1. Fig. 1 Characterization of hESC-RPE cells.
Characterization of hESC-RPE cells. (A) Timeline of the differentiation protocol for generating retinal pigment epithelial (RPE) cells from human embryonic stem cells (hESCs). (B) Quantitative reverse transcription polymerase chain reaction analysis was performed to measure mRNA expression of RPE markers [paired box 6 (PAX6), retinal pigment epithelium–specific 65-kDa protein (RPE65), bestrophin 1 (BEST1), and microphthalmia-associated transcription factor (MITF)] and pluripotency markers [POU class 5 homeobox 1 (POU5F1) and nanog (NANOG)] in three hESC-RPE cell differentiation batches at passage 1. Expression is presented relative to expression in undifferentiated hESCs. (C to E) Confocal images (maximal projections of zx planes) of hESC-RPE cells after immunostaining for the markers ZO-1 (zonula occludens-1) and BEST1 (C), PAX-6 and EZRIN (D), and MERTK and BEST1 (E). Nuclei were counterstained with DRAQ5 (white). Scale bars, 10 μm. (F) Evaluation by flow cytometry of the number of tyrosinase-related protein 1 (TYRP1)–positive and LIN28-positive cells before hESCs and after differentiation into RPE cells (hESC-RPE cells). Four hESC-RPE cell batches were tested in total. (G) Representative images of hESC-RPE cells after 3 hours of exposure or no exposure to fluorescein isothiocyanate (FITC)–labeled pig photoreceptor cell outer segment (FITC-POS; green). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (white). Scale bars, 10 μm. (H) Quantification of vascular endothelial growth factor (VEGF) secreted by hESC-RPE cells at different time points during culture using an enzyme-linked immunosorbent assay. Values plotted are means ± SD.
Karim Ben M’Barek et al., Sci Transl Med 2017;9:eaai7471
Published by AAAS