Single-Cell RNA Sequencing Reveals Molecular Features of Heterogeneity in the Murine Retinal Pigment Epithelium

Transcriptomic analysis of the mammalian retinal pigment epithelium (RPE) aims to identify cellular networks that influence ocular development, maintenance, function, and disease. However, available evidence points to RPE cell heterogeneity within native tissue, which adds complexity to global transcriptomic analysis. Here, to assess cell heterogeneity, we performed single-cell RNA sequencing of RPE cells from two young adult male C57BL/6J mice. Following quality control to ensure robust transcript identification limited to cell singlets, we detected 13,858 transcripts among 2667 and 2846 RPE cells. Dimensional reduction by principal component analysis and uniform manifold approximation and projection revealed six distinct cell populations. All clusters expressed transcripts typical of RPE cells; the smallest (C1, containing 1-2% of total cells) exhibited the hallmarks of stem and/or progenitor (SP) cells. Placing C1-6 along a pseudotime axis suggested a relative decrease in melanogenesis and SP gene expression and a corresponding increase in visual cycle gene expression upon RPE maturation. K-means clustering of all detected transcripts identified additional expression patterns that may advance the understanding of RPE SP cell maintenance and the evolution of cellular metabolic networks during development. This work provides new insights into the transcriptome of the mouse RPE and a baseline for identifying experimentally induced transcriptional changes in future studies of this tissue.

Automated summary

This study analyzed the transcriptome of mouse retinal pigment epithelium (RPE) using single-cell RNA sequencing, identifying six distinct cell populations and revealing cell heterogeneity within RPE. One cell population exhibited stem and/or progenitor characteristics. Analysis along a pseudotime axis showed a relative decrease in melanogenesis and SP gene expression and an increase in visual cycle gene expression during RPE maturation. Additional expression patterns were identified that may contribute to understanding RPE stem cell maintenance and cellular metabolic network evolution.