Abstract
Purpose :
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.
Methods :
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. Immunohistochemistry and time-lapse imaging were utilized to characterize gene expression within the subpopulations in eye tissue and to generate lineage trees from isolated primary cells.
Results :
We defined 13 distinct RPE subpopulations, demonstrating significant heterogeneity within the RPE layer. One cluster was identified as the RPESC based on a unique gene expression profile and validated by expression in expanding RPESCs in culture. Furthermore, this RPE subpopulation was shown to integrate into an established RPE monolayer using an in vitro model system. Finally, using the CITE-seq data, we were able to generate flow cytometry-based cell sorting schemes to isolate RPE subpopulations including the RPESC for future studies.
Conclusions :
Our results are consistent with a heterogeneous RPE monolayer containing cells with different gene signatures and functionality. Identifying the diversity of human RPE cells and RPESC gene expression signature broadens our understanding of retinal function and improves our potential to combat RPE dysfunction and vision loss.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.