Fig. 5
From: 3D organoid-derived human glomeruli for personalised podocyte disease modelling and drug screening
Temporal analysis of MAFB-expressing cells within organoids. a Differentiation of MAFB-BFP2 iPSC into kidney organoids was successful with blue fluorescent protein 2 (BFP2) expression observed in live organoids from d7 + 7 onwards. Scale bar 1000 µm. b FACS plot showing the BFP2-positive cell population isolated at d7 + 10. Representative plot of three biological replicates. c RT-PCR analysis of BFP2-positive and negative organoid cell fractions showed MAFB expression is only found in BFP2-positive cells. d Brightfield and BFP2-fluorescent live imaging of glomeruli isolated from d7 + 18 MAFB-reporter organoids at the time of plating and after 24 h culture. Strong BFP2 signal is observed within glomeruli when in suspension (0 h and 24 h arrow), but does not remain active when the glomeruli are adhered for culture, nor is it expressed in the migrating podocyte population (inset). Scale bar 200 µm. e A principle component analysis of RNA sequencing (RNA-Seq) data was performed on three biological replicates for d7 + 10, d7 + 14 and d7 + 19 BFP2-positive cell populations and compared to OrgPod samples. A clear separation between the 2D cultured organoid podocytes and MAFB BFP2-positive cells isolated from whole organoids was observed in dimension 1; in dimension 2 the variable of time was evident in the separation of the triplicates. f 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 of statistically significant upregulated genes was identified in the OrgGlom (d7 + 19) vs immature podocyte (d7 + 10) MAFB-BFP2 population. g Heatmap showing the top 50 upregulated differentially expressed genes between d7 + 19 and d7 + 10, with many enriched genes found to transcribe proteins in the human glomerular ECM proteome. Fold change of log-normalised gene expression levels for each of the triplicate samples presented. h Gene Ontology (GO) enrichment analysis of the top 100 upregulated genes differentially expressed between d7 + 19 and d7 + 10 found enrichment of GO terms associated with extracellular matrix (ECM) maturation, collagen maturation and cell adhesion. The top 10 most statistically significant GO term categories are shown. P-value of 0.05 depicted as a dotted line. i Top 15 most-significantly upregulated genes with time, identified using a human renal glomerulus-enriched gene expression dataset31. Significant upregulation of genes associated with the maturation of additional glomerular cells, including endothelium and mesangial cells alongside specific GBM components and associated proteoglycans. Two-way ANOVA p < 0.0001; error bars = SEM; significant difference between time points assessed by Tukey’s multiple comparisons test; n = 3 biological replicates shown by symbols; F-value(Interaction) = 27.16, DF (Interaction) = 28; F-value (Gene) = 37.58, DF (Gene) = 14; F-value(Time) = 495.7, DF (Time) = 2
