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Alberto Muniz, Mark Plamper, Jae Hyek Choi, Whitney Greene, Anthony Johnson, Andrew Tsin, Heuy-Ching Wang; Human iPS-RPE Synthesize and Release 11-cis Retinaldehyde from Exogenous All-trans Retinol. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3763.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelium (RPE) has recently been derived from human induced pluripotent stem (iPS) cells. Before newly derived iPS-RPE cells can be used for stem cell therapies, it is important to determine the functional ability of iPS-RPE. One crucial role of the RPE is uptake and processing of retinoids to supply photoreceptors with visual chromophore. However, the ability of iPS-RPE to support visual pigment regeneration has not been demonstrated. The purpose of this study is to investigate the retinoid processing ability of the visual cycle in cultured iPS-RPE.
RPE derived from human iPS cells were analyzed by RT-PCR and western blot analyses to detect expression of RPE visual cycle genes and proteins LRAT, RPE65, CRALBP and the RPE specific gene PEDF. To examine the retinoid processing ability of the iPS-RPE, all-trans retinol (all-trans ROL) was delivered to cultured iPS-RPE. After a 24 hour incubation period, retinoids were extracted from cell homogenates and the culture media. The extracts were analyzed by HPLC on a 0.2% to 10% dioxane/hexane gradient system. Retinoids were identified by retention time and respective absorbance spectrum.
Expression of the critical visual cycle components LRAT, RPE65, CRALBP and the RPE specific gene PEDF was confirmed by RT-PCR and western blot in iPS-RPE. In addition, following incubation with all-trans ROL, all-trans retinyl palmitate was detected from cell homogenates of iPS-RPE by HPLC . Furthermore, HPLC analysis of the culture media revealed a peak corresponding to the retention time and absorbance spectrum of 11-cis retinaldehyde (11-cis RAL). Retinoids were not detected in control samples.
iPS-RPE maintains expression of visual cycle proteins. The detection of all-trans retinyl palmitate in iPS-RPE and 11-cis RAL in the culture media demonstrates that the iPS-RPE can uptake, process, and secrete retinoids, confirming that the visual cycle proteins expressed in iPS-RPE are functional and can support visual pigment regeneration. This is the first study to demonstrate expression of visual cycle machinery and complete visual cycle activity in stem cell-derived RPE.
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