Persistence of reduced expression of putative stem cell markers and slow wound healing in cultured diabetic limbal epithelial cells

PURPOSE: To examine the expression of putative limbal epithelial stem cell (LESC) markers and wound healing rates in primary healthy and diabetic human limbal epithelial cells (LECs) cultured on different substrata. METHODS: Primary limbal epithelial cells were isolated from human autopsy corneas and discarded corneoscleral rims with dispase II treatment. LECs were cultured in EpiLife medium on human amniotic membrane (AM) denuded with mild alkali treatment, on plastic dishes and on glass slides coated with a mixture of human fibronectin, collagen type IV, and laminin (FCL). Cultured LECs were fixed in p-formaldehyde or methanol, and the expression of the putative LESC markers ΔNp63α, PAX6, and ABCG2 and keratins K12, K15, and K17 was examined with immunostaining. Wound healing was evaluated in scratch wound assay in LECs cultured on FCL-coated plates 20 h after wounding. RESULTS: LECs cultured on denuded AM expressed ΔNp63α, PAX6 (both showed nuclear staining), K15, K17 (cytoskeleton staining), and ABCG2 (cytoplasmic and/or plasma membrane staining). LECs cultured on FCL-coated slides also expressed these markers, whereas no expression was detected for differentiated corneal epithelial cell marker K12. Decreased expression of LESC markers was observed in diabetic LECs compared to healthy LECs cultured on the FCL-coated slides. This reduction was most prominent for K15 and K17. Diabetic LECs were found to heal scratch wounds slower than healthy cells in accordance with previous results in corneal organ cultures. CONCLUSIONS: Healthy human LECs cultured either on AM or FCL-coated slides preserved LESC marker expression. The observed reduction in LESC marker expression and slower wound healing in cultured diabetic LECs are in line with our earlier reports and may account for diabetic LESC dysfunction and clinically observed impaired corneal epithelial wound healing.