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. 2016 Dec 1;10:551.
doi: 10.3389/fnins.2016.00551. eCollection 2016.

Novel Methodology for Creating Macaque Retinas With Sortable Photoreceptors and Ganglion Cells

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Free PMC article

Novel Methodology for Creating Macaque Retinas With Sortable Photoreceptors and Ganglion Cells

Shreyasi Choudhury et al. Front Neurosci. .
Free PMC article

Abstract

Purpose: The ability to generate macaque retinas with sortable cell populations would be of great benefit to both basic and translational studies of the primate retina. The purpose of our study was therefore to develop methods to achieve this goal by selectively labeling, in life, photoreceptors (PRs) and retinal ganglion cells (RGCs) with separate fluorescent markers. Methods: Labeling of macaque (Macaca fascicularis) PRs and RGCs was accomplished by subretinal delivery of AAV5-hGRK1-GFP, and retrograde transport of micro-ruby™ from the lateral geniculate nucleus, respectively. Retinas were anatomically separated into different regions. Dissociation conditions were optimized, and cells from each region underwent fluorescent activated cell sorting (FACS). Expression of retinal cell type- specific genes was assessed by quantitative real-time PCR to characterize isolated cell populations. Results: We show that macaque PRs and RGCs can be simultaneously labeled in-life and enriched populations isolated by FACS. Recovery from different retinal regions indicated efficient isolation/enrichment for PRs and RGCs, with the macula being particularly amendable to this technique. Conclusions: The methods and materials presented here allow for the identification of novel reagents designed to target RGCs and/or photoreceptors in a species that is phylogenetically and anatomically similar to human. These techniques will enable screening of intravitreally-delivered AAV capsid libraries for variants with increased tropism for PRs and/or RGCs and the evaluation of vector tropism and/or cellular promoter activity of gene therapy vectors in a clinically relevant species.

Keywords: adeno associated virus (AAV); fluorescent activated cell sorting (FACS); lateral geniculate nuclei (LGN) injection; macaque; photoreceptors (PRs); retinal ganglion cells (RGCs); subretinal injection.

Figures

Figure 1
Figure 1
Details of tissue processing from OD and OS eyes of animal AV263. Anterior segment and vitreous were removed from both eyes and eyecups immersed in oxygenated Ames media. Retinas were carefully isolated from RPE. The OS retina was divided into four quadrants, each intended for purposes described within the top panel. During incubation in Ames media, optic nerve from the OD eyecup was incubated in 2.5 mg/mL micro-ruby™ diluted in oxygenated Ames media for 3 h at room temperature. The OD retina was then divided into four quadrants, each intended for purposes described within the bottom panel.
Figure 2
Figure 2
Experimental design and details of tissue processing from OD and OS eyes of animal SA76A. Subretinal injections of AAV5-hGRK1-GFP (1.0 × 1012 vg/ml) were performed in five different sites of both OD (480 μl total) and OS (250 μl total) eyes. At 20 days post-injection, in life images (fluorescence fundus) were taken to confirm GFP expression within the subretinal blebs. Three days later, injections of micro-ruby™ were performed at a total of 9 sites (0.6 μl per site) within both lateral geniculate nuclei (LGN). At 32 days post injection, eyes were enucleated, and anterior segment and vitreous were removed. Four millimeters punches were made to isolate the macula/fovea from both eyes. The remaining OS retina was divided into superior and inferior hemispheres. The remaining OD retina was divided into 4 quadrants. Quadrant 2 of the OD eye was fixed and mounted for imaging. Quadrant 3- OD was combined with superior OS retina. Quandrant 4- OD was combined with inferior OS retina. The macula/fovea punches from both eyes were combined. All pooled samples were subsequently dissociated and sorted.
Figure 3
Figure 3
Isolation and characterization of macaque retinal cells labeled post sacrifice. Scatter plots show the different gating conditions used for isolating the Ruby+, PNA+, and unlabeled cells (blue rectangles) following incubation with papain for either 20 min (A) or 45 min (B). The total numbers of sorted Ruby+, PNA+, and unlabeled cells are shown below each respective scatter plot. Expression of THY1, RHO, and GNAT2 in Ruby+ and PNA+ cells following 20 min (C) or 45 min (D) papain dissociation. Expression was normalized to GAPDH and is shown relative to expression in unlabeled cells. Error bars represent SEM.
Figure 4
Figure 4
Fluorescent fundus images taken 20 days post- subretinal injection with AAV5-hGRK1-GFP in SA76A. Upper panel: 50° field of view images reveal green fluorescent protein (GFP) fluorescence and the location of the macula/fovea in both eyes (white circles). Lower panel: Montages show the location of the five injection blebs placed in each eye. Scale bars = 500 μm.
Figure 5
Figure 5
Coronal cross-section through the thalamus showing the locations of bilateral micro-ruby™ injections into the LGN. The left medial LGN is clearly labeled, as is the adjacent optic tract. The entire right LGN is labeled. Yellow arrows denote two injection tracks that targeted the nucleus. Scale bar = 1 mm.
Figure 6
Figure 6
Representative fluorescent images of retinal flat mounts from the inferior temporal portion of SA76A's OD eye. The upper panel shows micro-ruby™ labeled retinal ganglion cells and the lower panel shows AAV5-hGRK1- mediated GFP expression in photoreceptors. RGC, retinal ganglion cells. Scale bar = 25 μm.
Figure 7
Figure 7
Representative macaque fundus image is shown in (A) to denote retinal locations from which dissociated cells were used for FACS analysis. Scatter plots showing the different gates used for isolating the Ruby+, GFP+, and unlabeled cells (blue rectangles) sorted from superior (B), macula/fovea (C), and inferior (D) retinal regions. The total number of sorted Ruby+, GFP+, and unlabeled cells isolated by FACS is shown beneath each respective scatter plot.
Figure 8
Figure 8
Expression of THY1, RHO, GNAT2, GLUL, and GRM6 in Ruby+, GFP+, and unlabeled cells sorted from superior (A), macula/fovea (B), and inferior (C) retina. Expression was normalized to GAPDH. Expression of THY1, RHO, GNAT2, GLUL, and GRM6 in Ruby+ and GFP+ populations relative to the unlabeled cell populations from superior (D), macula/fovea (E), and inferior (F) retina. Expression was normalized to GAPDH and is shown as relative to anatomically matched unlabeled cell population. Error bars represent SEM.
Figure 9
Figure 9
Expression of M/L Opsin (OPN1LW) (A) and S Opsin (OPN1SW) (B) in GFP+ and unlabeled cells sorted from superior, macula/fovea, and inferior retinal regions. Expression was normalized to GAPDH. Error bars represent SEM.

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