Abstract
Purpose :
RPE transplantation using grafts generated from adult RPE stem cells is a promising strategy to repair the damaged RPE tissue in patient with age-related macular degeneration. However, little is known about the RPE stem cells and their molecular characteristics. In this work, we performed single-cell RNA sequencing on adult human RPE cells to characterize heterogeneity in the RPE compartment 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 Gemonics 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 :
A total of ~6800 and ~5100 cells passed the quality check steps of the 10x pipeline and ICELL8 pipeline, respectively. Uniform Manifold Approximation and Projection (UMAP) analyses for the sequencing data showed at least 5 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, SPP1 and WIF1). Finally, ICELL8 results revealed a small subpopulation of RPE cells positive for RPE65 that also expressed a hepatic progenitor cell marker, EPCAM, 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. Using the single cell data, we have uncovered potential markers of this population and functional studies will further verify their identity.
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 express progenitor markers, suggesting that they may be the RPE stem cells.
This is a 2020 ARVO Annual Meeting abstract.