Ranaei Pirmardan Ehsan, Soheili Zahra-Soheila, Samiei Shahram, Ahmadieh Hamid, Mowla Seyed Javad, Ezzati Razie, Naseri Marzieh
Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
Exp Cell Res. 2016 Oct 1;347(2):332-8. doi: 10.1016/j.yexcr.2016.08.015. Epub 2016 Aug 21.
Retinal pigmented epithelium (RPE), the outermost layer of the retina, has a key role in maintaining retinal cells' functions. 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. Here we report generation of a spontaneously immortalized murine RPE cell line in primary mouse RPE cell culture. Founded colonized cells were picked up and expression of RPE and retinal progenitor cells' (RPC) markers were studied using immunocytochemistry (ICC). Emerged cells cultured over 35 passages and population doubling times in different serum concentrations were calculated. We also investigated the ability of cells for becoming transfected by calcium-phosphate method and for becoming infected by adeno-associated virus serotype 2 (AAV2) using flow cytometry. Data showed that the cobblestone constituent cells expressed RPE65, cytokeratin and ZO1 and moreover several progenitor markers such as Pax6, Sox2, Nestin and Chx10. It revealed that, despite primary RPE cells, the newly emerged cells were easily transfectable and were highly 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.
视网膜色素上皮（RPE）是视网膜的最外层，在维持视网膜细胞功能方面发挥着关键作用。RPE细胞培养的难度对体外和体内研究及其治疗应用都造成了诸多限制。因此，建立具有高增殖潜力的RPE细胞系可显著改善RPE细胞生物学研究。在此，我们报告在原代小鼠RPE细胞培养中生成了一种自发永生化的小鼠RPE细胞系。挑选出形成集落的细胞，并用免疫细胞化学（ICC）研究RPE和视网膜祖细胞（RPC）标志物的表达。计算在不同血清浓度下培养超过35代的细胞的群体倍增时间。我们还使用流式细胞术研究了细胞通过磷酸钙法转染以及被2型腺相关病毒（AAV2）感染的能力。数据显示，鹅卵石样组成细胞表达RPE65、细胞角蛋白和ZO1，此外还表达几种祖细胞标志物，如Pax6、Sox2、巢蛋白和Chx10。结果表明，与原代RPE细胞不同，新出现的细胞易于转染，并且与HEK293T细胞相比具有高度感染性。我们的数据表明，新出现的小鼠RPE细胞系呈现出类似RPC的表型，同时也表达RPE标志物。它将是一个用于RPE和RPC细胞前沿研究的有前景的模型，并充分证实了RPE的巨大可塑性及其在研究中的宝贵潜力。
