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Hideyuki Miyashita, Satoru Yoshida, Tetsuya Kawakita, Kazuo Tsubota, Shigeto Shimmura; Self Assembly of the Limbal Epithelial Stem Cell Niche in Vitro. Invest. Ophthalmol. Vis. Sci. 2013;54(15):986.
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© ARVO (1962-2015); The Authors (2016-present)
The corneal limbus contains cytokeratin 15 (K15) positive basal epithelial cells and melanocytes that form units with basal epithelial cells. We confirmed whether long-term cultivated epithelial cell sheets can reconstruct melanocytes-epithelial cell units.
Human limbal epithelial cells were isolated from eyebank eyes, seeded on fibrin-coated cell culture inserts, and 3 dimensional co-cultured with feeder cells. Cells were fed with supplemented hormonal epithelial medium with EGF or KGF and a ROCK inhibitor Y-27632 at day 3, day 5, and every day after day 7 for up to 3 months. Cryosections and whole cell sheets were fixed with ice-cold 4% paraformaldehyde in PBS, and stained with melanocyte marker MART-1 and epithelial precursor marker K15.
MART-1 positive melanocytes were observed in whole mounted epithelial cell sheets in all culture conditions. Immunohistochemisrty showed that melanocytes were scattered as MART-1 positive cells in the basal layer of cultured epithelium. Melanocytes showed dendritic morphology, some of which were proximal to K15 highly positive epithelial cell clusters. In sheets derived from dense-pigmented limbus, melanin pigments were observed in basal cells around the melanocytes.
Melanocytes and limbal epithelial cells can self-assemble melanin units in vitro, mimicking the limbal niche in vivo
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