ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells

Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and “pump” functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34), the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy.     

Expansion and cryopreservation of porcine and human corneal endothelial cells

Impairment of the corneal endothelium causes blindness that afflicts millions worldwide and constitutes the most often cited indication for corneal transplants. The scarcity of donor corneas has prompted the alternative use of tissue-engineered grafts which requires the ex vivo expansion and cryopreservation of corneal endothelial cells. The aims of this study are to culture and identify the conditions that will yield viable and functional corneal endothelial cells after cryopreservation. Previously, using human umbilical vein endothelial cells (HUVECs), we employed a systematic approach to optimize the post-thaw recovery of cells with high membrane integrity and functionality. Here, we investigated whether improved protocols for HUVECs translate to the cryopreservation of corneal endothelial cells, despite the differences in function and embryonic origin of these cell types. First, we isolated endothelial cells from pig corneas and then applied an interrupted slow cooling protocol in the presence of dimethyl sulfoxide (Me₂SO), with or without hydroxyethyl starch (HES). Next, we isolated and expanded endothelial cells from human corneas and applied the best protocol verified using porcine cells. We found that slow cooling at 1 °C/min in the presence of 5% Me₂SO and 6% HES, followed by rapid thawing after liquid nitrogen storage, yields membrane-intact cells that could form monolayers expressing the tight junction marker ZO-1 and cytoskeleton F-actin, and could form tubes in reconstituted basement membrane matrix. Thus, we show that a cryopreservation protocol optimized for HUVECs can be applied successfully to corneal endothelial cells, and this could provide a means to address the need for off-the-shelf cryopreserved cells for corneal tissue engineering and regenerative medicine.     

Discovery of Molecular Markers to Discriminate Corneal Endothelial Cells in the Human Body

The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs) on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs in vivo have a limited capacity in proliferation, and loss of a significant number of CECs results in corneal edema called bullous keratopathy which can lead to severe visual loss. Corneal transplantation is the most effective method to treat corneal endothelial dysfunction, where it suffers from donor shortage. Therefore, regeneration of CECs from other cell types attracts increasing interests, and specific markers of CECs are crucial to identify actual CECs. However, the currently used markers are far from satisfactory because of their non-specific expression in other cell types. Here, we explored molecular markers to discriminate CECs from other cell types in the human body by integrating the published RNA-seq data of CECs and the FANTOM5 atlas representing diverse range of cell types based on expression patterns. We identified five genes, CLRN1, MRGPRX3, HTR1D, GRIP1 and ZP4 as novel markers of CECs, and the specificities of these genes were successfully confirmed by independent experiments at both the RNA and protein levels. Notably none of them have been documented in the context of CEC function. These markers could be useful for the purification of actual CECs, and also available for the evaluation of the products derived from other cell types. Our results demonstrate an effective approach to identify molecular markers for CECs and open the door for the regeneration of CECs in vitro.     

Gene transfer of cyto-protective molecules in corneal endothelial cells and cultured corneas: analysis of protective effects in vitro and in vivo

The loss of corneal endothelial cells plays a critical role in many corneal diseases and is a common phenomenon following cornea transplantation. In addition, the non-regenerative capacity of human corneal endothelial cells (HCEC) ultimately requires appropriate protection of corneal tissues during ex vivo storage to ensure vitality of the cells. However, only 70% of donor corneas can be used for grafting because of endothelial deficiencies. Corneal endothelial cell loss during storage is mainly induced by apoptotic cell death. This study was undertaken, for proof of principle, to investigate whether over-expression of cyto-protective molecules Bcl-x(L), Bag-1, and HO-1 prevents the loss of corneal endothelial cells both in vitro and in vivo. We demonstrate that gene transfer of both Bcl-x(L) and HO-1 has cyto-protective effects on HCEC in vitro. However, gene transfer of a single cyto-protective molecule does not prevent its rejection upon transplantation in a MHC class I/II disparate rat model.     

Activation of RhoA-ROCK-BMP signaling reprograms adult human corneal endothelial cells

Currently there are limited treatment options for corneal blindness caused by dysfunctional corneal endothelial cells. The primary treatment involves transplantation of healthy donor human corneal endothelial cells, but a global shortage of donor corneas necessitates other options. Conventional tissue approaches for corneal endothelial cells are based on EDTA-trypsin treatment and run the risk of irreversible endothelial mesenchymal transition by activating canonical Wingless-related integration site (Wnt) and TGF-β signaling. Herein, we demonstrate an alternative strategy that avoids disruption of cell–cell junctions and instead activates Ras homologue gene family A (RhoA)–Rho-associated protein kinase (ROCK)–canonical bone morphogenic protein signaling to reprogram adult human corneal endothelial cells to neural crest–like progenitors via activation of the miR302b-Oct4-Sox2-Nanog network. This approach allowed us to engineer eight human corneal endothelial monolayers of transplantable size, with a normal density and phenotype from one corneoscleral rim. Given that a similar signal network also exists in the retinal pigment epithelium, this partial reprogramming approach may have widespread relevance and potential for treating degenerative diseases.     

Transplantation of cryopreserved human corneas in a xenograft model

An ideal model to test methods of corneal storage for transplantation would simulate the environment of the grafted human cornea and predict the success of clinical corneal transplants (human to human). In this study, we tested such a model, the corneal xenograft (human to cat). Nine pairs of human corneas were transplanted into both eyes of nine recipient cats. One cornea of each pair was cryopreserved at -196 degrees C in 2.5 M dimethyl sulfoxide while the other was stored in preservative medium at 4 degrees C (control) for 6 +/- 2 (mean +/- SD) days before transplantation. One week after transplantation, the cats were euthanized and the eyes were examined. Three of the grafts (all cryopreserved) were clinical failures and showed no survival of donor corneal endothelial cells on scanning electron microscopy. The remaining six pairs of grafts were examined with a specular microscope and showed endothelial cell losses of 48 +/- 16% in cryopreserved and 8 +/- 16% in control corneas (p < 0.05). This survival is similar to survival in an earlier corneal perfusion model. The nine cryopreserved grafts were thicker than the control grafts, had fewer surviving keratocytes in the central stroma, and had more apoptotic central keratocytes (TUNEL assay). This failure rate in cryopreserved corneas clearly shows that this technique of cryopreservation was not adequate for clinical use. The corneal xenograft model can be used to study cellular survival and apoptosis in vivo after preservation as well as to test new methods of corneal preservation before initiating clinical trials.     

Corneal stem cells and tissue engineering: Current advances and future perspectives

Major advances are currently being made in regenerative medicine for cornea. Stem cell-based therapies represent a novel strategy that may substitute conventional corneal transplantation, albeit there are many challenges ahead given the singularities of each cellular layer of the cornea. This review recapitulates the current data on corneal epithelial stem cells, corneal stromal stem cells and corneal endothelial cell progenitors. Corneal limbal autografts containing epithelial stem cells have been transplanted in humans for more than 20 years with great successful rates, and researchers now focus on ex vivo cultures and other cell lineages to transplant to the ocular surface. A small population of cells in the corneal endothelium was recently reported to have self-renewal capacity, although they do not proliferate in vivo. Two main obstacles have hindered endothelial cell transplantation to date: culture protocols and cell delivery methods to the posterior cornea in vivo. Human corneal stromal stem cells have been identified shortly after the recognition of precursors of endothelial cells. Stromal stem cells may have the potential to provide a direct cell-based therapeutic approach when injected to corneal scars. Furthermore, they exhibit the ability to deposit organized connective tissue in vitro and may be useful in corneal stroma engineering in the future. Recent advances and future perspectives in the field are discussed.     

Constructing an in vitro cornea from cultures of the three specific corneal cell types

Summary This paper presents a reliable method for establishing pure cultures of the three types of corneal cells. This is believed to be the first time, corneal cells have been cultured from fetal pig corneas. Cell growth studies were performed in different media. Subcultures of the three corneal cell types were passaged until the 30th generation without their showing signs of senescence. For engineering an in vitro cornea, corneal epithelial cells were cultured over corneal stromal cells in an artificial biomatrix of collagen with an underlying layer of corneal endothelial cells. The morphology, histology, and differentiation of the in vitro cornea were investigated to determine the degree of comparability to the cornea in vivo. The in vitro construct displayed signs of transition to an organotypic phenotype of which the most prominent was the formation of two basement membranes.     

High Throughput Gene Expression Analysis Identifies Reliable Expression Markers of Human Corneal Endothelial Cells

Considerable interest has been generated for the development of suitable corneal endothelial graft alternatives through cell-tissue engineering, which can potentially alleviate the shortage of corneal transplant material. The advent of less invasive suture-less key-hole surgery options such as Descemet’s Stripping Endothelial Keratoplasty (DSEK) and Descemet’s Membrane Endothelial Keratoplasty (DMEK), which involve transplantation of solely the endothelial layer instead of full thickness cornea, provide further impetus for the development of alternative endothelial grafts for clinical applications. A major challenge for this endeavor is the lack of specific markers for this cell type. To identify genes that reliably mark corneal endothelial cells (CECs) in vivo and in vitro, we performed RNA-sequencing on freshly isolated human CECs (from both young and old donors), CEC cultures, and corneal stroma. Gene expression of these corneal cell types was also compared to that of other human tissue types. Based on high throughput comparative gene expression analysis, we identified a panel of markers that are: i) highly expressed in CECs from both young donors and old donors; ii) expressed in CECs in vivo and in vitro; and iii) not expressed in corneal stroma keratocytes and the activated corneal stroma fibroblasts. These were SLC4A11, COL8A2 and CYYR1. The use of this panel of genes in combination reliably ascertains the identity of the CEC cell type.     

Macrophage Depletion Impairs Corneal Wound Healing after Autologous Transplantation in Mice

Purpose

Macrophages have been shown to play a critical role in the wound healing process. In the present study, the role of macrophages in wound healing after autologous corneal transplantation was investigated by depleting local infiltrated macrophages.

Methods

Autologous corneal transplantation model was used to induce wound repair in Balb/c mice. Macrophages were depleted by sub-conjunctival injections of clodronate-containing liposomes (Cl₂MDP-LIP). The presence of CD11b⁺ F4/80⁺ macrophages, α-smooth muscle actin⁺ (α-SMA⁺) myofibroblasts, CD31⁺ vascular endothelial cells and NG₂ ⁺ pericytes was examined by immunohistochemical and corneal whole-mount staining 14 days after penetrating keratoplasty. Peritoneal macrophages were isolated from Balb/c mice and transfused into conjunctiva to examine the recovery role of macrophages depletion on wound healing after autologous corneal transplantation.

Results

Sub-conjunctival Cl₂MDP-LIP injection significantly depleted the corneal resident phagocytes and infiltrated macrophages into corneal stroma. Compared with the mice injected with PBS-liposome, the Cl₂MDP-LIP-injected mice showed few inflammatory cells, irregularly distributed extracellular matrix, ingrowth of corneal epithelium into stroma, and even the detachment of donor cornea from recipient. Moreover, the number of macrophages, myofibroblasts, endothelial cells and pericytes was also decreased in the junction area between the donor and recipient cornea in macrophage-depleted mice. Peritoneal macrophages transfusion recovered the defect of corneal wound healing caused by macrophages depletion.

Conclusions

Macrophage depletion significantly impairs wound healing after autologous corneal transplantation through at least partially impacting on angiogenesis and wound closure.     

Establishment of a porcine corneal endothelial organ culture model for research purposes

Human corneas usually are not available for research, as they are used for transplantation only. At the same time, scientific studies on cultured human endothelial cells can produce misleading results due to inevitable dedifferentiation. Therefore, an organ-culture model of porcine corneas—displaying endothelial cell death rates comparable to those of cultured human corneas—would be very desirable. Fresh pig eyes were prepared under sterile conditions to obtain corneoscleral buttons, corneal buttons and so called “split corneal buttons” (new preparation method) and cultivated for 15 days. Morphology of the endothelial cell layer was observed by light microscopy on day 1, 8 and 15. On day 15 staining with trypan blue and alizarin red S was performed. Photographs were evaluated in a randomized, blinded manner. Here, the morphology of the corneal endothelium and the number of endothelial cells per mm² were analyzed. After 15 days of cultivation the endothelial cell layer was maintained only in corneal buttons and split corneal buttons. Alizarin red S stained areas and the existence of polymorphisms like rosette figures and reformation figures were significantly less frequent in split corneal buttons than in corneal buttons. Loss of endothelial cells was significantly greater in corneal buttons [575 ± 25/250 cells/mm² (median ± 25%/75%-quantile); 14.8%] than in split corneal buttons [417 ± 138/179 cells/mm² (median ± 25%/75%-quantile); 10.2%]. The new preparation method of split corneal buttons allows the cultivation of porcine corneas for 2 weeks with cell death rates comparable to those of the corresponding human tissue in cornea banks without the need to add de-swelling additives to the media. This is therefore a simple and highly reliable method model to be applied in intervention studies on corneal endothelial cells in their natural compound.     

The corneal epithelial stem cell

The aim of this paper was to develop a GFP-expressing transgenic mouse model for the keratoepithelioplasty and to use this to follow the outcome of this form of graft, when placed on an inflamed corneal surface. Further aims were to characterize both the graft and the epithelial surface of the mouse and rat cornea using putative stem cell markers (P63 and Telomerase) and marker of cell differentiation (14-3-3 sigma). Keratepithelioplasty was carried out using a GFP transgenic mouse cornea as donor tissue. Fluorescent epithelial outgrowth from each keratepithelioplasty was scored and quantified. Donor corneal graft tissue was obtained from the paracentral region or the anatomical limbal region of murine corneas. Paracentral donor grafts (n = 20) consistently demonstrated a significant increase in proliferative potential compared to grafts obtained from the anatomical limbal region of the mouse cornea (n = 25) (P = 0.000, Mann-Whitney U). Correspondingly, P63 expression was maximal in the paracentral region of the mouse cornea, in keeping with the demonstrated increased proliferative potential of donor grafts harvested from this region of the cornea. The murine corneal epithelium demonstrated decreased rather than increased cellular layers at the limbal region, in contrast to that of the rat or human epithelium. In addition, as a general finding in all species tested, there was an apparent increase noted in P63 expression in basal corneal epithelial cells in regions that had increased cellular layers (limbus in humans and rats and the paracentral corneal region in the mouse). Epithelium, which had migrated from donor grafts onto recipient corneas, retained P63 expression for the period of time examined (up to 3 days postengraftment). In addition, the conjunctival surface of an injured conjunctivalized displayed an abnormal pattern of P63 expression. Telomerase expression was widespread throughout many layers of both the murine and rat corneal epithelium. In the mouse and rat corneal epithelium P63 expression was maximal in areas of increased proliferative potential. Its expression, however, was not confined to stem cells alone. Migrating cells from transplanted keratoepithelial grafts retained P63 expression at least in the early stages post-transplantation. Finally, damaged conjunctivalized corneas displayed an abnormal P63 expression pattern when compared to either normal conjunctiva or normal cornea.     

Specular microscopy in glaucoma patients

The endothelial cells are one of the most important structures in a donor cornea. Morphology and concentration of endothelial cells must be carefully evaluated with a specular microscope before transplantation. The aim of this study is to evaluate the status of corneal endothelium in glaucoma patients. Prospective study included 50 patients suffering from glaucoma and 50 patients in control group. Patients had no corneal disease, ocular inflammation, previous trauma or ocular surgery. Patients were not contact lens wearers. They were also analyzed in groups according to type of glaucoma. Specular microscopy was performed on central corneas. This study showed that patients with glaucoma have lower central corneal endothelial cell density than those without glaucoma of the same age group. Also, patients with pseudoexfoliative glaucoma had lower values of central endothelial cell density comparing to patients with open angle or angle closure glaucoma.     

Different characteristics of endothelial cells from central and peripheral human cornea in primary culture and after subculture

Summary Several methods for isolation and cultivation of human corneal endothelial cells have been described during the last few decades. In contrast to the situation in vivo, the cultured cells show mitogenic activity but often lose their typical morphological appearance. In this paper, we describe a technique to isolate and cultivate morphologically unchanged endothelium from the human cornea. This method revealed different characteristics of endothelial cells according to their position within the human cornea. Endothelial cells isolated from the central part have a morphology similar to that of cells in vivo (i.e., they are densely packed and show no mitogenic activity). In contrast, endothelial cells derived from the peripheral part of the cornea are characterized by mitogenic activity but their cell-to-cell attachment seems to be less tight than in vivo. The significance of these two different endothelial cell types for wound healing in the human cornea is discussed.     

A human corneal equivalent constructed from SV40-immortalised corneal cell lines

Within the last decade, extensive research in the field of tissue and organ engineering has focused on the development of in vitro models of the cornea. The use of organotypic, three-dimensional corneal equivalents has several advantages over simple monolayer cultures. The aim of this study was to develop a corneal equivalent model composed of the same cell types as in the natural human tissue, but by using immortalised cell lines to ensure reproducibility and to minimise product variation. We report our success in the establishment of an SV40-immortalised human corneal keratocyte cell line (designated HCK). A collagen matrix, built up with these cells, displayed the morphological characteristics of the human stromal tissue and served as a biomatrix for the immortalised human corneal epithelial and endothelial cells. Histological cross-sections of the whole-cornea equivalents resemble human corneas in tissue structure. This organotypic in vitro model may serve as a research tool for the ophthalmic science community, as well as a model system for testing for eye irritancy and drug efficacy.     

Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty

Descemet’s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, along with its basement membrane (Descemet''s membrane) is introduced onto the recipient''s posterior stroma³. Unlike Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK), where additional donor stroma is introduced, no unnatural stroma-to-stroma interface is created. As a result, the natural anatomy of the cornea is preserved as much as possible allowing for improved recovery time and visual acuity⁴. Endothelial Keratoplasty (EK) is the procedure of choice for treatment of endothelial dysfunction. The advantages of EK include rapid recovery of vision, preservation of ocular integrity and minimal refractive change due to use of a small, peripheral incision¹. DSAEK utilizes donor tissue prepared with partial thickness stroma and endothelium. The rapid success and utilization of this procedure can be attributed to availability of eye-bank prepared precut tissue. The benefits of eye-bank preparation of donor tissue include elimination of need for specialized equipment in the operating room and availability of back up donor tissue in case of tissue perforation during preparation. In addition, high volume preparation of donor tissue by eye-bank technicians may provide improved quality of donor tissue. DSAEK may have limited best corrected visual acuity due to creation of a stromal interface between the donor and recipient cornea. Elimination of this interface with transplantation of only donor Descemet''s membrane and endothelium in DMEK may improve visual outcomes and reduce complications after EK⁵. Similar to DSAEK, long term success and acceptance of DMEK is dependent on ease of availability of precut, eye-bank prepared donor tissue. Here we present a stepwise approach to donor tissue preparation which may reduce some barriers eye-banks face in providing DMEK grafts.     

Corneal morphogenesis in the Indian garden lizard,Calotes versicolor (agamidae)

The present study traces corneal morphogenesis in a reptile, the lizard Calotes versicolor, from the lens placode stage (stage 24) until hatching (stage 42), and in the adult. The corneal epithelium separates from the lens placode as a double layer of peridermal and basal cells and remains bilayered throughout development and in the adult. Between stages 32– and 33+, the corneal epithelium is apposed to the lens, and limbic mesodermal cells migrate between the basement membrane of the epithelium and the lens capsule to form a monolayered corneal endothelium. Soon thereafter a matrix of amorphous ground substance and fine collagen fibrils, the presumptive stroma, is seen between the epithelium and the endothelium. Just before stage 34 a new set of limbic mesodermal cells, the keratocytes, migrate into the presumptive stroma. Migrating limbic mesodermal cells, both endothelial cells and keratocytes, use the basement membrane of the epithelium as substratum. Keratocytes may form up to six cell layers at stage 37, but in the adult stroma they form only one or two cell layers. The keratocytes sysnthesize collagen, which aggregates as fibrils and fibers organized in lamellae. The lamellae become condensed as dense collagen layers subepithelially or become compactly organized into a feltwork structure in the rest of the stroma. The basement membrane of the endothelium is always thin. Thickness of the entire cornea increases up to stage 38 and decreases thereafter until stage 41. In the adult the cornea is again nearly as thick as at stage 38.