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Shaomin Peng, Caihong Qiu, Lina Li, Ron A. Adelman, Lawrence J. Rizzolo; Tight Junctions of RPE Derived from Human Embryonic Stem Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2237.
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Age-related macular degeneration (AMD) is the leading cause of blindness in patients over 60. Human embryonic stem cells (hESC) may serve as unlimited donor source of retinal pigment epithelium (RPE) cells for transplantation. This report characterizes the tight junctions of RPE derived from hESC.
Embryoid bodies (EBs) from the H1 line were formed in knockout medium with 10 mM nicotinamide. A week later, EBs were plated on laminin-coated culture dishes for 6 weeks. To promote RPE differentiation 140 ng/ml activin A was added during the third and fourth weeks. Monolayers of pigmented epithelial cells were isolated and cultured on laminin-coated Transwell filters for 6-8 weeks. Some cultures were transferred to a serum-free medium. The transepithelial electrical resistance (TER) was used to assess the function of tight junctions. Gene expression of claudins and occludin was examined by quantitative real-time RT-PCR, and protein expression was examined by immunoblotting and confocal, immunofluorescence microscopy. Expression was compared to human fetal RPE (hfRPE) isolated from 15-16 week fetuses.
The hESC-derived RPE cells exhibit the polygonal monolayer morphology with melanin granules and RPE-specific gene markers such as RPE65, Bestrophin, CRALBP, Otx2, MITF, tyrosinase and PEDF. The TER was ~250 Ω×cm2. Like native fetal RPE, Claudin-19 mRNA was the most prominent mRNA and was >30× claudin-3, >100× claudin-1, and >900× claudin-2. Other claudins were evident that are not normally expressed by native hfRPE included claudins-6 and -14. Claudin-19 was evident by immunoblotting and localized to tight junctions in all cells by immunofluorescence. Unlike hfRPE, claudin-3 was only evident in some cells and often along the lateral membrane in addition to the tight junction. By switching to a serum-free medium, the amount of claudin-3 increased, all the cells were positive, and immunofluorescence was confined to tight junctions. Like hfRPE, claudin-2 was only evident in a few cells.
The hESC-derived RPE cells express appropriate RPE markers, including claudin-19 as the predominant claudin. Nonetheless, more native-like expression of the claudin family could be induced using specialized culture media.
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