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Claudia Müller, Silvia C Finnemann; F-actin organization as potency indicator for high-quality, functional human RPE cell culture. Invest. Ophthalmol. Vis. Sci. 2016;57(12):239. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelial (RPE) cell replacement therapies for treatment of age-related macular degeneration are under development. Cells used for transplantation must establish critical functional interactions with photoreceptors in the diseased retina. Phagocytosis of shed photoreceptor outer segment fragments (POS) is one important property of differentiated RPE. We investigated cellular markers that may predict robust phagocytic capacity.
RPE cell lines derived from adult human RPE stem cells (RPESC-RPE) passage 2 were used for experiments. Western blot analysis, immunostaining and confocal microscopy were used to study cell morphology and marker protein expression. Protein secretion was assessed by ELISA. Binding and internalization of purified POS were quantified to assess phagocytic capacity of RPE cells in culture.
Phagocytosis assays showed that RPE cell lines could be categorized as either highly phagocytic (with 55-80% of cells engulfing POS in our assay) or poorly phagocytic (with 5-40% of cells engulfing POS). Regardless of phagocytic capacity, all RPE lines tested expressed known proteins of the phagocytic pathway and secreted phagocytic ligands (MFG-E8 and ProteinS). However, in poorly phagocytic cells, POS surface binding and apical ezrin localization were dramatically reduced indicating abnormal microvilli. In highly phagocytic cells, the majority of cells showed cobblestone epithelial morphology with an F-actin-rich apical brush border, a circumferential apical adhesion belt and basal F-actin at substrate attachment sites. In poorly phagocytic cells, less than 10% of cells showed a circumferential F-actin belt, and most of these cells contained less apical F-actin and abundant central and basal F-actin stress fibers. Fewer than 1% of cells expressed the mesenchymal marker smooth muscle actin (SMA). Inhibition of Rho-associated, coiled-coil protein kinase (ROCK) was sufficient to improve epithelial F-actin characteristics and to increase the fraction of cells engulfing POS.
Our results suggest that F-actin cytoskeleton analysis is sufficient to assess epithelial morphology of RPE cells in culture. Moreover, alterations in F-actin directly correlate with and may contribute to diminished phagocytic function of RPE in culture.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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