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Caoimhe Normile, Mustapha Irnaten, David A Simpson, Oisín Cappa, Colm J O'Brien; Single-Cell RNA Sequencing of Lamina Cribrosa Fibroblasts in Glaucoma. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2002.
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Optic nerve cupping in glaucoma is associated extra-cellular matrix (ECM) changes in the lamina cribrosa (LC). Previously, using micro-array, we have demonstrated upregulation of several ECM genes, including collagen 1A1, alpha-SMA and TGFbeta. The aim of this study was to use single-cell RNA sequencing (scRNA seq) to explore differential ECM gene expression in LC fibroblasts in human samples.
Lamina cribrosa cells were obtained from 2 normal and 2 glaucoma donors. Cells were cultured and dissociated using papain dissociation method. The suspension viability was >90%. Cells from 4 individuals were pooled and the cell suspension processed on the 10x Genomics platform at the QUB Genomics Core Technology Unit to capture a total of ~5000 cells. The library was sequenced on an Illumina Novaseq generating ~800 million reads. The FastQ files were processed using CellRanger software and the resulting expression matrix analysed with the Seurat package. The individuals were demultiplexed based on SNP data using the Demuxlet package.
Individual transcriptome data was generated for a total of 500 cells. These were distributed in four distinct cell clusters (Fig.1). While all individuals were represented in each cluster, one cluster comprised cells predominantly from one individual. The gene expression in all clusters was characteristic of fibroblasts. Many of the most differentially (p<0.0001) expressed genes between clusters encoded ECM proteins, such as SBSPON, LUM, FBN2 in cluster 1; BGN, FBN1, Col 6A2, PEDF in cluster 2; HGF, BLAM, Col4 A6 in cluster 3; and FGF7, GPC3, NCAM1 in cluster 4 (Fig.1).
Primary cultures derived from Lamina cribrosa are heterogeneous and comprise cells of at least four distinct types. Analysis of differential gene expression in cultures from different donors and following experimental manipulations must take into account differences in the relative proportions of these sub-types. ScRNAseq has identified 4 LC cell clusters each showing a differential pattern of ECM and fibrosis related genes that are relevant to the pathogenesis of glaucoma cupping.
This is a 2020 ARVO Annual Meeting abstract.
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