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M. Kawashima, T. Kawakita, S. Shimmura, K. Higa, J. Shimazaki, K. Tsubota; Caracterization of Human Limbal Melanocytes Expanded in vitro. Invest. Ophthalmol. Vis. Sci. 2007;48(13):479.
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© ARVO (1962-2015); The Authors (2016-present)
On the ocular surfaces, limbal epithelial stem cells are maintained in undifferentiated and quiescent state in specialized microenvironment termed stem cell niche. Such niche was different from central cornea, and one of which is the existence of pigmentation in limbal epithelium. Those pigmentations could be transferred from melanocytes, which settled in limbal basal epithelial layers, however little was known about limbal melanocytes. Therefore to investigate whether limbal melanocytes can expand in defined medium or not, and whether melanocytes exsistent in cultivated epithelial sheet or not, experiments were performed.
Human corneoscleral rims from donors were obtained from the Northwest Lions Eye bank within 4 hours after penetrating keratoplasty. A fresh corneoscleral tissue was cut into 4 symmetric segments in a 60 mm culture dish containing HBSS, and the remaining iris and endothelial cells were rubbed off. Each segment was digested with 10 mg/ml Dispase II in KSFM medium at 4 0C for 16 h, and separated from the stroma of each segment. KSFM medium was made of defined KSFM (Gibco), additional 10 ng/ml EGF and 30 ng/ml cholera toxin A subunit. Such limbal epithelial sheets were rendered into single cells by 0.25% trypsin and 1 mM EDTA for 10 min. Single cells as a density of 2.5 x 104/cm2 were seeded on plastic dishes in SHEM, KSFM, and KSFM+5%FBS for 1 week, and immunostained against MART1, vimentin, Pan-cytokeratin, and MITF. Residual cultivated epithelial sheets after clinically applied and intact human limbal tissue were also immunostained by same markers to determine whether melanocytes exist or not.
In human limbal tissue, melanocytes expressing melan A, vimentin, and MITF were observed in basal layers with closely attached with K19(+)-limbal epithelial basal cells. However, no melan A and MITF was observed in cultivated limbal epithelial sheet. KSFM+5%FBS showed many dendritic melanocytes in monolayer in the culture of limbal epithelia, but there are no melanocytes in KSFM and SHEM.
Melan A (+)-melanocytes were not observed in cultivated limbal epithelial sheet. We need to further investigate whether limbal melanocytes were necessary to create better limbal epithelial microenvironment in vitro or not.
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