podocytes), (J) CUBN (proximal tubules), and (K) GATA3 (distal tubules and collecting duct) was significantly downregulated in CEP83−/− organoids. n=3 clones per group. Data are mean ± SD. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. ns = not significant.