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Expression of Connexins 26 and 43 in Human Limbal and Corneal Epithelial Cells Cultured on Amniotic-membranes

1Meller D., 1Hernandez Galindo E. E., 2Theiss C., 3Steuhl K. P.,
1Universität-Gesamthochschule Essen, Zentrum für Augenheilkunde (Essen)
2Ruhr-Universität Bochum, Abteilung für Zytologie (Bochum)
3Universität-Gesamthochschule Essen, Zentrum für Augenheilkunde, Abt. für Erkrankungen des vorderen Augenabschnitts (Essen)

Purpose: To determine the expression of connexins 26 and 43 in human limbal (HLEC) and corneal (HPCEC) epithelial cells during ex vivo expansion on preserved human amniotic membrane (AM).
Methods: HLEC and HPCEC from limbal and peripheral corneal explants were cultured with SHEM either on intact AM or plastic. After 4 to 5 weeks, cell cultures were terminated and processed for immunofluorescence. Formations of gap junctions were analyzed with a rabbit polyclonal antibody to connexin 26 (Cx26) and a mouse monoclonal antibody against connexin 43 (Cx43). Sections of human limbus and cornea served as positive control. Lucifer yellow (LY) known to be a gap junction permeant dye was used to analyze functionality of gap junctions (GJ). Microinjection of LY into single cells was performed with a pressure microinjection device.
Results: In vivo, labeling for Cx26 was observed in all cell layers of the human corneal epithelium and a punctate pattern of Cx43 was typically found in basal and suprabasal corneal epithelial cells, however, subpopulations of limbal basal epithelial cells were negative for both connexins. In HLEC cultured on AM, a scanty immunolabeling for Cx26 and Cx43 was noted, but HPCEC cultured on AM as well as HLEC cultured on plastic showed a higher labeling index for Cx26 and Cx43. Gap junctional communication was evidenced more frequently in HLEC cultured on plastic (50.96%, p<0.05) in contrast to HLEC cultures on AM, which exhibited a limited dye spread in 29.70% of injected cells. A significant difference in dye coupling was also evidenced between HPCEC on AM (52,88%; p<0.05) and HLEC on AM. However, no dramatic difference in dye coupling was found between HPCEC cultured either on AM or plastic (57,84%).
Conclusions: Expression of Cx26 was reported for the first time in vivo and vitro in the human cornea. Subpopulations of HLEC cultured on AM remain Cx26 and Cx43 negative and without functional gap junctions indicating that characteristic features of limbal epithelial progenitor cells might be preserved during ex vivo expansion on AM. This data provides support to the use of the ex vivo expansion of HLEC as an alternative therapeutic strategy for corneal surface reconstruction in distinct ocular surface diseases.

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