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B.S. McKay; Tissue Specific Protein Expression in Cultured Adult Human RPE . Invest. Ophthalmol. Vis. Sci. 2003;44(13):387.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Cultured RPE cells do not form regular hexagonal arrays of pigmented cells, but can be stimulated to do so by coordinated induction of adhesion. To investigate the 'tissue-type' properties of RPE cultures produced from adult donors and stimulated to re-establish a monolayer after multiple passages, here we test whether these pigmented RPE monolayers express proteins typically lost in cultured RPE. Methods: Human RPE cultures were isolated by standards methods from donor eyes, then propagated for at least 6 passages prior to exposiure to the experimental paradigm (Calcium (Ca++) switch). Cells were plated in 50 micromolar Ca++ to inhibit cadherin adhesion and create contact naive cells. At high density the cells are changed to normal Ca++ medium to coordinately induce cell adhesion. Cells were maintained at confluency without passage then for up to 4 months. Cell proteins were harvested for western blot analysis to test for expression of CRABP, bestrophin and tyrosinase. Results: CRABP expression could be detected in repigmented cultures of adult human RPE cells beginning 2 months after the Ca switch, but the protein remained undetectable in parallel control cultures (paired cultures maintained at confluency but not subjected to the Ca-switch paradigm). Tyrosinase expression occurs early after the Ca switch in the experimental cells (days) as opposed to parallel cultures, in which expression could only be detected at very late time points (>2 months). Bestrophin was not detected in any culture tested up to 4 months after the Ca-switch. Conclusions: Our results suggest that the coordinated adhesion paradigm used to create pigmented RPE monolayers also leads to expression of some but not all proteins lost when RPE are cultured. The timing of the expression of individual proteins may be related to the development of a 'tissue-type' monolayer, and expression of 'differentiation' markers is not an all or none phenomena.
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