Purchase this article with an account.
Alberto Muniz, Mark L. Plamper, Brandi S. Betts, Anthony J. Johnson, Heuy-Ching H. Wang; Visual Cycle Machinery in Human Induced Pluripotent Stem Cell-Derived RPE. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5903.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Recently RPE cells have been derived from human induced pluripotent stem (iPS) cells. These newly derived iPS-RPE cells are promising for stem cell therapies. Therefore it is of interest to determine the functional ability of iPS-RPE cells. One of the main roles of the RPE in-vivo is to uptake and process retinol to supply photoreceptors with visual chromophore. However the ability of iPS-RPE to uptake and metabolize retinol is unknown. In this study we investigate the expression of visual cycle proteins and the ability of iPS-RPE to uptake and process retinol.
The iPS-RPE cells were derived from iPS cell line IMR90-1 in differentiation media. RPE cells were then manually isolated, reseeded and subcultured up to passage five. For experiments, iPS-RPE cells at passage 5 were cultured for 29 days. The cells were then serum starved for 8 hours followed by a 24 hour incubation period with 10 µM all-trans retinol with 2% BSA in MEM. Control cells were cultured in the absence of all-trans retinol. The cells were then harvested, homogenized and retinoids were extracted with hexane. Retinoids were separated by HPLC and identified by retention time and absorbance spectrum. RPE65 Immunocytochemistry was performed on cells cultured for 17 days. Western blots against LRAT, RPE65, and CRALBP were performed on whole cell homogenate from iPS-RPE cultured for 29 days.
iPS-RPE cells showed all-trans retinol uptake. The cells also showed synthesis and accumulation of all-trans retinyl palmitate. No detectable retinoids were observed in the control samples. 11 cis- retinoids were not detected under these conditions. Western Blot analysis showed the presence of LRAT and CRALBP. RPE65 protein was also observed by Western blot and immunocytochemistry.
We have derived RPE cells from human iPS cells. These iPS-RPE cells have the ability to uptake all-trans retinol and synthesized all-trans retinyl ester in culture. These retinyl esters in human iPS-RPE cells are important since all-trans retinyl esters are the substrate for isomerohydrolase. Furthermore, the detection of LRAT, CRALBP, and RPE65 by Western Blot and cytochemisty in these iPS-RPE cells suggest they possess the machinery to synthesize 11-cis retinoid for support of the visual cycle. Lack of 11-cis retinoid detection may be due to thermo isomerization during processing of samples or a lack of sensitivity in our detection method. This study is the first to demonstrate functional retinol uptake and retinyl ester synthase activity in iPS-RPE cells.
This PDF is available to Subscribers Only