Subconfluent ARPE-19 Cells Display Mesenchymal Cell-State Characteristics and Behave like Fibroblasts, Rather Than Epithelial Cells, in Experimental HCMV Infection Studies

Abstract

Human cytomegalovirus (HCMV) has a broad cellular tropism and epithelial cells are important physiological targets during infection. The retinal pigment epithelial cell line ARPE-19 has been used to model HCMV infection in epithelial cells for decades and remains a commonly used cell type for studying viral entry, replication, and the cellular response to infection. We previously found that ARPE-19 cells, despite being derived from an epithelial cell explant, express extremely low levels of canonical epithelial proteins, such as E-cadherin and EpCAM. Here, we perform comparative studies of ARPE-19 and additional epithelial cell lines with strong epithelial characteristics. We find that ARPE-19 cells cultured under subconfluent conditions resemble mesenchymal fibroblasts, rather than epithelial cells; this is consistent with previous studies showing that ARPE-19 cultures require extended periods of high confluency culture to maintain epithelial characteristics. By reanalyzing public gene expression data and using machine learning, we find evidence that ARPE-19 cultures maintained across many labs exhibit mesenchymal characteristics and that the majority of studies employing ARPE-19 use them in a mesenchymal state. Lastly, by performing experimental HCMV infections across mesenchymal and epithelial cell lines, we find that ARPE-19 cells behave like mesenchymal fibroblasts, producing logarithmic yields of cell-free infectious progeny, while cell lines with strong epithelial character exhibit an atypical infectious cycle and naturally restrict the production of cell-free progeny. Our work highlights important characteristics of the ARPE-19 cell line and suggests that subconfluent ARPE-19 cells may not be optimal for modeling epithelial infection with HCMV or other human viruses. It also suggests that HCMV biosynthesis and/or spread may occur quite differently in epithelial cells compared to mesenchymal cells. These differences could contribute to viral persistence or pathogenesis in epithelial tissues.

ARPE-19 Epithelial Cells Fail To Initiate Type-I Interferon Signaling in Response to Human Cytomegalovirus Infection

“…The ARPE-19 cell line is a commonly used retinal epithelial cell line for biochemical and virological studies [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] . Despite being derived from a retinal epithelium explant 20 , ARPE-19 cells exhibit cell culture characteristics that diverge significantly from primary retinal epithelial cells [21][22][23][24][25] . Most importantly, ARPE-19 cells exhibit strong density-dependent changes in their epithelial phenotype 20,21,26,27 .…”

“…A number of studies have found that long periods of high confluency culture are required for ARPE-19 cells to establish a functional epithelial phenotype 20,21,23,[27][28][29] . After two to four months of high density culture, ARPE-19 cells turn on expression of epithelial (e.g., CDH1 and EPCAM) and visual cycle (e.g., RPE1, LRAT, RDH5, and RDH10) genes 21,23,27 ; assume a cobblestone epithelial morphology 20,23,27 ; establish apico-basal polarization 20,28,29 ; and display high trans-epithelial resistance (a measure of barrier function) 20,[27][28][29] .…”

“…Despite being derived from a retinal epithelium explant 20 , ARPE-19 cells exhibit cell culture characteristics that diverge significantly from primary retinal epithelial cells [21][22][23][24][25] . Most importantly, ARPE-19 cells exhibit strong density-dependent changes in their epithelial phenotype 20,21,26,27 . At low cell density, typical of subcultured, or "passaged" cells in monolayer cell culture, ARPE-19 cells exhibit a mesenchymal morphology and express low levels of genes involved in epithelial functional and retinal biogenesis 21,27 .…”

“…Most importantly, ARPE-19 cells exhibit strong density-dependent changes in their epithelial phenotype 20,21,26,27 . At low cell density, typical of subcultured, or "passaged" cells in monolayer cell culture, ARPE-19 cells exhibit a mesenchymal morphology and express low levels of genes involved in epithelial functional and retinal biogenesis 21,27 . A number of studies have found that long periods of high confluency culture are required for ARPE-19 cells to establish a functional epithelial phenotype 20,21,23,[27][28][29] .…”

“…At low cell density, typical of subcultured, or "passaged" cells in monolayer cell culture, ARPE-19 cells exhibit a mesenchymal morphology and express low levels of genes involved in epithelial functional and retinal biogenesis 21,27 . A number of studies have found that long periods of high confluency culture are required for ARPE-19 cells to establish a functional epithelial phenotype 20,21,23,[27][28][29] . After two to four months of high density culture, ARPE-19 cells turn on expression of epithelial (e.g., CDH1 and EPCAM) and visual cycle (e.g., RPE1, LRAT, RDH5, and RDH10) genes 21,23,27 ; assume a cobblestone epithelial morphology 20,23,27 ; establish apico-basal polarization 20,28,29 ; and display high trans-epithelial resistance (a measure of barrier function) 20,[27][28][29] .Despite their known density-dependent phenotype, ARPE-19 cells are frequently used as a simple epithelial cell culture model for experimental infection studies with a variety of human viruses [30][31][32][33][34][35][36][37] , including human cytomegalovirus (HCMV) 1,[3][4][5][6][7][8][10][11][12][13][15]…”