A Uveal Single Cell Atlas Reveals Stromal Cell Heterogeneity: The Stromal Basis of Degenerative Eye Disease

Abstract

Purpose : Single-cell RNA sequencing (scRNAseq) has revealed heterogeneity in cell populations often considered homogenous, such as fibroblasts. We use scRNAseq to show intra and inter-tissue heterogeneity of fibroblasts in the adult and the developing human uvea.

Methods : 66,967 cells from the iris, ciliary body and choroid of adult donors with no history of eye disease (n=3), and 95,943 cells from the anterior and posterior uvea of normal foetal eyes (n=8) were profiled by 3’ scRNAseq. Quality control, pre-processing and clustering was performed using the Scanpy suite of bioinformatics tools. Integration of donors was performed using Harmony. Cell annotation was manual using canonical cell type markers. Spatial localisation used RNA in situ hybridisation and multiplexed immunofluorescence staining. For functional experiments, primary fibroblast cell lines were stimulated for 24 hours with PEDF, gene expression was quantified using qPCR

Results : Significantly differentially expressed genes can be defined in the iris, ciliary body and choroid fibroblasts. Overrepresentation analysis shows upregulated pathways differ between sites, demonstrating fibroblast specialisation to support specific tissue functions. Fibroblast subtypes are discernible in the choroid, two key populations are named based on expression of PI16 & VEGFB and ANGPT1 & CXCL12 respectively. Overrepresentation analysis of these show upregulation of pathways such as the cyclooxygenase pathway and angiogenesis related pathways respectively, making them of interest in choroidal neovascularisation. The PI16+ population is specifically localised in the suprachoroid region, demonstrating a microanatomical niche within the tissue. Stimulation of cultured choroidal fibroblasts with PEDF, a factor produced by the RPE, significantly reduces PI16 production (p=0.037, one way ANOVA), indicating that proximity to the RPE is a driver of choroidal fibroblast heterogeneity. The fibroblasts of the foetal uvea also show differences between the anterior and posterior uvea, a key differentially expressed gene in anterior uveal fibroblasts is TFAP2B, a transcription factor linked to anterior segment dysgenesis in mouse models.

Conclusions : Distinct fibroblast subtypes within the uveal tract may be linked to disorders of development such as anterior segment dysgenesis, as well as degenerative diseases of the adult such as choroidal neovascularisation

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.