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Jody A Summers Rada, Salman Hamid, Angelica Harper, Lynn Forest-Smith, Jonathan Wren; Isolation and Transcriptome Analyses of Choroidal Retinaldehyde Dehydrogenase-2 (RALDH2) Expressing Cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1100.
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© ARVO (1962-2015); The Authors (2016-present)
Choroidal expression of RALDH2 by an unknown cell type is responsible for all-trans-retinoic acid (atRA) synthesis during visually guided eye growth. Therefore, the objective of this study is to identify the cell type(s) responsible for choroidal atRA synthesis in the chick and human eye.
Choroidal cells were isolated following digestion of isolated choroids with collagenase/trypsin. Cells were stained with Ghost Dye UV 450 (to label non-viable cells), fixed, and immunolabeled with anti-chick RALDH2 and anti-rabbit IgG-Alexafluor 488 (2○ antibody). RALDH2(+) cells were isolated by fluorescence activated cell sorting (FACS) using gates for Alexfluor 488 and Ghost Dye UV 450. RNA was isolated from the Ghost(-)/RALDH2(+) cell population as well as from unsorted choroidal cells, RNAseq libraries were constructed, and transcripts sequenced using an Illumina MiSeq. Raw data were analyzed using CLC Genomics Workbench and mapped to the Gallus gallus genome. The choroidal RALDH2(+) cell transcriptome was analyzed using: 1) the computational algorithm GAMMA, 2) expression of cell-type specific signature genes, and 3) Ingenuity Pathway Analysis.
Following FACS isolation and RNAseq, a total of 13,579 genes were detected in RALDH2(+) cells; 2131 were upregulated (≥ 2 fold) and 589 were downregulated (≤ 0.5 fold), relative to unsorted cells, (p ≤ 0.01, Baggerley’s Beta-binomial test). Among the most highly expressed (8,464 – 15,773 RPKM) and significantly overexpressed (↑6 – 526 fold) genes in the RALDH2(+) population included: Ig lambda chain-C region; ferritin heavy chain; apolipoprotein A-1; C-reactive protein; and hemoglobin subunit gamma-2. GAMMA analyses of overexpressed genes suggests that RALDH2+ cells resemble T-cells and monocytes. Additionally, choroidal RALDH2+ cells expressed cell type-specific signature genes associated with myofibroblasts, smooth muscle cells and pericytes. RALDH2(+) cells were similarly isolated by FACS from the choroids of two human eyes, representing approximately 29% of the total cell population.
Choroidal RALDH2(+) cells can be isolated from the chick and human choroid and utilized for transcriptome analyses. At present, it is unclear as to whether these cells are endogenous choroidal cells or represent a population of cells that migrate into the choroid from the vasculature or neighboring tissues.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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