Fig. 6. FSGS patient’s IPSC-kidney organoid-derived podocytes exhibit defective cell adhesion and mitochondria.

(A) FSGS patient’s IPSC characterization. DNA sequencing analysis of the INF2-DID region showed a heterozygous missense mutation causing serine (S) to proline (P) at amino acid position 186 in INF2-CAAX. (B) S186P INF2 IPSCs formed kidney organoids with glomeruli and tubular structures. There is no apparent difference in the distribution of glomeruli (nephrin stained) and tubules (E-cadherin stained). (C) Marker protein analysis of S186P organoids in basal and PAN-treated condition. The basolateral localization pattern of nephrin is altered to a punctate pattern by the S186P mutation. PAN injury affected nephrin staining pattern in normal and S186P organoids. The E-cadherin staining pattern remained unaffected in tubular structures. (D and E) Mitochondria assessments. Outgrown podocytes were examined for mitochondrial filament length. PAN-treated S186P podocytes showed a shorter filament length (*P > 0.001, PAN-treated heterozygous knock-in S186P INF2 podocytes versus other groups; one-way ANOVA and Tukey’s multiple comparison test; n, 100 for each group). (F) Cell adhesion assessments. Heterozygous S186P knock-in podocytes lack lamellipodial cortactin (white arrow) and are defective in cell adhesion on a crossbow micropattern in basal and PAN-treated conditions. The percentage of cells with lamellipodial cortactin was indicated (*P > 0.001, percentage of heterozygous knock-in S186P INF2 podocytes versus other groups; one-way ANOVA and Tukey’s multiple comparison test; n, 100 for each group). Scale bars, 10 μm.