Single-Cell RNA Sequencing Reveals the Heterogeneity of the Human Retinal Pigment Epithelium

Purpose: In this work, we performed single-cell RNA sequencing on adult human RPE cells to characterize heterogeneity in human RPE and identify the RPE stem cells. Methods: RPE cells were isolated from four adult human donor eyes. Single-cell RNA sequencing for cells from one donor eye was performed using 10X Genomics pipeline to survey the RPE subpopulations. For RPE cells from the other three donor eyes, the ICELL8 pipeline was used to obtain a broader and more in-depth survey of gene expression. RNA sequencing results were analyzed using the Seurat package for R. Results: Uniform Manifold Approximation and Projection (UMAP) analyses for the 10x data showed 8 different RPE subpopulations. Within the different RPE subpopulations we found a cluster with cones-specific transcripts (ARR3, PDE6H and OPN1MW), clusters with rod RNA (RHO and PDE6A), and a cluster with transcripts involved in vascular endothelial growth factor signaling (TF and SPP1). ICELL8 results revealed a small subpopulation of RPE cells positive for RPE65 that also expressed early retinal developmental genes (VIM, GNL3), and MKI67, a marker of actively cycling cells, making the cells of this cluster strong candidates for being the RPE stem cells. Our immunostaining for potential markers of clusters uncovered in our single-cell RNA-Seq results, further verify the identity of RPE populations. Conclusions: Our single-cell RNA sequencing results show that RPE is a heterogeneous tissue. Of the varied subpopulations, we have identified a small population with the potential to proliferate and expressing stem cell markers, suggesting that they may be the RPE stem cells.