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Andrei A Kramerov, Mehrnoosh Saghizadeh, Ezra Maguen, Yaron S Rabinowitz, Alexander V Ljubimov; Expression of Putative Stem Cell Markers in Human Limbal Epithelial Cells Cultured on Different Substrata. Invest. Ophthalmol. Vis. Sci. 2014;55(13):509.
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© ARVO (1962-2015); The Authors (2016-present)
To examine the expression of putative limbal epithelial stem cell (LESC) markers in primary normal and diabetic human limbal epithelial cells (LEC) and to validate the preservation of LESC marker expression in LEC cultivated on different substrata for further LESC studies and expansion.
Primary limbal epithelial cells were isolated from discarded human corneoscleral rings by dispase II treatment. LEC were cultured in EpiLife medium on human amniotic membrane (AM) denuded by mild alkali treatment, on plastic dishes and on glass slides coated with a mixture of human fibronectin, collagen type IV and laminin (FNCL). Cultured LEC were fixed in p-formaldehyde or methanol and expression of putative LESC markers ΔNp63α, PAX6, keratins K15 and K17, and ABCG2 was examined by immunostaining.
LEC cultured on denuded AM expressed ΔNp63α, PAX6 (both showing nuclear staining), K17 (cytoskeleton staining), and ABCG2 (cytoplasmic and/or plasma membrane staining). Similarly, LEC cultured on FNCL-coated slides expressed these LESC markers, whereas no expression was detected for differentiated corneal epithelial cell marker K12. Interestingly, decreased expression of LESC markers was observed in diabetic LEC as compared to normal LEC cultured on FNCL-coated slides. This reduction was most prominent for K15 and K17.
Human LEC cultured either on AM or FNCL-coated dishes preserved LESC marker expression. The observed reduction in LESC marker expression in cultured diabetic LEC is in line with our earlier reports on alterations of LESC markers in human diabetic corneas, which may account for diabetic LESC dysfunction and impaired corneal epithelial wound healing.
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