Fig. 3

Organoid-derived glomeruli display improved podocyte identity. a Principle component analysis of RNA sequencing (RNA-Seq) data was performed on three biological replicates of OrgGloms, OrgPods and ciPods in both differentiated and undifferentiated states and compared to previously published data (Lab 2)³⁰. b Venn diagram displaying the intersections of each comparison, upregulated genes are shown in red and downregulated genes are shown in blue. This shows the greatest number statistically significant upregulated genes were identified in the OrgGloms versus differentiated ciPods. There was little to no overlap of differentially expressed genes in differentiated ciPods versus undifferentiated ciPods compared with all other pairwise comparisons. c Heatmap showing the top 50 differentially expressed genes of each sample triplicate. A stark contrast in expression levels between the 3D OrgGloms and the 2D podocyte cultures is observed, particularly in genes associated with the podocyte. Log-normalised gene expression levels depicted. d Gene Ontology (GO) enrichment analysis of the top 100 upregulated genes differentially expressed between OrgGloms and differentiated ciPods found enrichment of GO terms associated with developmental processes, and slit diaphragm components including genes associated with podocyte foot processes and those of the podocyte actin cytoskeleton. The top 10 most statistically significant GO term categories are shown. P-value of 0.05 depicted as dotted line. e Heatmap representation of key podocyte-associated genes showed low expression levels in both undifferentiated and differentiated ciPods, regardless of the laboratory in which they were cultured. By contrast, significantly elevated gene expression levels were displayed in the OrgGlom samples. Log-normalised gene expression levels depicted. f Quantitative PCR analysis of key podocyte-associated genes validated the RNA-seq data showing enhanced gene expression in OrgGloms compared to 2D podocyte models. Two-way ANOVA p < 0.0001; error bars = SEM; significant difference was assessed by Tukey’s multiple comparisons test; n = 3 biological replicates shown by symbols; F-value (Interaction) = 58.22, DF (Interaction) = 21; F-value (Gene) = 56.82, DF (Gene) = 7; F-value (Cell Type) = 544.4, DF (Cell Type) = 3. g A glomerular expression score was determined for each sample by calculating the average log expression across the top 100 upregulated genes identified from a human renal glomerulus-enriched gene expression dataset³¹ (GSE21785). OrgGlom samples were found to have the highest scores for this gene set, showing greatest congruence to human glomerular isolates