Single-cell transcriptomics of the human retinal pigment epithelium and choroid in health and macular degeneration

Abstract

The human retinal pigment epithelium (RPE) and choroid are complex tissues that provide crucial support to the retina. Disease affecting either of these supportive tissues can lead to irreversible blindness in the setting of age-related macular degeneration. In this study, single-cell RNA sequencing was performed on macular and peripheral regions of RPE-choroid from 7 human donor eyes in 2 independent experiments. In the first experiment, total RPE/choroid preparations were evaluated and expression profiles specific to RPE and major choroidal cell populations were identified. As choroidal endothelial cells represent a minority of the total RPE/choroidal cell population but are strongly implicated in age-related macular degeneration (AMD) pathogenesis, a second single-cell RNA-sequencing experiment was performed using endothelial cells enriched by magnetic separation. In this second study, we identified gene expression signatures along the choroidal vascular tree, classifying the transcriptome of human choriocapillaris, arterial, and venous endothelial cells. We found that the choriocapillaris highly and specifically expresses the regulator of cell cycle gene (RGCC), a gene that responds to complement activation and induces apoptosis in endothelial cells. In addition, RGCC was the most up-regulated choriocapillaris gene in a donor diagnosed with AMD. These results provide a characterization of the human RPE and choriocapillaris transcriptome, offering potential insight into the mechanisms of choriocapillaris response to complement injury and choroidal vascular disease in age-related macular degeneration.

Keywords: age-related macular degeneration; choriocapillaris; choroid; single cell.

Fig. 1.

Identification of RPE and choroidal cell populations. (A) UMAP dimensionality reduction was performed to visualize the 11 identified RPE and choroidal clusters. A total of 4,335 cells were recovered after filtering. (B) An overview schematic of the cell populations found within the choroid. SC, Schwann cell; SMC, smooth muscle cell. (C) For each cluster, the average RNA expression of each gene was calculated. The top 100 most enriched transcripts in each cluster were identified and compiled, and the average expression of these transcripts was used as input for hierarchical clustering analysis. Violin plots of genes previously reported to be enriched in RPE and choroidal cell populations are plotted alongside the dendrogram.

Fig. 2.

Differential expression of macular versus peripheral pericytes and RPE cells. Differential expression was performed between smooth muscle cells/pericytes (A) and RPE cells (B) of macular and peripheral origin. For each gene, the average log fold change and the percentage of cells that express the gene above background are compared between the 2 clusters (Right). For example, a delta percent of +0.25 indicates that 25% more cells of macular pericytes express the gene above background than peripheral pericytes. Expression patterns from the most enriched genes were compared to bulk RNA expression from pooled RPE/choroid cell lysates (19), which largely corroborates the regional expression differences observed in cell populations identified by single-cell RNA sequencing.

Fig. 3.

Retinal versus choroidal endothelial cell gene expression. (A) Two of the donors in this investigation (donors 1 and 2) were included in a single-cell RNA-sequencing study of human retina (donors 2 and 3 of that report). The RPE/choroid and retinal data were aggregated for these donors. Two clusters of endothelial cells (clusters 5 and 6) consisting of 413 total cells were identified. (B) The clusters were largely distinct based on tissue of origin, with retinal endothelial cells colored in blue and choroidal endothelial cells colored in red. (C and D) Differential expression between retinal and choroidal endothelial cells was performed. For each gene, the average log fold change and the percentage of cells that express the gene above background are compared between the 2 clusters (Right). For example, a delta percent of +0.25 indicates that 25% more cells of choroidal endothelial cells express the gene above background than retinal endothelial cells.

Fig. 4.

Identification of choroidal vascular cell populations. (A) A second single-cell RNA-sequencing study was performed on 4 additional human donors. Endothelial cells were enriched prior to sequencing. A total of 14,234 cells were recovered after filtering, of which 8,521 (clusters 5 to 8) were classified as endothelial cells. (B) Choroidal vasculature consists of large caliber arteries and veins and an intermediary superficial capillary system known as the choriocapillaris. (C) The vast majority of cells in clusters 5 to 8 express the endothelial-specific transcript VWF. (D–F) Differential expression was performed to identify genes enriched in arterial (D), choriocapillaris (E), and vein (F) clusters. For each gene, the average log fold change and the percentage of cells that express the gene above background are compared between the 2 clusters (Right). For example, a delta percent of +0.25 indicates that 25% more cells of arterial endothelial cells express the gene above background than vein and choriocapillaris endothelial cells. (G) Hierarchical clustering of the top 100 most highly expressed genes in each cluster reveals that the choriocapillaris cluster has a more similar expression pattern to veins opposed to arteries. Expression of known endothelial (VWF), vein (DARC, MMRN1), choriocapillaris (CA4, PLVAP), and arterial (HEY1, SEMA3G) genes are compared across each endothelial cluster. (H) Expression of RGCC, 1 of the top 10 most enriched genes in the choriocapillaris cluster, was assessed by qPCR in an immortalized human choroidal endothelial cell line after treatment with 10% normal human serum (NHS, black) and 10% heat-inactivated serum (Hi NHS, gray) in 3 experiments (N1, N2, and N3). RGCC expression is increased in response to NHS compared to Hi NHS. **P < 0.01 and ***P < 0.001. (I) Immunohistochemical localization of RGCC. A section through the macula of an 87-y-old donor with type I neovascular AMD was colabeled with RGCC, the endothelial-specific fucose binding lectin Ulex europaeus agglutinin-I (UEA-I), and DAPI. RGCC localizes to an endothelial cell (yellow arrow) within a choroidal neovascular membrane (CNVM). BrM, Bruch’s membrane. Red fluorescence in the RPE is due to lipofuscin (an autofluorescent pigment); RGCC expression was not detected in the RPE cluster. (Scale bar: 25 µm.)