Abstract
Purpose: Retinal pigment epithelium (RPE) is fundamental for vision and xanthophyll transport from the circulation to photoreceptors. RPE dysfunction occurs in numerous eye diseases, especially ischemic and neovascular retinopathies. This study is evaluates telomerase immortalized normal human RPE cells for xanthophyll vision research.Methods: Human RPE cells (hTERT‐RPE‐1) were cultured on porous membranes for 2 days to 6 weeks. Differentiation into a polarized RPE monolayer was evaluated by confocal imaging and transepithelial resistance (TER) measurement. RPE gene expression was analyzed by microarray profiling; expression changes in specific genes were verified by qRT‐PCR and western blotting.Results: RPE differentiation was confirmed by localization of the tight junction protein, ZO1, actin organization and development of high TER. Microarray analysis yielded 1,412 differentially expressed genes, including 852 down‐regulated genes and 560 up‐regulated genes. Gene set enrichment analysis and functional annotation clustering revealed emergence of specific gene pathways, including: retinal pigment synthesis, visual cycle, tight junctions, and transcellular transport. We also identified regulated expression of carotenoid/retinoid metabolic, transport and signaling genes, as well as of genes regulating angiogenesis, vascular remodeling, immunity and stress response. These findings indicate hTERT‐RPE‐1 cells achieve a high degree of RPE functional maturation. Initial experiments exposing RPE cells to oxidant/actinic stress + lutein indicate lutein protects RPE integrity.Conclusions: This study demonstrates morphological and gene‐expression profile changes in hTERT‐RPE‐1 cells during terminal differentiation. This model’s structural and functional properties characteristic of RPE cells in vivo suggest it’s unique, to date, utility and value for in vitro studies of RPE pathophysiology, including lutein/zeaxanthin effects and metabolism.
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