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Abstract
Retinal pigment epithelial (RPE) cells in vivo have a polarized structure with specialized apical and basal faces. Isolated RPE cells lose but eventually regain their epithelial morphology under appropriate culture conditions. We evaluated the ability of isolated feline, primate, and human RPE cells to regain this morphology in culture with scanning electron, transmission electron, phase contrast, and immunofluorescence microscopy. In culture, isolated RPE cells lose their cuboidal shape, their apical microvilli, and their in vivo cytoskeletal organization. Stress fibers from in these cells; microtubules radiate from the cells' center to their periphery; and vimentin filaments radiate from the cells' nucleus to their periphery. As cultures become confluent, RPE cells aggregate into small groups, gradually regaining a cuboidal shape and acquiring microvilli on their apical surface. Filamentous actin redistributes to the apical face where it presumably forms the cytoskeletal core normally present in RPE microvilli. Stress fibers disappear and are replaced by a circumferential microfilament bundle (CMB). Confluent cells surrounding the colonies of differentiated RPE attain a cuboidal shape but do not show complete cytoskeletal redifferentiation. Such cells, while appearing to be differentiated by phase contrast microscopy, fail to develop a compacted CMB. In these cells, f-actin is organized as a loose peripheral band within the cell cytoplasm. Our observations indicate that confluency cannot be equated with the end stage of morphologic differentiation, and that cytoskeletal organization provides a more accurate gauge of RPE maturation in culture.