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Fawzia Bardag-Gorce, Joan Oliva, Andrew Makalinao, Imara Meepe, Julio Garcia, Amanda M. Laporte, Robert Niihara, Hyo Jung Ha, Richard H. Hoft, Alan L. Felsenfeld, Yutaka Niihara; Barrier function of cultured autologous oral mucosal epithelial cell sheets. Invest. Ophthalmol. Vis. Sci. 2016;57(12):895.
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© ARVO (1962-2015); The Authors (2016-present)
Cultured autologous oral mucosal epithelial cell sheet (CAOMECS) has been successfully used to regenerate corneal epithelium with limbal stem cell deficiency (LSCD). However, it is uncertain if corneal epithelium derived from CAOMECS has most of the barrier functions observed in normal corneal epithelium. Barrier functions include a barrier to conjunctivalization of the cornea, a barrier to external toxic and infectious agents, and barriers to stromal edema or dehydration. The present study reports the barrieral characterization of carrier free human CAOMECS (hCAOMECS).
Following informed consent procedures, a donated small biopsies was used to engineer hCAOMECS. Temperature responsive culture ware was used to harvest hCAOMECS with preserved extra cellular matrix, which permits a quick adherence to corneal stroma, and plays the role of conjunctival barrier (previously reported in our rabbit study (Bardag-Gorce et al, Ocul Surf. 2015)).
Immuno-histological analysis showed that hCAOMECS is a multilayered epithelial tissue-like cell sheet with apico-basal polarity evidenced by basal expression p63 and Ki67, and apical expression of K4/K13. hCAOMECS also expressed pro-angiogenic factors, such as matrix metalloproteinase (MMP-3) in balance with the anti-angiogenic tissue inhibitors of metalloproteinases (TIMP-3), which suggest that hCAOMECS has characteristics of a normal corneal epithelium. hCAOMECS epithelial markers were also investigated; the results showed positive expression of E-cadherin, alpha-catenin, and beta-catenin at the cell borders. While beta-catenin levels were similar, E-cadherin and Occludin levels were found significantly higher in hCAOMECS compared to cultured monolayer human corneal epithelia cells, indicating functional adherens and tight junctions. These results were repeatedly obtained in our protocols of engineering rabbits or human CAOMECS. E-cadherin has been used to examine potential cell tumorigenicity and is essential for organization of the cytoskeleton and junctional complexes. In our studies, we use E-cadherin expression as an indicator of a functional epithelial barrier because it solidifies intercellular adhesion, recruits catenins to cell borders, and recruits actin filaments to increase barrier function.
hCAOMECS has high expression of epithelial markers necessary for normal corneal epithelial barrier function.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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