fig2

Figure 2. Characterization of iPSC-RPE cells. *Indicates statistically significant differences < 0.05, Mann-Whitney U-test. (A) TEER was measured once a week while iPSC-RPE cells were matured on Transwell inserts and reached a stable plateau after week 2. Data were pooled from all cell lines. (B) Protein expression of RPE-specific markers BEST1 and RPE65 was assessed by Western blot and was uniform in all cell lines. ACTB served as a loading control. (C) iPSC-RPE morphology was assessed by immunocytochemistry with anti-BEST1 and anti-ZO-1 antibodies (scale bar: 20 µm). (D) VEGF secretion into the apical and basal compartment of the Transwell inserts was measured by ELISA and pooled data from all cell lines show a polar secretion of VEGF with higher protein amounts found in the basal compartment. (E) POS phagocytosis was followed by an uptake and degradation assay. Successful uptake of POS by iPSC-RPE cells is indicated by prominent rhodopsin (RHO) staining (37 kDa) at 0 h of degradation while efficient protein degradation can be seen after 2 and 4 h. ACTB served as a loading control. iPSC: Induced pluripotent stem cell; TEER: transepithelial electrical resistance; RPE: retinal pigment epithelium; VEGF: vascular endothelial growth factor; POS: photoreceptor outer segments; ELISA: enzyme-linked immunosorbent assay.