GENE EXPRESSION WITHIN A HUMAN CHOROIDAL NEOVASCULAR MEMBRANE USING SPATIAL TRANSCRIPTOMICS

Purpose: The goals of this study were to identify expression patterns in human macular neovascularization with spatial transcriptomics Methods: Independent libraries were prepared two human donors: one with macular neovascularation (MNV) and one healthy control. Three tissue sections were taken from the MNV donor and two from the control donor. From the right eyes of both donors, a 4 mm diameter macular punch was collected centered on the fovea centralis, as was a 4 mm peripheral punch approximately 12 mm superotemporal from the macula. Both central and peripheral punches (spanning retina, choroid and sclera) were placed in optimal cutting temperature compound (OCT) and were rapidly snap frozen in liquid nitrogen and stored at -80C. Final libraries were sequenced on an Illumina NovaSeq system. Sequenced reads were mapped to the human genome build hg38 with CellRanger(v7.0.0). Feature selection was performed with the variance stabilizing transformation with Seurat (v4). Results: Spots from the MNV and control donor were deconvoluted with CSIDE, which estimated the percent RNA contribution to each spot from different cell types. Differential expression was performed between the MNV and control donors. Within the area of neovascularization, endothelial cells were predicted to increase expression of genes related to Rho family GTPase signaling and integrin signaling. Likewise, VEGF and TGFB1 were identified as potential upstream regulators that could drive the observed gene expression changes produced by endothelial and retinal pigment epithelium cells in the macular neovascularization donor. Conclusions: Overall, this study spatially analyzes gene expression across the retina, retinal pigment epithelium, and choroid in health and describes a set of candidate molecules that become dysregulated in macular neovascularization. Overall design: mRNA profiles were spatially acquired for one donor with MNV and one control donor using the 10X Visium system followed by sequencing on an Illumina NovaSeq.