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

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.

The Axial Organ and the Pharynx Are Sites of Hematopoiesis in the Sea Urchin.

Background: The location of coelomocyte proliferation in adult sea urchins is unknown and speculations since the early 1800s have been based on microanatomy and tracer uptake studies. In adult sea urchins (Strongylocentrotus purpuratus) with down-regulated immune systems, coelomocyte numbers increase in response to immune challenge, and whether some or all of these cells are newly proliferated is not known. The gene regulatory network that encodes transcription factors that control hematopoiesis in embryonic and larval sea urchins has not been investigated in adults. Hence, to identify the hematopoietic tissue in adult sea urchins, cell proliferation, expression of phagocyte specific genes, and expression of genes encoding transcription factors that function in the conserved regulatory network that controls hematopoiesis in embryonic and larval sea urchins were investigated for several tissues. Results: Cell proliferation was induced in adult sea urchins either by immune challenge through injection of heat-killed Vibrio diazotrophicus or by cell depletion through aspiration of coelomic fluid. In response to either of these stimuli, newly proliferated coelomocytes constitute only about 10% of the cells in the coelomic fluid. In tissues, newly proliferated cells and cells that express SpTransformer proteins (formerly Sp185/333) that are markers for phagocytes are present in the axial organ, gonad, pharynx, esophagus, and gut with no differences among tissues. The expression level of genes encoding transcription factors that regulate hematopoiesis show that both the axial organ and the pharynx have elevated expression compared to coelomocytes, esophagus, gut, and gonad. Similarly, an RNAseq dataset shows similar results for the axial organ and pharynx, but also suggests that the axial organ may be a site for removal and recycling of cells in the coelomic cavity. Conclusions: Results presented here are consistent with previous speculations that the axial organ may be a site of coelomocyte proliferation and that it may also be a center for cellular removal and recycling. A second site, the pharynx, may also have hematopoietic activity, a tissue that has been assumed to function only as part of the intestinal tract.