Condition Dependancy of The Morphologic Changes and The Expressivity of Cytokeratin Subcloneg, Vimentin, and GFAP in Cultured Retinal Pigment Epithelial Cells

ABSTRACT

To describe and evaluate the morphologic changes and the different expression of cell-specific or correlating protein molecules during cell growth, immunocytochemistry and morphologic observations were done on retinal pigment epithelial(RPE) cells obtained from several culture conditions. These include culture time, spatial or cell density, transdifferentiation, and presence of growth factors. The human fetal and porcine RPE cells were cultured with and without individual growth factor or in combinations inchlding extracellular matrix (ECM), Insulin, basic fibroblatio growth factor (bFGF) and epidermal growth factor (EGF). Mouse monoclonal anti-human, or anti-mouse antibodieg with or without species cross reactlvity against the intermediate filament proteins (cytokeratin, vimentin, GFAP) were used. To determine RPE-specific molecules of cytokeratin, nine commercially available antibodies, representing subclones of Moll's catalog number 1, 5, 7, 8, 10, 14, 17, 18, 19 were applied. The morphological changes and the proliferation of cells started after their attachment on the culture plate as soon as they lost pigment granules. The epithelial cells like fibroblasts occurred in the area where the cellular density was low, and finally, their shape was restored to their original phenotype when the cellular connuency was achieved. The degree of proliferation and the duration of achieving confluency of cells were dependent on whether ECM and growth factors were added in media or not. Cells with the epithelial morphology were positively stained with anticytokeratine antibodies, especially with clone 19, 18, 17, 8 and 7 in human RPE cells; with 19, CAM 5.26 (8/18) in porcine cells. The fusiform or digitating cells of sparse density also expressed vimentin strongly through out all stages, whereas GFAP was not expressed at any stage in either species.