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

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.

Implication of specific retinal cell-type involvement and gene expression changes in AMD progression using integrative analysis of single-cell and bulk RNA-seq profiling

“…Retina was carefully separated from the underlying retinal pigment epithelium and choroidal vasculature (Fig. 1 A, B) and dissociated according to published protocols 27 . Dissociated cells, 36,959 and 55,426 cells from macula and periphery were processed through a 10X Chromium platform (Supplementary Fig.…”

A Novel Retinal Ganglion Cell Promoter for Utility in AAV Vectors

“…Whilst gene expression profiles of RGCs are available, the promoters that drive this expression are ill defined. We chose several key criteria to identify candidate promoters using microarray data for RGCs (Kim et al, 2006 ; Choudhury et al, 2016 ; Struebing et al, 2016 ). Conservation data of regions upstream of the most enriched RGC candidate genes were obtained from the UCSC genome browser database (UCSC, mm10).…”

“…Using these data, we assessed promoter conservation between mammalian species for genes that were highly expressed and enriched in RGCs, using data drawn from the UCSC database (mm10; Kent et al, 2002 ). Conservation of non-coding DNA sequence across species was used as an indicator of potential function, and a number of highly conserved promoter upstream sequences were identified from genes shown to be both highly expressed and enriched in RGCs (Kim et al, 2006 ; Choudhury et al, 2016 ; Struebing et al, 2016 ). The lead candidate promoter, upstream of the Nefh gene, was evaluated and compared to CMV -driven gene expression in RGCs in vivo .…”

A universal protocol for isolating retinal ON bipolar cells across species via fluorescence-activated cell sorting

“…This limitation holds true for other cell types in the retina as well, leading to the establishment of alternative cell isolation strategies. 34 To address this unmet need, we developed a method that is applicable to a broad range of in vivo models, in particular primate models.…”