Single-cell RNA sequencing reveals transcriptional changes of human choroidal and retinal pigment epithelium cells during fetal development, in healthy adult and intermediate age-related macular degeneration

Abstract

Age-related macular degeneration (AMD) is the most prevalent cause of blindness in the developed world. Vision loss in the advanced stages of the disease is caused by atrophy of retinal photoreceptors, overlying retinal pigment epithelium (RPE) and choroidal endothelial cells. The molecular events that underline the development of these cell types from in utero to adult as well as the progression to intermediate and advanced stages AMD are not yet fully understood. We performed single-cell RNA-sequencing (RNA-Seq) of human fetal and adult RPE-choroidal tissues, profiling in detail all the cell types and elucidating cell type-specific proliferation, differentiation and immunomodulation events that occur up to midgestation. Our data demonstrate that progression from the fetal to adult state is characterized by an increase in expression of genes involved in the oxidative stress response and detoxification from heavy metals, suggesting a better defence against oxidative stress in the adult RPE-choroid tissue. Single-cell comparative transcriptional analysis between a patient with intermediate AMD and an unaffected subject revealed a reduction in the number of RPE cells and melanocytes in the macular region of the AMD patient. Together these findings may suggest a macular loss of RPE cells and melanocytes in the AMD patients, but given the complex processing of tissues required for single-cell RNA-Seq that is prone to technical artefacts, these findings need to be validated by additional techniques in a larger number of AMD patients and controls.

Keywords: Schwann cells; age related macular degeneration (AMD); choroid; choroid endothelial cells; embryonic and fetal eye; melanocytes; pericytes; retinal pigment epithelium; single cell RNA-Seq; smooth muscle cells.

Figure 1

Single-cell RNA-Seq of fetal human RPE–choroid tissue. (A) Integrated UMAP of RPE–choroid tissue from 12 to 21 PCW revealing the presence of 26 cell clusters. (B) Violin plots showing the expression of key cell type-specific markers.

Figure 2

Volcano plots showing differentially expressed genes between early and late fetal development in RPE cells (A), CECs (B), Schwann cells (C), melanocytes (D), pericytes (E) and smooth muscle cells (F). Top 10 upregulated and downregulated genes are shown in green and red colour, respectively. A false discovery rate (FDR) threshold of <0.05 was applied.

Figure 3

Single-cell RNA-Seq of adult human RPE–choroid tissue. (A) Integrated UMAP (left) revealing the presence of 18 cell clusters in the adult RPE–choroid tissue and violin plots (right) showing the expression of key cell type specific markers. (B) Cell type representation in RPE–choroid tissue across five different donors of different ages. The age is shown on the x axis and cluster number on top of each panel.

Figure 4

Volcano plots showing differentially expressed genes between fetal development and adult in RPE cells (A), CECs (B), Schwann cells (C), melanocytes (D), pericytes (E) and smooth muscle cells (F). Top 10 upregulated and downregulated genes are shown in green and red colour, respectively. An FDR threshold of <0.05 was applied.

Figure 5

Single-cell RNA-Seq of human RPE–choroid tissues of an intermediate AMD patient and unaffected subject. (A) and (C) Integrated UMAP of macular and peripheral single-cell RNA-Seq of intermediated AMD patient (A) and unaffected subject (C) revealing the presence of 17 cell clusters; (B) and (D) Differential expression of macular versus peripheral pericytes, and RPE cells in the intermediate AMD patient (B) and unaffected subject (D). For each gene, the average log fold change is shown on the x axis. Genes enriched in the peripheral region and macular region are shown in red and green colour, respectively.

Figure 6

Comparative single-cell RNA-Seq of macular and peripheral region of the intermediate AMD patient versus unaffected subject. (A) and (B) Integrated UMAP of macular (A) and peripheral (B) regions showing the presence of 16 cell clusters. (C) and (D) Cell type representation in RPE–choroid tissue in the macular (C) and peripheral (D) region. The subject identity is shown on the x axis and cluster number on top of each panel.

Figure 7

Comparative single-cell RNA-Seq of RPE–choroid macular regions obtained from 12 AMD patients [one intermediate AMD presented in Figure 6 + nine early atrophic AMD (53) + two neovascular AMD (60)] and 11 unaffected controls [one control presented in Figure 6 + 10 additional controls (60)]. (A) Integrated UMAP of macular regions showing the presence of various cell clusters. (B) Cell type representation in RPE–choroid tissue in the macular region. The subject identity is shown on the x axis and cluster number on top of each panel.