Dynamic changes of endothelial and stromal cilia are required for the maintenance of corneal homeostasis.

Primary cilia are distributed extensively within the corneal epithelium and endothelium. However, the presence of cilia in the corneal stroma and the dynamic changes and roles of endothelial and stromal cilia in corneal homeostasis remain largely unknown. Here, we present compelling evidence for the presence of primary cilia in the corneal stroma, both in vivo and in vitro. We also demonstrate dynamic changes of both endothelial and stromal cilia during corneal development. In addition, our data show that cryoinjury triggers dramatic cilium formation in the corneal endothelium and stroma. Furthermore, depletion of cilia in mutant mice lacking intraflagellar transport protein 88 compromises the corneal endothelial capacity to establish the effective tissue barrier, leading to an upregulation of α-smooth muscle actin within the corneal stroma in response to cryoinjury. These observations underscore the essential involvement of corneal endothelial and stromal cilia in maintaining corneal homeostasis and provide an innovative strategy for the treatment of corneal injuries and diseases.

Effects of human platelet lysate on the growth of cultured human corneal endothelial cells.

Human platelet lysate (hPL) as a replacement for foetal bovine serum (FBS) in culturing human corneal endothelium is an emerging area of interest, although there are limited studies evaluating the quality of the hPL being used. Our study aimed to evaluate variations between sources of hPL and to explore the efficacy of hPL (with and without heparin) as a replacement for FBS in culturing human corneal endothelial cells in vitro. Immortalized human corneal endothelial cells (B4G12) and primary human corneal endothelial cells (PHCEnCs, n = 11 donors, age from 36 to 85 years old) were cultured with 5% hPL or FBS. A full characterisation of the effects of hPL and FBS on cell growth was conducted using IncuCyte Zoom (percentage cell confluence and population doubling time, PDT) to analyse cell proliferation. AlamarBlue assays were used to measure cell viability. The concentration of fibrinogen, PDGF, hEGF, VEGF and bFGF in two sources of hPL were analyzed by Enzyme-linked immunosorbent assay. Expression and localization of Na+/K+-ATPase, ZO-1 and CD166 on PHCEnCs and B4G12 cells were assessed with immunofluorescence and immunoblotting. Our results showed that a significant difference in fibrinogen, hEGF and VEGF concentrations was found between two sources of hPL. Heparin impaired the positive effect of hPL on cell growth. PDT and alamarBlue showed that hPL significantly increased proliferation and viability of PHCEnCs in two of three donors, and immunostaining indicated that hPL increased ZO-1 and CD166 expression but not Na+/K+-ATPase on PHCEnCs. In addition, heterogeneities on immunopositivity of Na+/K+-ATPase and ZO-1 and morphology were found on PHCEnCs derived from an individual donor cultured with hPL medium. In conclusion, hPL showed positive effect on primary corneal endothelial cell growth, and maintenance of their cellular characteristics compared to FBS. hPL can be considered as a supplement to replace FBS in PHCEnC culture. However, the variation observed between different hPL sources suggests that a standard quality control monitoring system such as storage time and a minimal concentration of growth factors may need to be established.

Primary cell culture of canine corneal endothelial cells.

OBJECTIVE: To establish a primary cell culture and clarify the characteristics of canine corneal endothelial cells in vitro. PROCEDURES: The eyes were enucleated from dogs that were euthanized for reasons unrelated to this study. Enucleated canine eyes were dissected, and the intact corneas were isolated from the globes. Using enzymes, the corneal endothelial cells were dispersed from the cornea. The obtained canine corneal endothelial cells were cultured in a cell culture dish. Cultured corneal endothelial cells were morphologically evaluated using phase-contrast microscopy. Immunohistochemical analysis of the cultured cells, particularly of the corneal endothelial cell marker, zonula occludens-1 (ZO-1), Na+ /K+ -ATPase, and vimentin, was performed to clarify whether the cultured cells were actually corneal endothelial cells. Furthermore, the post-passage morphology of cultured cells was evaluated. RESULTS: Canine primary cultured corneal endothelial cells showed morphologically small, cobblestone-like structures. The isolated cells had proliferative ability in vitro and demonstrated positive expression of the corneal endothelial cell markers, ZO-1, Na+ /K+ -ATPase, and vimentin. However, repeated passages resulted in larger cell sizes as assessed by phase-contrast microscopy. Repeated passages also resulted in lower cell density. CONCLUSIONS: This study demonstrated the successful culture of canine corneal endothelial cells. This might enhance the understanding of corneal endothelial cell characteristics in dogs.

Reconstruction of a tissue-engineered cornea with porcine corneal acellular matrix as the scaffold.

Interest in developing tissue-engineered cornea has increased with the decrease in the supply of donor tissue. The aim of the present study was to investigate the feasibility and method of reconstructing corneal equivalents with porcine corneal acellular matrix as the scaffold in a dynamic culturing system. Applying the detergent Triton X-100 (1%) and a freeze-drying process, porcine corneas were decellularized and prepared as a scaffold, and hematoxylin-eosin staining and scanning electron microscopy showed no cells in the decellularized stroma. In order to measure the in vivo biocompatibility, part of the scaffold was transplanted into a pocket of rabbit corneal stroma and observed for 3 months. No sign of rejection were observed, and the acellular matrix gradually integrated in the rabbit cornea, indicating that the scaffold had good biocompatibility. To reconstruct a tissue-engineered cornea, cultured rabbit keratocytes were seeded into the scaffold. After 1 week of culture in a culturing vessel, rabbit epithelial and endothelial cells were seeded on both sides of the stroma, respectively. The reconstructed cornea consisted of three layers in histological structure: the epithelium, stoma and endothelium. Stratified epithelial cells formed on the surface, which were cytokeratin 3 positive in the cytoplasm; endothelial cell monolayers were located on the inner side, and pump-related aquaporin 1 was found in the cells. These results confirmed that the corneal acellular matrix can be used as a scaffold for tissue-engineered cornea, and a biological corneal equivalent can be reconstructed in a dynamic culturing system.

Use of specular microscopy to determine corneal endothelial cell morphology and morphometry in enucleated cat eyes.

The purpose of this study was to investigate the effect of age on endothelial morphology and morphometry in cats. The corneal endothelium was studied using a contact specular microscope. A total of 18 cats (Felis catus Linnaeus, 1758) were evaluated in this study. The subjects were divided into three groups of six cats each in function of age: G1 (1 to 3 months old), G2 (5 to 12 months old), and G3 (24 to 40 months old). The examination presented data as endothelial cell density (ECD), average cell area, corneal thickness, polymegathism, and pleomorphism. Results revealed ECD decrease in corneas of normal cats with age, as well as a corresponding increase in endothelial cell area and pleomorphism. The present work suggests that the endothelial parameters evaluated change with advancing age.

Revisiting Existing Evidence of Corneal Endothelial Progenitors and Their Potential Therapeutic Applications in Corneal Endothelial Dysfunction.

PURPOSE: A recent successful clinical trial demonstrated that a less invasive cell-injection procedure is a viable medical modality for treating corneal endothelial dystrophy. This medical advance still relies on human corneal endothelial cell (HCEC) sources derived from rare cornea donations. The progenitor of the corneal endothelium, which has the characteristics of active proliferation and lineage restriction, will be an ideal cell source for expansion ex vivo. However, the distribution of progenitor-like cells in the corneal endothelial sheet has been under debate for more than a decade. METHODS: This paper re-examines the scientific evidence of the existence of human corneal endothelial progenitors (HCEPs) from the aspects of (1) the origin of cornea formation during ocular development, (2) manifestations from clinical studies, and (3) the distinctive properties of ex vivo-cultured subpopulations. RESULTS: The discrepancies regarding different types of progenitor-like cells in various locations of the cornea are based on the fact that the corneal endothelium is derived from different cell types with multiple origins during corneal formation. CONCLUSIONS: Resolving this long-standing issue in corneal biology will enable various types of progenitors to be isolated and their potencies regarding the formation of functional endothelial cells to be examined. Additionally, an effective niche system for quantitatively producing therapeutic cells can be formulated to satisfy the burning need associated with corneal endothelial dystrophy in the future.

In Vitro Expansion of Corneal Endothelial Cells for Transplantation.

The corneal endothelium forms a leaky barrier between the corneal stroma and the aqueous humor of the anterior chamber. This cell monolayer maintains the corneal stroma in a state of relative dehydration, a process called deturgescence, which is required in order to obtain corneal stromal transparency. Endothelial dysfunctions lead to visual impairment that ultimately can only be treated surgically via the corneal transplantation of a functional endothelium. Shortages of corneas suitable for transplantation has motivated research toward new alternatives involving in vitro corneal endothelial cell (CEC) expansion.This chapter describes current methods that allow isolate and culture CECs. In brief, Descemet membrane is peeled out of the cornea and digested in order to obtain CECs. Cells are then seeded and cultured.

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes.

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet's membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

Functional human corneal equivalents constructed from cell lines.

Human corneal equivalents comprising the three main layers of the cornea (epithelium, stroma, and endothelium) were constructed. Each cellular layer was fabricated from immortalized human corneal cells that were screened for use on the basis of morphological, biochemical, and electrophysiological similarity to their natural counterparts. The resulting corneal equivalents mimicked human corneas in key physical and physiological functions, including morphology, biochemical marker expression, transparency, ion and fluid transport, and gene expression. Morphological and functional equivalents to human corneas that can be produced in vitro have immediate applications in toxicity and drug efficacy testing, and form the basis for future development of implantable tissues.

In Vivo-Like Culture Conditions in a Bioreactor Facilitate Improved Tissue Quality in Corneal Storage.

The cornea is the most-transplanted tissue worldwide. However, the availability and quality of grafts are limited due to the current methods of corneal storage. In this study, a dynamic bioreactor system is employed to enable the control of intraocular pressure and the culture at the air-liquid interface. Thereby, in vivo-like storage conditions are achieved. Different media combinations for endothelium and epithelium are tested in standard and dynamic conditions to enhance the viability of the tissue. In contrast to culture conditions used in eye banks, the combination of the bioreactor and biochrom medium 1 allows to preserve the corneal endothelium and the epithelium. Assessment of transparency, swelling, and the trans-epithelial-electrical-resistance (TEER) strengthens the impact of the in vivo-like tissue culture. For example, compared to corneas stored under static conditions, significantly lower optical densities and significantly higher TEER values were measured (p-value <0.05). Furthermore, healing of epithelial defects is enabled in the bioreactor, characterized by re-epithelialization and initiated stromal regeneration. Based on the obtained results, an easy-to-use 3D-printed bioreactor composed of only two parts was derived to translate the technology from the laboratory to the eye banks. This optimized bioreactor facilitates noninvasive microscopic monitoring. The improved storage conditions ameliorate the quality of corneal grafts and the storage time in the eye banks to increase availability and reduce re-grafting.

RCE1 corneal epithelial cell line: its variability on phenotype expression and differential response to growth factors.

BACKGROUND: By serial transfer of rabbit corneal epithelial cells, the spontaneous RCE1 cell line was previously established. These cells mimic the stage-dependent differentiation of the corresponding cell type. METHODS: RCE1 cells were cultured either on plastic culture dishes or on collagen rafts to compare the epithelial ultrastructure after growth on these substrata. Phenotypic variability was studied after subcloning of cells. The differentiation ability of each subclone was determined by Western blot with antibodies against the differentiation-linked keratin pair K3/K12 and by measuring LDH activity and LDH isozymes in cytosolic extracts. The proliferative response of RCE1 cells to EGF, TGFalpha, amphiregulin, bFGF or IL-6 was determined under serum-free culture conditions. RESULTS: Cells grown on collagen rafts formed 5- to 7-layered epithelia with characteristics closer to those found in normal corneal epithelium than cells cultivated on plastic substrata, which formed 3- to 5-layered epithelia. Subcloning experiments demonstrated that every proliferative cell is able to grow and constitute stratified epithelia expressing K3/K12 keratins. LDH levels in RCE1 epithelia were similar to those of cultured or freshly harvested corneal epithelia; however, they showed a slightly altered LDH isozyme set, with prevalence of LDH-3 isoform. Whereas EGF and TGF-alpha were equipotent, amphiregulin elicited a 4-fold lower proliferative response. Also, bFGF was 10-fold less mitogenic than EGF, and IL-6 had the lowest effect with an ED(50) 20-fold lower than EGF. CONCLUSIONS: The results demonstrate that every RCE1 proliferative cell has the ability to generate epithelial sheets. We conclude that EGF and TGF-alpha are the major effectors of RCE1 cell proliferation.

Construction of tissue-engineered full-thickness cornea substitute using limbal epithelial cell-like and corneal endothelial cell-like cells derived from human embryonic stem cells.

The aim of this study was to construct a full-thickness artificial cornea substitute in vitro by coculturing limbal epithelial cell-like (LEC-like) cells and corneal endothelial cell-like (CEC-like) cells derived from human embryonic stem cells (hESCs) on APCM scaffold. A 400 µm thickness, 11 mm diameter APCM lamella containing Bowman's membrane was prepared as the scaffold using trephine and a special apparatus made by ourselves. LEC-like cells and CEC-like cells, derived from hESCs as our previously described, were cocultured on the scaffold using a special insert of 24-well plates that enabled seeding both sides of the scaffold. Three or four layers of epithelium-like cells and a uniform monolayer of CEC-like cells could be observed by H&E staining. The thickness, endothelial cell density, and mechanical properties of the construct were similar to that of native rabbit corneas. Immunofluorescence analysis showed expression of ABCG2 and CK3 in the epithelium-like cell layers and expression of N-cadherin, ZO-1 and Na+/K + ATPase in the CEC-like cells. The corneal substitutes were well integrated within the host corneas, and the transparency increased gradually in 8-week follow-up after transplantation in the rabbits. These results suggest that the strategy we developed is feasible and effective for construction of tissue-engineered full-thickness cornea substitute with critical properties of native cornea.

A novel strategy for corneal endothelial reconstruction with a bioengineered cell sheet.

Cellular organization of foreign grafts constructed from cultivated cells is critical to successful graft-host integration and tissue repair. This study described a novel human corneal endothelial cell (HCEC) therapeutic method, where cultivated adult HCEC sheet with uniform orientation was prepared and transplanted to a rabbit cornea. Having a correct morphology and intact barriers, the HCEC sheet was made by the temperature-modulated detachment of monolayered HCECs from thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm)-grafted surfaces and was delivered with proper polarity to the corneal posterior surface by a bioadhesive gelatin disc. Results of the in vivo studies, including the follow-up clinical observations and histological examinations, showed the laminated HCEC sheet was successfully integrated into rabbit cornea denuded with endothelial layer after the biodegradation of gelatin carrier. These data indicate the feasibility of the proposed procedure in cell therapy for corneal endothelial cell loss.

Comparison of rabbit corneal endothelial cell precursors in the central and peripheral cornea.

PURPOSE: To compare the distribution and self-renewal capacity of rabbit corneal endothelial cell precursors in the central and peripheral regions of the cornea. METHODS: The corneal endothelium (CE) and Descemet's membrane of New Zealand White rabbit corneas were divided into a peripheral region (6.0-10.0 mm in diameter) and a central region (6.0 mm in diameter). Then a sphere-forming assay was performed to isolate precursors from the CE of each region. Numbers of primary and secondary sphere colonies and sizes of primary spheres were compared between the central and peripheral regions. RESULTS: Primary spheres were isolated from the peripheral and the central regions of the CE. The rate of primary sphere formation in the peripheral region (34.4 +/- 10.4/10,000 cells) was significantly higher than in the central cornea (26.8 +/- 6.6/10,000 cells; P = 0.0042), but there was no significant difference in the size of primary spheres between the two regions. Self-renewal capacity was higher in the peripheral region than in the central region, as evidenced by a significantly higher secondary sphere formation rate for cells from the periphery (39.0 +/- 8.8/10,000 cells) compared with that for cells from the central region (25.4 +/- 4.2/10,000 cells; P = 0.00028). CONCLUSIONS: These findings demonstrate that peripheral and central rabbit corneal epithelia contain a significant number of precursors but that the peripheral endothelium contains more precursors and has a stronger self-renewal capacity than the central region.

Molecular description of eye defects in the zebrafish Pax6b mutant, sunrise, reveals a Pax6b-dependent genetic network in the developing anterior chamber.

The cornea is a central component of the camera eye of vertebrates and even slight corneal disturbances severely affect vision. The transcription factor PAX6 is required for normal eye development, namely the proper separation of the lens from the developing cornea and the formation of the iris and anterior chamber. Human PAX6 mutations are associated with severe ocular disorders such as aniridia, Peters anomaly and chronic limbal stem cell insufficiency. To develop the zebrafish as a model for corneal disease, we first performed transcriptome and in situ expression analysis to identify marker genes to characterise the cornea in normal and pathological conditions. We show that, at 7 days post fertilisation (dpf), the zebrafish cornea expresses the majority of marker genes (67/84 tested genes) found also expressed in the cornea of juvenile and adult stages. We also characterised homozygous pax6b mutants. Mutant embryos have a thick cornea, iris hypoplasia, a shallow anterior chamber and a small lens. Ultrastructure analysis revealed a disrupted corneal endothelium. pax6b mutants show loss of corneal epithelial gene expression including regulatory genes (sox3, tfap2a, foxc1a and pitx2). In contrast, several genes (pitx2, ctnnb2, dcn and fabp7a) were ectopically expressed in the malformed corneal endothelium. Lack of pax6b function leads to severe disturbance of the corneal gene regulatory programme.

Maturation of the corneal endothelial tight junction.

Apical tight junctional formation of the rabbit corneal endothelium was examined by freeze-fracture analysis and measurement of paracellular permeability to 5(6)-carboxyfluorescein. Freeze-fracture analysis indicated that apical tight junction formation of the rabbit corneal endothelium is a dynamic process. At birth, there are few tight junctional strands present and a minimal barrier for paracellular diffusion. As the rabbit matures, a more complex network of anastomosing tight junctional strands begins to encircle the cell perimeter under the apical folds. However, even in the mature animal (3 months), there are discontinuities and free ends in the network, thus suggesting that the barrier is not complete even at this stage. Paracellular permeability measurements using 5(6)-carboxyfluorescein as a tracer corroborate these anatomic findings. Endothelial paracellular flux measurements steadily decrease as the rabbit matures from birth to young adult. This indicates that the tight junctional network is increasing in complexity and progressively limiting the flow of substances through the intercellular space.

Changes in extracellular matrix proteins and actin during corneal endothelial growth.

Basement membranes influence growth, shape and differentiation of cells and tissues. However, the role and influence of Descemet's membrane during corneal development is not understood. To address this question, the relationships between cell growth and fibronectin, laminin and actin distribution in the developing rat corneal endothelium in vivo has been examined. During fetal development, rat corneal endothelial cells undergo DNA synthesis and mitosis. However, at day 14 of gestation both processes begin to decline and neither can be detected in endothelium of 1-month-old animals. By this time cell number has increased to approximately 100,000 and tissue area has increased 25-fold. However, as the tissue area increased, cell density decreased, indicating that cell spreading occurred in order to maintain tissue integrity. Changes in endothelial growth were accompanied by changes in the distribution of laminin, fibronectin and actin. Laminin and fibronectin were diffusely localized within endothelial cells in newborn animals. By 4 weeks of age, no proliferation was demonstrated and both extracellular matrix proteins were localized in pericellular patterns. Actin, on the other hand, which appeared diffuse at 16 days in utero, was distributed at or near the cell membrane by 19 days in utero. Thus, the reorganization of extracellular matrix glycoproteins and actin may indicate important roles for these components in regulating the growth and formation of the corneal endothelium in vivo.

Involvement of p27KIP1 in the proliferation of the developing corneal endothelium.

PURPOSE: To examine the involvement of p27(KIP1) in the regulation of the proliferation of the developing corneal endothelium. METHODS: Central and peripheral corneas in C57Bl6 mice at postnatal day (P)1, P11, and 12 weeks after birth were analyzed by immunocytochemistry with anti-p27(KIP1), -p57(KIP2), and -proliferating cell nuclear antigen (PCNA) antibodies. Nuclear staining was performed with 4',6'-diamino-2-phenylindole (DAPI) in wholemounts of corneal endothelium of the center and peripheral cornea in wild-type and p27(KIP1) knockout (-/-) mice at 12 weeks of age. p27(KIP1-/-) and control mice were injected with bromodeoxyuridine (BrdU) once on P7, twice per day on P8 and P9, and once on P10 and then were analyzed by a BrdU cell-proliferation assay on P11. RESULTS: On P1, p27(KIP1) immunoreactivity was detected in a small number of corneal endothelial cells, and many endothelial cells expressed PCNA. At P11 and 12 weeks after birth, p27(KIP1) immunoreactivity was detected in many corneal endothelial cells. PCNA-positive cells in the endothelium were rare on P11 and completely absent at 12 weeks after birth. p57(KIP2) was not detected in either corneal epithelium or endothelium at P1, P11, or 12 weeks after birth. In wholemounts of corneal endothelium at 12 weeks of age, the number of endothelial nuclei in the p27(KIP1-/-) mice was significantly higher than that in wild-type mice in both the center and peripheral regions of the cornea. In the BrdU assay, positive cells were abundant in the corneal endothelium of p27(KIP1-/-) mice, whereas there were few positive cells in control mice. PCNA immunoreactivity in the endothelium of the p27(KIP1-/-) mice was completely absent at 12 weeks after birth. CONCLUSIONS: These results suggest that p27(KIP1) is involved in the regulation of proliferation in the endothelium of the developing cornea.

Corneal endothelium modulation factor released by polymorphonuclear leukocytes. Partial purification and initial characterization.

Polymorphonuclear leukocytes produce a polypeptide factor that is released into the medium. This factor is partially purified 83-fold by ammonium sulfate precipitation followed by chromatography on a DEAE-Sephadex or heparin-Sepharose column. The partially purified factor is trypsin-sensitive. This factor affects a population of rabbit corneal endothelial cells by modulating them to fibroblastlike cells and by further stimulating their growth, leading to the formation of colonies of multilayered modulated cells. There is a dose-dependent phenotypic modulation of corneal endothelial cells by the partially purified corneal endothelium modulation factor (CEMF); cell shape is changed and type I collagen synthesis is increased with greater concentrations of CEMF. Since the fully modulated endothelial cells have collagen phenotypes distinct from those of normal cells, collagen synthesized by the first-passaged cells (a mixture of normal and modulated cells) was determined by immunoblot analysis with antibodies specific against types I and IV collagens. The first-passaged cells, in the presence of CEMF, contained a large amount of type I collagen (modulated phenotype) and a dramatically reduced amount of type IV collagen (physiologic type), whereas the normal endothelial cells demonstrated strongly positive staining only with antibodies to type IV collagen. Using cloned cDNA probes, the relative quantities of the transcripts of these collagens were determined by slot-blot hybridization; the first-passaged cells contained type IV collagen RNA in an amount similar to the normal cells, but a slightly larger amount of type I mRNA. These results demonstrate a functional involvement of a protein factor released by polymorphonuclear leukocytes in modulating cell shape and collagen gene expression in corneal endothelial cells.