The Expression of Native and Cultured Human Retinal Pigment Epithelial Cells Grown in Different Culture Conditions

Abstract

Abstract: : Purpose: The established retinal pigment epithelial (RPE) cell line ARPE–19 is commonly used to study RPE cell behavior. While this cell line has the potential to develop differentiated properties, the optimal global gene expression changes under different culture conditions have not been established. The purpose of this work was to determine the proximity of expression profiles by RPE cells grown under different culture conditions to native RPE. Methods: Morphologically normal RPE cells overlying nonthickened Bruch’s membrane (n=5000 cells) from 10 eyes (45–95 yrs) were laser capture microdissected (LCM).ARPE–19 cells were grown in subconfluent, visually confluent, and high confluent for 2.5 months with or without serum. Total RNA was used to synthesize first and second strand 33P–dATP and 33P–dCTP cDNA probes which were hybridized to a ResGen array (5353 genes), and the signal was visualized by phosphorimager analysis. The TIFF images were analyzed by ResGen’s Pathways 3 software. Experiments were repeated twice (n=3) for each condition. The data were normalized to a mean signal reference array, and subsequently analyzed using Cluster/Treeview and SAM. Real time PCR was used to confirm differential expression of selected genes. Results: The expression profiles of ARPE–19 cells were grown under 5 culture conditions were compared to morphologically normal native RPE cells that were laser capture microdissected from 5 eyes. While 78% of genes on the array were expressed by native RPE, 45.3–47.7% of genes were expressed by ARPE–19 cells, depending on culture condition. The most abundant genes were commonly expressed by native and cultured cells, but significant differences in low abundance genes were seen between native and cultured RPE. Unsupervised and supervised cluster analysis showed that confluent and differentiated, serum withdrawn cultures clustered closest to native RPE, and that serum segregated cultured cells away from native RPE. The number of differentially expressed genes, function of differentially expressed genes, and profile of expressed and unexpressed genes demonstrate significant differences between native and ARPE–19 cells with the culture conditions chosen for this study. Conclusions: While cultured RPE cells and in particular ARPE–19 cells, have significant value for studying RPE behavior, investigators must be aware of how culture conditions can influence the mRNA phenotype of the cell.

Keywords: retinal pigment epithelium • gene/expression