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Lucero J. Vivar, Yueying Liu, Patrice Goletz, Mohammad Dahrouj, Peter H. Tang, Zsolt Ablonczy; Human Retinal Pigment Epithelium Cells As Functional Models for Retinoid Metabolism. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4295.
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© ARVO (1962-2015); The Authors (2016-present)
A culture model of the retinal pigment epithelium (RPE) is fetal human RPE (fhRPE) cells. Under correct culture conditions, these cells express considerable levels of RPE markers and can develop monolayers of highly pigmented cells that are uniform in size and shape. They also develop transepithelial potential and high (> 400 Ωcm2) transepithelial resistance (TER). Although these cells exhibit many functional charateristics of the native RPE, their ability to participate in retinoid metabolism and convert vitamin-A into 11-cis retinal has received only limited attention.
fhRPE cells were cultured on transwell filters by established methods until they formed monolayers of cuboid pigmented cells with a TER > 400 Ωcm2. The expression of RPE65, CRALBP, and CRBP were examined by immunoblotting and immunohistochemical analysis. mRNA expression levels of RDH 5, RDH 8, RDH 10, RDH were determined by RT-PCR. Some cultures were administered vitamin-A or all-trans retinal in phosphatidylcholine vesicles at a concentration of 5nM/L for 2 days, then the cells and the media were harvested and analyzed by HPLC to determine the respective retinoid profiles. Select cultures received bleached rod outer segments (ROS), or human albumin after the vesicles were absorbed.
fhRPE cells express the full complement of RPE proteins, which facilitate the isomerization of 11-cis retinal. Western blots provided evidence on the presence of substantial quantities of RPE65, CRALBP, and CRBP. RT-PCR showed the expression of RDH5, RDH8, RDH10, and RDH12. Immuno-histochemistry indicated that the proteins were localized in their native compartments. Feeding the cells with all-trans retinal or vitamin-A encapsulated in phosphatidylcholine vesicles resulted in the appearance of mainly retinyl esters and a small quantity of 11-cis retinal in the cells, while the administered retinoid disappeared from the media. Administering bleached rod outer segments or human albumin after the retinoid-filled vesicles were absorbed by the cells enhanced the quantity of detected 11-cis retinal.
Fully differentiated fhRPE cells not only express all the enzymes which facilitate the visual cycle, but these enzymes are in the correct cellular compartments, and together, they can also functionally convert vitamin-A to 11-cis retinal. The efficiency of the conversion is relatively little compared to the eye, but it is mostly a direct consequence of missing photoreceptors. When a sink, which can absorb 11-cis retinal, is introduced the conversion speeds up. These data demonstrate that fhRPE cells are a suitable functional model for vitamin-A metabolism.
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