title: Retinoid uptake, processing, and secretion in human iPS-RPE support the visual cycle.
abstract: OBJECTIVE
  Retinal pigmented epithelium derived from human induced pluripotent stem (iPS) cells (iPS-RPE) may be a source of cells for transplantation. For this reason, it is essential to determine the functional competence of iPS-RPE. One key 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. Immunocytochemistry, RT-PCR, and Western blot analysis were used to detect expression of RPE genes lecithin-retinol acyl transferase (LRAT), RPE65, cellular retinaldehyde-binding protein (CRALBP), and pigment epithelium-derived factor (PEDF). All-trans retinol was delivered to cultured cells or whole cell homogenate to assess the ability of the iPS-RPE to process retinoids.
  
  
  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 N-ethylmaleimide (NEM) prevented retinyl ester synthesis. Significantly, 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 classic 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 iPS-RPE.
authors.affiliation: Ocular Trauma, U.S. Army Institute of Surgical Research, JBSA Fort Sam Houston, Houston, Texas.
authors.forename: Alberto
authors.initials: A
authors.lastname: Muñiz
authors.forename: Whitney A
authors.initials: WA
authors.lastname: Greene
authors.forename: Mark L
authors.initials: ML
authors.lastname: Plamper
authors.forename: Jae Hyek
authors.initials: JH
authors.lastname: Choi
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Johnson
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Tsin
authors.forename: Heuy-Ching
authors.initials: HC
authors.lastname: Wang
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D005136:Eye Proteins / Q000096:biosynthesis / Q000235:genetics*; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D009414:Nerve Growth Factors / Q000096:biosynthesis / Q000235:genetics*; D012313:RNA / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*; D012176:Retinoids / Q000378:metabolism*; D015843:Serpins / Q000096:biosynthesis / Q000235:genetics*; D014785:Vision, Ocular / Q000235:genetics*; D000099174:Pigment Epithelium-Derived Factor
chemical_list: D005136:Eye Proteins; D009414:Nerve Growth Factors; D012313:RNA; D012176:Retinoids; D015843:Serpins; D000099174:Pigment Epithelium-Derived Factor
keywords: induced-pluripotent stem cell; retinal pigment epithelium; visual cycle