June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Isolation of retinal ganglion cells for single cell transcriptomics in a model of dominant optic atrophy
Author Affiliations & Notes
  • Michael James Gilhooley
    Institute of Ophthalmology, University College London, London, London, United Kingdom
    Moorfields Eye Hospital NHS Foundation Trust, London, London, United Kingdom
  • Nicholas Owen
    Institute of Ophthalmology, University College London, London, London, United Kingdom
  • Mariya Moosajee
    Institute of Ophthalmology, University College London, London, London, United Kingdom
    Moorfields Eye Hospital NHS Foundation Trust, London, London, United Kingdom
  • Patrick Yu-Wai-Man
    Mitochondrial Biology Unit, University of Cambridge, Cambridge, Cambridgeshire, United Kingdom
    Institute of Ophthalmology, University College London, London, London, United Kingdom
  • Footnotes
    Commercial Relationships   Michael James Gilhooley None; Nicholas Owen None; Mariya Moosajee None; Patrick Yu-Wai-Man None
  • Footnotes
    Support  NIHR Grant CL-2019-18-004
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 1212 – A0212. doi:
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    • Get Citation

      Michael James Gilhooley, Nicholas Owen, Mariya Moosajee, Patrick Yu-Wai-Man; Isolation of retinal ganglion cells for single cell transcriptomics in a model of dominant optic atrophy. Invest. Ophthalmol. Vis. Sci. 2022;63(7):1212 – A0212.

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

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Abstract

Purpose : Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) survive late in dominant optic atrophy (DOA), long after other RGC sub-types (and vision) have been lost: the underlaying mechanism is unclear. Our purpose here was to optimise a pathway for the preparation and enrichment of single RGC suspensions from an experimental mouse model of DOA and wildtype retinas. This will facilitate single cell RNA sequencing (scRNAseq) approaches to determine if differentially upregulated neuroprotective genes or pathways in ipRGCs are the source of this resistance.

Methods : Retinas of C57/BL6J (P21) mice of both sexes were compared: four groups underwent papain enzyme treatment for 20, 30, 40 or 45 minutes respectively while a final group underwent mechanical disruption [N=3 mice (n=6 retinas) / group]. Fluorescence Assisted Cell Sorting (FACS) on the resulting single cell suspensions was used to enrich the samples for viable RGCs [CD90(thy1.2)+,CD48-,CD57-,CD15-,DAPI-]. This was validated by FACS-post-sort-purity-check, RGC marker (SNCG) immunocytochemistry (ICC) & RT-qPCR for RGC specific genes (Thy1, Brn3a, Sncg). This optimised protocol was used to isolate RGCs from both Opa1+/TTAG_del mice and littermate (wildtype) controls (5 months old, N=18 mice per group) for subsequent scRNAseq analysis.

Results : Viability post papain digestion was (mean±SEM) 60.8±2.4%, 54.4±4.3%, 60.2±5.9%, 59.3±3.3% at 20,30,40,45 minutes respectively with no difference between timepoints (one- way ANOVA F(1.28, 2.56)=1.84 p=0.30)). Mechanical dissociation led to lower viability (10.41±2.90%; t-test p<0.0001 vs. papain (30m) group). Following FACS processing, papain (30m) suspensions showed RGC enrichment of 78.0±6.6% on post-sort-purity-check and viability of 96.5±3.2% maintained (90.1±1.8%) 5 hours later. ICC showed 75.2±10.6% of cells in the RGC enriched sample staining for SNCG, compared to 13.1±3.1% in the remaining suspension (t-test p<0.0001); across the same comparison, expression of RGC specific genes demonstrated logfold increases by RT-qPCR of: Thy1 25.3±0.9, Brn3a 61.4±0.8, Sncg 7.5±0.9.

Conclusions : This optimised protocol has allowed for enrichment of RGCs from wildtype and DOA model retina for use in scRNAseq. Such investigations will contribute to a greater understanding of the disease resistance of ipRGCs, potentially opening the way for novel neuroprotective therapies for DOA.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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