Corneal transplantation (CT) is the most frequently performed type of transplant worldwide. It restores visual function when impairment caused by corneal damage is deemed too severe to provide acceptable quality of life in the country where it is performed. Corneal blindness is the third leading cause of blindness worldwide after cataract and glaucoma,¹ with 10 million people having bilateral corneal blindness.²

The control of corneal transparency depends on the integrity of its endothelial monolayer, which is considered nonregenerative in adult humans. In pathological situations, endothelial cell (EC) loss, not offset by mitosis, can lead to irreversible corneal edema and blindness. However, the hypothesis of a slow, clinically insufficient regeneration starting from the corneal periphery remains debatable. The authors have re-evaluated the microanatomy of the endothelium in order to identify structures likely to support this homeostasis model. Whole endothelia of 88 human corneas (not stored, and stored in organ culture) with mean donor age of 80 ± 12 years were analyzed using an original flat-mounting technique. In 61% of corneas, cells located at the extreme periphery (last 200 μm of the endothelium) were organized in small clusters with two to three cell layers around Hassall-Henle bodies. In 68% of corneas, peripheral ECs formed centripetal rows 830 ± 295 μm long, with Descemet membrane furrows visible by scanning electron microscopy. EC density was significantly higher in zones with cell rows. When immunostained, ECs in the extreme periphery exhibited lesser differentiation (ZO-1, Actin, Na/K ATPase, CoxIV) than ECs in the center of the cornea but preferentially expressed stem cell markers (Nestin, Telomerase, and occasionally breast cancer resistance protein) and, in rare cases, the proliferation marker Ki67. Stored corneas had fewer cell clusters but more Ki67-positive ECs. We identified a novel anatomic organization in the periphery of the human corneal endothelium, suggesting a continuous slow centripetal migration, throughout life, of ECs from specific niches. STEM CELLS2012;30:2523–2534

Corneal endothelial cells (CECs) are terminally differentiated cells, specialized in regulating corneal hydration and transparency. They are highly polarized flat cells that separate the cornea from the aqueous humor. Their apical surface, in contact with aqueous humor is hexagonal, whereas their basal surface is irregular. We characterized the structure of human CECs in 3D using confocal microscopy of immunostained whole corneas in which cells and their interrelationships remain intact. Hexagonality of the apical surface was maintained by the interaction between tight junctions and a submembraneous network of actomyosin, braced like a drum. Lateral membranes, which support enzymatic pumps, presented complex expansions resembling interdigitated foot processes at the basal surface. Using computer-aided design and drafting software, we obtained a first simplified 3D model of CECs. By comparing their expression with those in epithelial, stromal and trabecular corneal cells, we selected 9 structural or functional proteins for which 3D patterns were specific to CECs. This first 3D map aids our understanding of the morphologic and functional specificity of CECs and could be used as a reference for characterizing future cell therapy products destined to treat endothelial dysfunctions.

Research from JAMA Ophthalmology — Prospective, Randomized Clinical and Endothelial Evaluation of 2 Storage Times for Corneal Donor Tissue in Organ Culture at 31°C

Aim: To examine the differences in manual endothelial cell counting methods in French eye banks and to analyse whether these differences could explain some substantial discrepancies observed in endothelial cell density (ECD) for corneas made available for transplant. Methods: A questionnaire was sent to the 22 eye banks asking for details of the technical features of the light microscopes used, the microscope calibration, strategy for cell counting, the technical staff, and the method of presenting endothelial data. Results: All eye banks responded and 91% (20/22) used only manual counting methods, in real time, directly through a microscope, and 62 different technicians, with varying experience, were involved in such counting. Counting of cells within the borders of a grid that were in contact with two adjacent borders was the most common method (17/22, 77%). Of the eight banks (8/22, 36%) that did not calibrate their microscopes, six reported the highest ECD values. Of the 14 others (64%), six applied a “magnification correcting factor” to the initial cell counts. In five of these cases, the corrected ECD was lower than estimated on initial count. Most of the banks (12/22, 55%) counted 100 cells or less in one to six non-adjacent zones of the mosaic. 14 of the banks (14/22, 64%) also graded cell polymegethism while seven (7/22, 32%) also graded pleomorphism (“hexagonality”). Conclusions: Lack of microscope calibration appears to be the leading cause of variance in ECD estimates in French eye banks. Other factors such as differences in counting strategy, the evaluation of smaller numbers of cells, and the different extent of experience of the technicians may also contribute to intraobserver and interobserver variability. Further comparative studies, including cross checking and the outcome of repeated counts from manual methods, are clearly needed with cross calibration to a computer based image archiving and analysis system.

Purpose: Engineered corneal endothelial grafts able to provide numerous functional endothelial cells for the restoration of corneal transparency would be a worthwhile way of replacing donor tissue, which is extremely scarce. The grafts are simply constructed: a biocompatible thin and transparent carrier colonized by a monolayer of cultured endothelial cells (ECs). Here we describe a process able to obtain appropriate carriers by recycling human corneas unsuitable for graft in their original state, but liable to provide multiple thin lamellae when cut with a femtosecond laser as used in refractive surgery. Methods: We selected a robust method of stromal decellularization. To demonstrate that neither this process nor long-term storage hindered cell adherence, lamellae were endothelialized with an EC line. Results: The constructs achieved up to very high EC density (the main quality criterion for regular donor corneas) while remaining transparent and thin. We verified that they could be inserted in the anterior chamber of a human eye, like a conventional endothelial graft. Human decellularized cornea will likely be directly compatible with the recipient cornea and comply with the requirements of health regulatory authorities. Conclusions: This study demonstrates that thin human corneal lamellae could have high potential as carriers in next-generation therapy for endothelial dysfunctions.