Fig. 1: L-ORD-iRPE replicates disease cellular phenotype in a dish.

a Sanger sequencing confirms the presence of p.Ser163Arg variant in L-ORD-iPSCs. The heterozygous single nucleotide variant in C1QTNF5 (c.489 C > C/G) appears as a peak within a peak. b Boxplots of ΔCt values for RPE signature genes are comparable in healthy and L-ORD-iRPE (boxplots represent n = 3 independent experiments from at least two donors; 25th, median, and 75th percentiles correspond to the bottom, middle, and top of each box). c TEM images of healthy and L-ORD-iRPE monolayers show polarized RPE structure including abundant apical processes (orange arrow), melanosomes (magenta arrow), and basally located nuclei (white arrow). Scale bar: 2 µm. d SEM images of healthy-iRPE and L-ORD-iRPE demonstrate hexagonal morphology and abundant apical processes. Scale bar: 2 µm. e Boxplot of cell area reveals larger cell size in L-ORD-iRPE than heathy-iRPE. iRPE monolayers were immunostained with membrane marker (ADIPOR1) to outline their hexagonal shape (healthy-iRPE: n = 9; L-ORD-iRPE: n = 6). Total of n > 10,000 cells counted. f Scatterplot of dedifferentiation-related genes shows no difference in L-ORD-iRPE (n = 2 donors) compared to healthy-iRPE (n = 2 donors). g Transepithelial resistance (TER) measurements were performed using an epithelial voltohmmeter in L-ORD-iRPE (n = 264 inclusive of all clones, two donors) compared to healthy-iRPE (n = 266 inclusive of all clones, 2 donors). (h) Comparative analysis of sub-RPE APOE (red)-positive deposits (n = 25) in healthy-iRPE (n = 11) and L-ORD-iRPE (n = 14) show 1.3-fold (p = 0.01) increase in APOE staining. Hoechst 33342 staining confirms the lack of cellular debris contributing towards APOE staining. Scale bar: 100 µm. i Apical and basal VEGF secretion is mispolarized in L-ORD-iRPE (white, n = 13) compared with healthy-iRPE (gray, n = 17). For validation of iPSC-derived lines and karyotyping analysis: See Table 1 and Fig. S1. ∗p < 0.05; ∗∗∗p < 0.001; ns not significant.