Retinal pigmented epithelium (RPE), the outermost layer of the retina, has a key role in maintaining retinal cells’ functions. RPE dysfunction can lead to many ocular disorders. Besides, plasticity of these cells has been previously demonstrated. Severity of the culture of RPE cells has exerted many limitations to both in vitro and in vivo studies and its therapeutic applications. Therefore, establishment of RPE cell lines with high proliferative potential can considerably improve study of RPE cell biology and its potential in cell replacement therapy and regenerative medicine. Here we report generation of a spontaneously immortalized murine RPE cell line in primary mouse RPE cell culture.

Mouse RPE cells were isolated, characterized and cultured according to standard protocols. In the third passage a sub-population of colonized cells, with very different morphology, was detected in cultures. Founded colonized cells were picked up and expression of RPE and retinal progenitor cells’ (RPC) markers was studied using immunocytochemistry (ICC) and Real-Time PCR. Emerged cells cultured over 35 passages and population doubling times in 2.5%, 5% and 10% fetal bovine serum (FBS) were calculated. We also investigated the ability of cells to be transfected by calcium-phosphate method and to be infected by adeno-associated virus serotype 2 (AAV2) using flow cytometry.

Our results showed that the cobblestone constituent cells expressed RPE65, cytokeratin and ZO1 and moreover several progenitor markers as like as PAX6, SOX2, nestin and CHX10. We revealed that despite primary RPE cells, the newly emerged cells, were easily transfectable and were more infectable when compared with HEK293T cells.

Our data indicated that the emerged mouse RPE cell line pretended RPC-like phenotype and also simultaneously expressed RPE markers. It would be a promising model for leading studies on RPE and RPC cells and substantially confirmed the great RPE plasticity and its invaluable potential in research studies.