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Purpose: The corneas preserved in bioreactor (BR) had been shown to have not only a better endothelial viability, but also a more differentiated and stratified epithelium than corneas preserved in organoculture. Purpose: By using BR, we would analyse the respective contribution of corneal (C), limbal (L), and conjunctival (Conj) epithelia in corneal epithelial regeneration. Methods: Five pairs of corneas from body donation to Science were used with a death‐to‐collection time < 20 h. A 3‐ to 5‐mm‐wide conjunctival flange was kept intact. Five patterns were set up by fully mechanical removal of 1, 2, or 3 epithelia: C‐L + Conj+, C‐L‐Conj+, C‐L + Conj‐, C + L‐Conj‐, C‐L‐Conj‐ (control) n = 2 for each pattern. The limbus was destroyed by scraping and thermocoagulation. Corneas were then kept in BR (21 mmHg, 2.5 μl/min of Corneamax Eurobio, 31°C) for 3 weeks to allow epithelial regeneration. The epithelium was then analysed using immunofluorescence (IF) on flat mounted cornea by targeting CK12 (corneal epithelium) and CK15 (limbal epithelium). Cell nuclei were counterstained with DAPI. Corneal transparency was quantified using a transparometer. Results: No epithelium was reconstituted in the controls. In the other 4 models including the C‐L‐Conj+ group, the cornea was transparent and covered by a pluristratified corneal epithelium, characterized by CK12 expression. Conclusions: In this BR model, conjunctival epithelial cells allowed the regeneration of a typical corneal epithelium in model C‐L‐Conj+. The corneal epithelium can also migrate to the limbus and conjunctiva. We hypothesize that all 3 ocular surface epithelia (including the conj) contain stem cells or progenitors capable of migrating throughout the cornea and restoring the corneal epithelium independently of each other. The main difference between our ex vivo model and in vivo situation is absence of neovascularization. This suggests that the main cause of limbic insufficiency is due to the loss of the anti‐angiogenic barrier rather than the loss of limbic stem cells. [ABSTRACT FROM AUTHOR]

Purpose: Fuchs Endothelial Corneal Dystrophy (FECD) is characterized by the progressive slow accumulation of extra cellular matrix (ECM) forming Descemetic excrescents named guttae and by an increase in the thickness of the Descemet membrane (DM). Nevertheless, FECD pathophysiology remains only partially understood. Aim: To present the expression of newly identified proteins, using proteomics methods. Methods: DM were obtained from FECD patients undergoing DMEK and from donor corneas (controls) (University Hospital St‐Etienne). Some samples were analysed using MALDI imaging and shotgun proteomics (Kyoto, Doshisha University) to identified proteins selectively under or over expressed in FECD vs controls. Other samples were used for immunostaining (IS) of the top over‐expressed proteins. We used IS on flat mounts of DM in order to study the differences between the center and the periphery and cross sections to study the DM layer affected. Cross sections were performed using a cryostat on DM embedded in a mixture of gelatin, optimal cutting temperature compound and sucrose. Expression of each protein was observed using a fluorescence microscope. At least 3 different samples were used for each protein. Results: MALDI imaging and shotgun analysis identified 11 potential targets overexpressed in FECD, belonging to different families: mainly collagen, proteogylcans, and TGF beta signalling. Using IS, none of the 11 proteins was expressed in healthy samples, except for fibronectin. In FECD samples we identified proteins selectively expressed in different structures of the diseased DM: embryonic layers, Guttae, posterior fibrous layers. Notable differences were also observed between the periphery and the center. Conclusions: These new potential FECD markers located in different ECM abnormal structures suggest a dynamics phenomenon where different proteins are successively expressed and accumulated with time. [ABSTRACT FROM AUTHOR]