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LE Lamb, JD Simon, BS McKay; Coordinated Stimulation of Cell Junctions and Pigmentation in Human RPE Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4576.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: When culturing human RPE cells changes in the distinctive tissue characteristics are observed, including loss of functional aspects such as apical-basal polarity and pigmentation. Loss of these characteristics limits comparisons between the in vivo and cultured systems. The development of epithelial morphology in tissues is thought to be related to cadherin signal transduction and formation of cell junctions. A Ca2+ switching protocol was used to induce rapid formation of such junctions. This protocol also tested for additional changes in the functional tissue characteristics. Recovery of such functional aspects is necessary to obtain a cultured model system more similar to that seen in vivo. Methods: RPE cell cultures devoid of pigment displaying a variety of morphologies were used between passages five and sixteen. The cultures were trypsinized and split into paired groups. One group was maintained in low (50 µM) Ca2+ DMEM plus 10% dialyzed FBS while the other was maintained in normal (1.8 mM) Ca2+ DMEM plus 10% FBS. Once the low Ca2+ group reached 100% confluency, the media was switched to the normal Ca2+ DMEM. Cells were maintained at confluency for up to 16 weeks. Monolayers were evaluated for cadherin distribution, tyrosinase expression, pigment formation and overall level of organization. Results: Initially, cadherins appeared cytoplasmic in the low group, but one week after the media switch they became zonular. In contrast, the normal group had discontinuous border staining as well as diffuse staining. After the media switch, the low group showed tyrosinase expression, as observed by fluorescence microscopy, and by one week was prominent in western blots. Tyrosinase expression increased during weeks four through eight while little if any expression was detectable in cells from the normal group. In the low group, pigmentation was successfully induced. By eight weeks clusters of pigmented cells were evident, and after twelve weeks gross pigmentation was visible. In contrast, in the normal group pigmented cells were only occasionally observed. In addition, the morphology of the two monolayers was quite different. The low group exhibited a high degree of epithelial organization with arrays of polygonal cells. The normal group morphology varied but was not similar to the low group or the in vivo tissue. Conclusion: Human RPE cells respond to coordinated cadherin stimulation by initiating development of a tissue-type monolayer. This response includes induction of tyrosinase expression and eventual pigmentation.
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