FIGURE 1.

Polarised EVs secretion in iPSC‐RPE. (A) EVs were purified using size exclusion chromatography from CCM of human iPSC derived RPE grown on porous membrane supports that allow the cells to develop apical (A) and basal (B) polarity. A representative TEM image of apical EVs is shown. Scale bar 500 nm. (B) TRPS was used to quantify the EV samples, with four independent experiments carried out to characterise the rate of EVs secretion in high‐risk compared to low‐risk RPE. Cell DNA contents were used for normalisation. The same data sets were used to calculate average polarised and cumulative rate of secretion. Overall, high‐risk RPE secrete statistically significantly more EVs compared to the control low‐risk RPE. (C) High‐risk cells apical EV secretion is statically significantly enhanced compared to low‐risk apical and basal EV secretion. *p < 0.05. Mean with SEM shown. Statistical analysis by means of normality test, followed by unpaired t test or ordinary one‐way ANOVA with Tukey's multiple comparisons test. (D) EVs samples were immunoblotted for markers of exosomes (vesicles of endosomal origin) and ectosomes, with Alix, CD63 and CD81 being detected at various levels and indicating their particular enrichment in the apical fractions compared to the basal counterparts, a feature of RPE EVs described previously (Klingeborn et al., 2017). Note equal volumes of apical and basal EVs were loaded per sample to visualise the polarisation of EVs load and contents. (E) Equal EV protein amounts were screened by western blotting for the levels of exosomal (CD63 and LAMP1) and ectosomal (CD9, CD81, SLC3A2 and BSG) markers, as proposed previously (Mathieu et al., 2021).