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. 2020 Jul 6;10:11074. doi: 10.1038/s41598-020-68120-0

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Corneal epithelial tissue engineering using limbal melanocytes and LN-511-E8. (A) Live/dead viability assay and immunofluorescence double labeling studies after 24 h of cultivation of limbal melanocytes on fibrin hydrogels pretreated with LN511-E8 or untreated fibrin gels (control). In both conditions, cells are viable and co-express melanocyte markers Melan-A (green) in association with HMB-45 and tyrosinase related protein 1 (TRP1) (red), respectively; nuclear counterstaining with 4′,6‐diamidino‐2‐phenylindole (DAPI, blue). (B) Light microscopic analysis of fibrin-based epithelial constructs formed by limbal epithelial progenitor cells (LEPC) without or with co-cultured melanocytes after 10 days of cultivation (top). Transmission electron microscopy images of epithelial constructs without or with basally located melanocytes (center). Immunofluorescence analysis of epithelial constructs showing epithelial cells positive for the epithelial marker pan-keratin (green) in association with Melan-A-positive melanocytes (red) localized within the basal cell layer (bottom); nuclear counterstain with DAPI (blue).