April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Retinoid Uptake, Processing and Secretion in Human iPS-RPE Support the Visual Cycle
Author Affiliations & Notes
  • Alberto Muniz
    Ocular Trauma, United States Army Institute of Surgical Research, Fort Sam Houston, TX
  • Whitney Greene
    Ocular Trauma, United States Army Institute of Surgical Research, Fort Sam Houston, TX
  • Mark Plamper
    Ocular Trauma, United States Army Institute of Surgical Research, Fort Sam Houston, TX
  • Jae Hyek Choi
    Ocular Trauma, United States Army Institute of Surgical Research, Fort Sam Houston, TX
  • Anthony James Johnson
    Ocular Trauma, United States Army Institute of Surgical Research, Fort Sam Houston, TX
  • Andrew T C Tsin
    Department of Biology, The University of Texas at San Antonio, San Antonio, TX
  • Heuy-Ching Hetty Wang
    Ocular Trauma, United States Army Institute of Surgical Research, Fort Sam Houston, TX
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 3467. doi:
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      Alberto Muniz, Whitney Greene, Mark Plamper, Jae Hyek Choi, Anthony James Johnson, Andrew T C Tsin, Heuy-Ching Hetty Wang; Retinoid Uptake, Processing and Secretion in Human iPS-RPE Support the Visual Cycle. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3467.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Retinal pigmented epithelium (RPE) derived from human induced pluripotent stem (iPS) cells (iPS-RPE) may be a source of cells for transplantation, in-vitro disease models, and drug screening. For this reason it is essential to determine the functional competence of iPS-RPE. One essential role of the RPE is uptake and processing of retinoids via the visual cycle. The purpose of this study is to investigate the expression of visual cycle proteins and the functional ability of the visual cycle in iPS-RPE.

Methods: iPS-RPE was derived from human iPS cells. RT-PCR, immunocytochemistry and western blot analysis were used to detect expression of RPE- genes LRAT, RPE65, CRALBP and PEDF. All-trans retinol was delivered to cultured cells or whole cell homogenate to assess the ability of the iPS-RPE to process retinoids. Retinoids were extracted and analyzed by HPLC.

Results: Cultured iPS-RPE expresses visual cycle genes LRAT, CRALBP, and RPE65. After incubation with all-trans retinol iPS-RPE synthesized up to 2942 ± 551 pmol/mg protein all-trans retinyl esters. Inhibition of LRAT with NEM prevented retinyl ester synthesis. Additionally, after incubation with all-trans retinol, iPS-RPE released 188 ± 88 pmol/mg protein 11-cis retinaldehyde into the culture media.

Conclusions: iPS-RPE develops classical RPE characteristics and maintains expression of visual cycle proteins. The results of this study confirm that iPS-RPE possesses the machinery to process retinoids for support of visual pigment regeneration. Inhibition of all-trans retinyl ester accumulation by NEM confirms LRAT is active in iPS-RPE. Finally, the detection of 11-cis retinaldehyde in the culture medium demonstrates the cells’ ability to process retinoids through the visual cycle. This study demonstrates expression of key visual cycle machinery and complete visual cycle activity in induced pluripotent stem cell-derived RPE.

Keywords: 721 stem cells • 701 retinal pigment epithelium • 592 metabolism  
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