Characterization of RPE generated from human embryonic stem cells (hESC-RPE) derived on Synthemax II-SC. (A): Phase-contrast and bright-field images show the typical pigmentation and cobblestone morphology of H9 hESC-RPE cultured on different surfaces are shown. Scale bar = 200 μm. (B): Representative flow cytometry histograms are shown for the premelanosome pigmentation marker PMEL17 and the pluripotency marker Oct4 (not detected) in H9-RPE at passage 2 day 28 on the indicated substrate. (C): Expression of RPE marker genes in H9 hESC-RPE on the indicated substrates. fRPE served as a positive control (n = 3). ∗, p < .05, t test. (D): Normal karyotype of H9 hESC-RPE after spontaneous differentiation, enrichment, and three passages on Synthemax II-SC. (E): Epifluorescent images are shown of H9 hESC-RPE derived on Synthemax II-SC stained for the tight junction marker, ZO-1, RPE markers Otx2 and PMEL17, and the pluripotency marker Sall4 (not detected). Nuclei were detected using Hoechst (blue, merged right panels). Scale bar = 50 μm. Abbreviations: fRPE, fetal retinal pigmented epithelium; Mg-SR, condition of culturing and differentiating hESCs on Matrigel and then enriching and propagating the hESC-RPE on Synthemax-R; RPE, retinal pigmented epithelium.