Fig. 3

Differentiation and characterization of the retinal pigment epithelium (RPE). a–c Bright-field images during the course of differentiation. a Epithelial population starting to show pigmentation and typical hexagonal morphology. b RPE culture plates showing the incremental levels of visible pigmentation at progressive day points. c Morphology of pigmenting RPE cultures at different day points. d–h Immunofluorescence images representing the protein expression during different stages of RPE differentiation. d RPE progenitor markers Ezrin and RX at day 35. e Tight junction protein ZO1 on day 35 and day 70. f RPE commitment factor MITF at day 35 and day 70; RPE maintenance factor OTX2 at day 35 and day 50. g Mature RPE markers RPE65 at day 50 and day 70 and PMEL17 and Tyrosinase at day 70. h RPE ciliation markers β-catenin at day 50 and day 70 and Phalloidin, ARL13B, and acetylated tubulin at day 70. i Gene quantification profile using real-time PCR of selected markers represented as fold change in 40-, 50-, and 70-day RPE with respect to undifferentiated iPSC. j Flow cytometry analysis of MITF, RPE65, PMEL17, FOXG1, and Ki67 in day 70 RPE. Images and graphs are representative of a minimum of three independent experiments. Scale bars represent 100 μm