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Roni A Hazim, Stefanie Volland, David S Williams; A Rapid protocol for the differentiation of human ARPE-19 cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3980. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
ARPE-19 is an immortalized cell line of retinal pigment epithelium (RPE) currently used to investigate RPE cell biology. Continuous passaging over the past two decades has resulted in the cell line losing several key properties of native human RPE. Nonetheless, many laboratories continue to use undifferentiated ARPE-19 cells to make inferences about the RPE in the context of normal and patho- physiology. Published protocols have shown that ARPE-19 cells can reach an in vivo-like state, but only after several months in culture. Here, we describe a simple protocol to rapidly differentiate ARPE-19 cells, producing homogenous cultures that reacquire many of the characteristics of their in vivo counterparts. We propose that these cells, when cultured using this protocol, can serve as a valuable in vitro model of human RPE.
ARPE-19 cells were grown on plastic surfaces, then differentiated on Transwell inserts for 2-6 weeks, in a previously published medium with modifications. Morphology of the cells was examined using brightfield and electron microscopy. RNA was harvested from the cells and RT-PCR was used to test for expression of RPE-specific genes. Protein expression and subcellular localization was analyzed using western blot and immunofluorescence. Functional tests included phagocytosis assays and polarized protein secretion.
Differentiated ARPE-19 cells formed a monolayer with cobblestone morphology, and possessed elaborate microvilli protruding from their apical surface. The cytoskeleton consisted of cortical actin, and microtubules that were horizontally-oriented in the apical region and vertically-oriented throughout the cell body. Immunolabeling of acetylated tubulin revealed primary cilia emanating from the apical surface of the cells. Gene and protein analyses demonstrated that the ARPE-19 cells have an expression profile similar to that observed in native human RPE. In phagocytosis assays, the cells were capable of phagocytizing photoreceptor outer segments, with kinetics similar to that in vivo, demonstrating an essential function of RPE. Finally, the cells were shown to secrete factor H preferentially from their apical surface, suggesting proper apical-basal polarity.
Our results indicate that ARPE-19 cells can rapidly acquire a differentiated state in which they display several key epithelial characteristics of native RPE.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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