Characterization of RPE subpopulation gene expression and cell surface protein composition using single cell sequencing

The retinal pigment epithelium (RPE) has traditionally been considered a homogenous monolayer, however differences in morphology, gene expression and disease susceptibility of RPE cells in different retinal regions has recently been described. To further catalog RPE subpopulation diversity, we have isolated RPE cells from both donated adult human eyes and RPE derived from RPE stem cells (RPESC) to characterize transcriptomic and cell surface differences within the native and clinically relevant cultured RPE subpopulations using CITE-Seq. Using the ICELL8 well-based single cell system, we undertook deep single cell RNA-sequencing (median library size ~130,000 reads) and CITE-Seq with 154 antibodies (median library size ~225,000 reads) to investigate RPE subpopulation identity using ~12,000 cells from six donors. Approximately half of the cells were isolated directly from primary tissue, while the remainder came from RPESC-RPE cultures. scRNA-seq data was analyzed with the Seurat package and the Cite-seq was analyzed by in-house scripts.