Proliferation Of Human Corneal Endothelial Cells Research Articles

Repair effects of thermosensitive hydrogels combined with iPSC-derived corneal endothelial cells on rabbit corneal endothelial dysfunction

Considering the limitations of human corneal endothelial cell proliferation as well as the severe shortage of corneal donations, it is imperative to develop improved methods of corneal endothelial cell transplantation. The purpose of this study was to construct a modified corneal endothelial cell transplantation approach using thermosensitive hydrogels combined with induced pluripotent stem cells (iPSCs)-derived human corneal endothelial cells (hCECs). In this study, thermosensitive hydrogels hydroxypropyl chitin/carboxymethyl chitosan (HPCH/CMCS) were fabricated, and their hydrogels properties and biocompatibility were investigated. Our results demonstrated that HPCH/CMCS hydrogels exhibited superior transparency, appropriate mechanical properties and favorable biocompatibility. A two-step induction method of small molecule compounds was employed, by which iPSCs were differentiated into hCECs via neural crest cells (NCCs). Additionally, a rabbit corneal endothelial dysfunction model was established in vivo, aiming to evaluate the safety and effectiveness of the combined method. Slit lamp microscope results indicated that significant transparency improvement could be noted in HPCH/CMCS/hCECs group (P = 0.006), whereas the corneal transparency was not homogeneous in different areas. Moreover, histological examinations and immunofluorescence analysis revealed that HPCH/CMCS/hCECs group showed a higher density of corneal endothelial cells and positive expressions of related markers. This study may provide ideas and experimental basis for the combined application of hydrogels and iPSC-derived corneal endothelial cells for corneal endothelial dysfunction. Statement of SignificanceCorneal transplantation is the most effective treatment for corneal endothelial dysfunction, which is challenged by issues such as corneal donor shortages and immune rejection. In this study, we proposed a combined transplantation method of cells and hydrogels for corneal endothelial dysfunction. We modified the protocols to obtain corneal endothelial cells from iPSCs by a two-step induction method. Besides, thermosensitive hydrogels with satisfactory biocompatibility and degradability were fabricated as fixation and support carriers of iPSC-derived corneal endothelial cells for in vivo transplantation. Experimental results demonstrated that this method could locally repair corneal endothelial dysfunction in rabbits, with the repaired corneas expressing relevant markers. This study presented a preliminary attempt to combine hydrogels and cells for corneal endothelial dysfunction.

Effects of Autologous Serum and Platelet-Rich Plasma on Human Corneal Endothelial Cell Regeneration: A Comparative Study.

To investigate the effects of autologous serum (AS) and platelet-rich plasma (PRP) on human corneal endothelial cell (HCEC) proliferation and apoptosis in comparison to Y-27632 as the commonly studied Rho-associated kinase (ROCK) inhibitor. The human corneal endothelial primary cell line was used for this study. As the treatment groups, HCECs were incubated with AS, PRP, and Y-27632, whereas the control group received no treatment. Cell proliferation (measured by 5-bromo-2'-deoxyuridine [BrdU] incorporation) and apoptosis (based on the caspase-3 level) were compared between the control, Y-27632, AS, and PRP groups. In the Y-27632, AS, and PRP groups, the ratios of BrdU-incorporated cells were significantly higher (115±0.2%, 125±0.2%, 122±0.4% at 24 hr, and 138±2.4%, 160±0.2%, 142±0.2% at 48 hr, respectively) than in the control group (100±18.4% at 24 hr, 100±1.1% at 48 hr) ( P <0.05 for all). Furthermore, AS provided a higher HCEC proliferation ratio compared with the Y-27632 group at 24 and 48 hr ( P <0.05 for all). Caspase-3 was significantly lower in the AS group (60.3±3.3%) than in the control (100±2.3%), Y-27632 (101.9±5.2%), and PRP (101±6.8%) groups ( P <0.05 for all). The results of this study demonstrated for the first time that AS and PRP promoted HCEC proliferation and AS significantly decreased apoptosis in HCECs. A superior effect on HCEC proliferation was also observed with AS compared with Y-27632. Future "autologous" regenerative therapeutic options for corneal endothelial failure may involve the utilization of AS and PRP owing to their accessibility, simplicity in preparation, immunologic compatibility, and donor-free nature.

Brief incubation of corneal grafts in activated platelet rich plasma enhances corneal endothelial cell survival and regeneration

Corneal transplantation is the most frequent organ transplantation worldwide. Unfortunately, corneal graft failure is common and endothelial decompensation is considered the major cause. Corneal endothelial cells (CECs) lack the capacity to reproduce, and perioperative and postoperative endothelial cell loss remains a significant challenge associated with corneal graft viability. Therefore, strategies to preserve CEC density are critical to extend graft survival. Activated platelet rich plasma (aPRP), a product extracted from autologous blood, has both antioxidant and regenerative properties. aPRP eye drops have shown effectiveness in the treatment of corneal pathologies such as ulcers, dry eye, and burns. Our purpose is to determine the protective and regenerative effect of aPRP on corneal grafts by evaluating aPRP's effect on the survival and proliferation of human CECs. Human corneal grafts were incubated in aPRP for 15 min to assess the activation of the CEC pAkt survival pathway as measured by ELISA. Evaluation of the protective effect of aPRP was made using an apoptotic model, which simulated oxidative stress conditions. Expression of apoptotic markers was measured using ELISA and endothelial cell viability was determined by optical microscopy. The CEC proliferation rate was measured in vitro with Ki-67 staining. Corneal graft gross structure was evaluated by Hematoxylin & Eosin and Masson trichrome staining. Our results indicate that a short incubation of human corneal grafts in aPRP protects CECs from apoptosis by upregulating the pAkt survival pathway and promoting CEC proliferation. Additionally, aPRP incubation does not induce histological changes in the grafts. A brief pre-treatment of human corneal grafts in aPRP may be beneficial for transplant longevity, as it protects CECs from apoptosis by upregulating intracellular survival pathways and promoting proliferation. In addition, this approach appears to be safe and has the potential to improve surgical outcomes following corneal transplantation.

Effect of Stem Cell-Derived Extracellular Vesicles on Damaged Human Corneal Endothelial Cells.

Purpose Human corneal endothelial cells (HCECs) are essential to visual function; however, since they have limited proliferative capacity in vivo, they are prone to corneal endothelial dysfunction. At present, the only treatment is a corneal transplantation from donor cadavers. Also, due to a global shortage of donor corneas, it is important to find alternative strategies. Recent studies highlight that stem cell–derived extracellular vesicles (EVs) play a relevant role in stem cell-induced regeneration by reprogramming injured cells and inducing proregenerative pathways. The aim of this work is to evaluate whether EVs derived from mesenchymal stem cells (MSC-EVs) are able to promote regeneration of damaged HCECs. Methods We isolated HCECs from discarded corneas in patients undergoing corneal transplantation or enucleation (N = 23 patients). Bone marrow mesenchymal stem cells (MSCs) were obtained from Lonza, cultured, and characterized. MSC-EVs were obtained from supernatants of MSCs. In order to establish a valid in vitro damage model to test the regenerative potential of EVs on HCECs, we evaluated the proliferation rate and the apoptosis after exposing the cells to serum-deprived medium at different concentrations for 24 hours. We then evaluated the HCEC migration through a wound healing assay. Results In the selected serum deprivation damage conditions, the treatment with different doses of MSC-EVs resulted in a significantly higher proliferation rate of HCECs at all the tested concentrations of EVs (5‐20 × 103 MSC-EV/cell). MSC-EVs/cell induced a significant decrease in number of total apoptotic cells after 24 hours of serum deprivation. Finally, the wound healing assay showed a significantly faster repair of the wound after HCEC treatment with MSC-EVs. Conclusions Results highlight the already well-known proregenerative potential of MSC-EVs in a totally new biological model, the endothelium of the cornea. MSC-EVs, indeed, induced proliferation and survival of HCECs, promoting the migration of HCECs in vitro.

Effect of a p38 Mitogen-Activated Protein Kinase Inhibitor on Corneal Endothelial Cell Proliferation

We have performed clinical research on cell-based therapy for corneal endothelial decompensation since 2013. The purpose of this study was to investigate the usefulness of a p38 MAPK inhibitor for promoting proliferation of human corneal endothelial cells (HCECs). HCECs were cultured in media supplemented with various low-molecular-weight compounds to screen for the effect of those compounds on cell proliferation. Activation of substrates of p38 MAPK and cell cycle regulatory proteins were evaluated by western blotting. Corneal endothelial wounds were created in a rabbit model, and p38 MAPK was applied in eye drop form, followed by evaluation of cell proliferation in the corneal endothelium by Ki67-immunostaining. HCECs cultured with SB203580 exhibited hexagonal morphology and similar size and morphology, whereas control HCECs cultured without inhibitor exhibited monolayer morphology and varied in size and morphology. Flow cytometry demonstrated that cell proliferation was significantly increased by SB203580. Western blotting showed activation of ATF2 and HSP27 (substrates of p38 MAPK), and upregulation of cyclin D and downregulation of p27 were induced by inhibiting p38 MAPK. In the rabbit model, promotion of wound healing of the corneal endothelium was associated with significant upregulation of Ki67-positive proliferating cells following topical administration of SB203580 when compared with untreated endothelium (50.9% and 36.1%, respectively). Activation of p38 MAPK signaling due to culture stress might suppress the proliferation of HCECs, whereas a p38 MAPK inhibitor can counteract this activation and enable efficient in vitro HCEC expansion.

The presence of a feeder layer improves human corneal endothelial cell proliferation by altering the expression of the transcription factors Sp1 and NFI

Based on the use of tissue-cultured human corneal endothelial cells (HCECs), cell therapy is a very promising avenue in the treatment of corneal endothelial pathologies such as Fuchs' dystrophy, and post-surgical corneal edema. However, once in culture, HCECs rapidly lose their phenotypic and physiological characteristics, and are therefore unsuitable for the reconstruction of a functional endothelial monolayer. Expression of NFI, a transcription factor that can either function as an activator or a repressor of gene transcription, has never been examined in endothelial cells. The present study therefore aimed to determine the impact of a non-proliferating, lethally irradiated i3T3 feeder layer on the maintenance of HCEC's morphological characteristics, and both the expression and stability of Sp1 (a strong transcriptional activator) and NFI in such cells. The typical morphology of endothelial cells was best maintained when 8 × 103/cm2 HCECs were co-cultured in the presence of 2 × 104 cells/cm2 i3T3. HCECs were found to express both Sp1 and NFI in vitro. Also, the presence of i3T3 led to higher levels of Sp1 and NFI in HCECs, with a concomitant increase in their DNA binding levels (assessed by electrophoretic mobility shift assays (EMSA)). Specifically, i3T3 increased the expression of the NFIA, NFIB and NFIC isoforms, without a noticeable increase in their mRNAs (as revealed by gene profiling on microarray). Gene profiling analysis also identified a few feeder layer-dependent, differentially regulated genes whose protein products may contribute to improving the properties of HCECs in culture. Therefore, co-culturing HCECs with an i3T3 feeder layer clearly improves their morphological characteristics by maintaining stable levels of Sp1 and NFI in cell culture.

The ROCK inhibitor, thiazovivin, inhibits human corneal endothelial‑to‑mesenchymal transition/epithelial‑to‑mesenchymal transition and increases ionic transporter expression.

Corneal diseases exhibit a high prevalence and are prone to cause blindness; furthermore, maintaining the morphology and ionic transporter expression in corneal endothelial cells (CECs) is crucial for treatment of these diseases. This study aimed to investigate the effects of the novel Rho associated coiled-coil containing protein kinase (ROCK) inhibitor, thiazovivin (2,4-disubstituted thiazole, TZV), on human corneal endothelial-to-mesenchymal transition/epithelial-to-mesenchymal transition (EndMT/EMT), cell morphology, junction proteins and ionic transporter expression in human CECs (HCECs) in vitro and then to clarify the mechanisms of action of TZV. In the present study, primary HCECs were cultured in vitro and passaged. The expression levels of adhesion proteins (E-cadherin and N-cadherin), the EndMT/EMT marker, α smooth muscle actin (α-SMA), the tight junction protein, Zonula occludens-1 (ZO-1), and the ionic transporter, Na+/K+-ATPase, were detected by immunofluorescence. The proliferative ability of the HCECs was determined by CCK-8 assay. The mRNA expression of the EndMT/EMT-inducing gene, Snail, was examined by RT-PCR. The protein expression levels of ROCK1/2 were evaluated by western blot analysis. The HCECs were cultured with TZV at various concentrations (2, 4, or 6 µM) for different periods of time (24 or 48 h). We found that the the cell states of the HCECs co-cultured with 4 µM TZV for 48 h reached the optimum, and corneal EndMT/EMT was inhibited, as evidenced by the significantly upregulated expression of ZO-1 and E-cadherin, and the markedly downregulated expression of N-cadherin and α-SMA. Furthermore, the cells exhibited a normal, tightly connected hexagonal or pentagonal morphology. Additionally, the protein expression of ROCK1/2 and the mRNA expression of Snail were significantly decreased. However, there was no significant difference between the TZV-treated and the control groups as regards HCEC proliferative ability. These findings suggested that the ROCK inhibitor, TZV (4 µM), was effective in improving the morphology, cell junctions and ionic transporter expression of HCECs by inhibiting EndMT/EMT, but had no effect on HCEC proliferation.

Micro- and nano-topography to enhance proliferation and sustain functional markers of donor-derived primary human corneal endothelial cells

One of the most common indications for corneal transplantation is corneal endothelium dysfunction, which can lead to corneal blindness. Due to a worldwide donor cornea shortage, alternative treatments are needed, but the development of new treatment strategies relies on the successful in vitro culture of primary human corneal endothelial cells (HCECs) because transformed cell lines and animal-derived corneal endothelial cells are not desirable for therapeutic applications. Primary HCECs are non-proliferative in vivo and challenging to expand in vitro while maintaining their characteristic cell morphology and critical markers. Biochemical cues such as growth factors and small molecules have been investigated to enhance the expansion of HCECs with a limited increase in proliferation. In this study, patterned tissue culture polystyrene (TCPS) was shown to significantly enhance the expansion of HCECs. The proliferation of HCECs increased up to 2.9-fold, and the expression amount and localization of cell–cell tight junction protein Zona Occludens-1 (ZO-1) was significantly enhanced when grown on 1μm TCPS pillars. 250nm pillars induced an optimal hexagonal morphology of HCEC cells. Furthermore, we demonstrated that the topographical effect on tight-junction expression and cell morphology could be maintained throughout each passage, and was effectively ‘remembered’ by the cells. Higher amount of tight-junction protein expression was maintained at cell junctions when topographic cues were removed in the successive seeding. This topographic memory suggested topography-exposed/induced cells would maintain the enhanced functional markers, which would be useful in cell-therapy based approaches to enable the in situ endothelial cell monolayer formation upon delivery. The development of patterned TCPS culture platforms could significantly benefit those researching human corneal endothelial cell cultivation for cell therapy, and tissue engineering applications.

Corneal Endothelial Expansion Promoted by Human Bone Marrow Mesenchymal Stem Cell-Derived Conditioned Medium

Healthy corneal endothelium is essential for maintaining corneal clarity, as the damage of corneal endothelial cells and loss of cell count causes severe visual impairment. Corneal transplantation is currently the only therapy for severe corneal disorders. The greatly limited proliferative ability of human corneal endothelial cells (HCECs), even in vitro, has challenged researchers to establish efficient techniques for the cultivating HCECs, a pivotal issue for clinical applications. The aim of this study was to evaluate conditioned medium (CM) obtained from human bone marrow-derived mesenchymal stem cells (MSCs) (MSC-CM) for use as a consistent expansion protocol of HCECs. When HCECs were maintained in the presence of MSC-CM, cell morphology assumed a hexagonal shape similar to corneal endothelial cells in vivo, as opposed to the irregular cell shape observed in control cultures in the absence of MSC-CM. They also maintained the functional protein phenotypes; ZO-1 and Na+/K+-ATPase were localized at the intercellular adherent junctions and pump proteins of corneal endothelium were accordingly expressed. In comparison to the proliferative potential observed in the control cultures, HCECs maintained in MSC-CM were found to have more than twice as many Ki67-positive cells and a greatly increased incorporation of BrdU into DNA. MSC-CM further facilitated the cell migration of HCECs. Lastly, the mechanism of cell proliferation mediated by MSC-CM was investigated, and phosphorylation of Akt and ERK1/2 was observed in HCECs after exposure to MSC-CM. The inhibitor to PI 3-kinase maintained the level of p27Kip1 for up to 24 hours and greatly blocked the expression of cyclin D1 and D3 during the early G1 phase, leading to the reduction of cell density. These findings indicate that MSC-CM not only stimulates the proliferation of HCECs by regulating the G1 proteins of the cell cycle but also maintains the characteristic differentiated phenotypes necessary for the endothelial functions.

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.

In vitro evaluation of the interactions between human corneal endothelial cells and extracellular matrix proteins

The corneal endothelium is the innermost cell layer of the cornea and rests on Descemet's membrane consisting of various extracellular matrix (ECM) proteins which can directly affect the cellular behaviors such as cell adhesion, proliferation, polarity, morphogenesis and function. The objective of this study was to investigate the interactions between the ECM environment and human corneal endothelial cells (HCECs), with the ultimate goal to improve cell proliferation and function in vitro. To evaluate the interaction of HCECs with ECM proteins, cells were seeded on ECM-coated tissue culture dishes, including collagen type I (COL I), collagen type IV (COL IV), fibronectin (FN), FNC coating mix (FNC) and laminin (LM). Cell adhesion and proliferation of HCECs on each substratum and expression of CEC markers were studied. The results showed that HCECs plated on the COL I, COL IV, FN and FNC-coated plates had enhanced cell adhesion initially; the number for COL I, COL IV, FN and FNC was significantly higher than the control (P < 0.05). In addition, cells grown on ECM protein-coated dishes showed more compact cellular morphology and CEC marker expression compared to cells seeded on uncoated dishes. Collectively, our results suggest that an adequate ECM protein combination can provide a long-term culture environment for HCECs for corneal endothelium transplantation.

Involvement of P38MAPK in human corneal endothelial cell migration induced by TGF-β2

Because human corneal endothelial cells do not proliferate once the endothelial monolayer is formed, corneal wound healing is thought to be mediated by cell enlargement or migration rather than proliferation. However, the cellular mechanisms involved in corneal wound healing have not been fully determined. Because transforming growth factor-β2 (TGF-β2) isoform is present in high concentrations in normal human aqueous humor, it may play a role in human corneal endothelial cell wound healing. The purpose of this study was to determine the effect of TGF-β2 on the proliferation and migration of cultured human corneal endothelial cells (HCECs). To achieve this, we first examined the effect of TGF-β2 on the wound closure rate in an in vitro HCEC wound healing model. However, unexpectedly TGF-β2 had no effect on the wound closure rate in this model. Therefore, a real-time cell electronic sensing (RT-CES) system and the BrdU incorporation assay were used to determine the effect of TGF-β2 (0.1–10 ng/ml) on cultured HCEC proliferation during in vitro wound healing. The specificity of this effect was confirmed by adding the TGF-β receptor I kinase inhibitor. TGF-β2 inhibited the proliferation of HCECs in a dose dependent way and was blocked by TGF-β receptor I kinase inhibitor. Next, the Boyden chamber assay was used to determine how TGF-β2 (10 ng/ml) affect HCEC migration. Exposure to TGF-β2 increased cell migration, and a synergistic effect was observed when FGF-2 was added. To determine whether the mitogen-activated protein kinase (MAPK) signaling pathway is involved in the migration of HCECs, western blot analysis and Bio-Plex™ suspension array were used to detect phosphorylation of Erk1/2, p38, and JNK in HCECs stimulated by TGF-β2 and/or FGF-2. The effect of the p38 MAPK inhibitor, SB239063 (10 μM), on TGF-β2 and/or FGF-2-induced cellular migration was determined by the Boyden chamber assay. Both TGF-β2 and FGF-2-induced p38 phosphorylation, and a synergistic effect was observed with exposure to both growth factors. SB 239063 inhibited TGF-β2 and FGF-2-induced migration of HCECs. These results indicate that TGF-β2 reduces proliferation but stimulates migration of cultured HCECs. In addition, TGF-β2 and FGF-2 may have synergistic effects on the migration of HCECs mediated by p38 MAPK phosphorylation.

Regulation of functional corneal endothelial cells isolated from sphere colonies by Rho-associated protein kinase inhibitor

Human corneal endothelial cells (HCECs) form a monolayer covering the inner side of the cornea. Since HCECs have lower mitotic activity after birth, damage or disease of HCECs will lead to failure of cellular pump functions and inevitably to corneal stroma oedema and loss of vision. Ex vivo cultured HCEC transplantation is the most promising therapy used to avoid donor tissue shortages. However, proliferation of functional adult HCECs is difficult to achieve using standard cell culture techniques. In this study, we isolated sphere colonies from HCECs, which were characterised as HCEC precursor cells. HCECs from these spheres were incubated with the Rho-associated protein kinase (ROCK) inhibitor Y-27632 (30 μM for 1 h at 37°C) to observe cell pump functions and the levels of several cell markers. Cells from the spheres were immunostained positive for nestin (a marker of immature cells) and for an immature neuronal marker (β3-tubulin). There were no significant differences in the expression of genes associated with cell adhesion or ion transport channels, between the cells with or without Y-27632. However, immunostaining revealed a higher density of Na+/K+-ATPase in cell nuclei from HCECs (sphere-forming) and HCECs (sphere-forming/Y-27632) than normal HCECs. There was a higher resting potential difference and resting short circuit in the HCEC (sphere-forming) and HCEC (sphere-forming/Y-27632) cell lines, compared with normal HCECs. In this study, we successfully cultured functional HCEC cell lines, which possessed several important characteristics required to maintain the transparency and refractive parameters of the human cornea. These included hexagonal cell morphology, higher cell adhesion and proliferation ability, HCEC pump function and positive expression of several cell adhesion markers.

Increased Proliferation and Replicative Lifespan of Isolated Human Corneal Endothelial Cells withl-Ascorbic acid 2-phosphate

To explore an alternative culture method for human corneal endothelial cells (HCECs) and to examine the effect of l-ascorbic acid 2-phosphate (Asc-2P) on the growth of these cells. The influence of various mitogens, extracellular matrices (ECMs), and Asc-2P on growth of cultured HCECs was examined. HCECs were obtained from donors ranging in age from 12 to 74 years, and primary cultures and subcultures were performed with or without Asc-2P. Expanded HCECs were characterized with immunostaining and reverse transcription polymerase chain reaction (RT-PCR) and evaluated for generation of 8-hydroxy-2-deoxyguanosine (8-OHdG) with immunostaining and an enzyme-linked immunosorbent assay (ELISA). Culture with Asc-2P and bFGF on atelocollagen promoted the proliferation of HCECs in both primary cultures and subcultures as efficiently as conventional culture using ECM derived from bovine corneal endothelial cells. Zonula occludens-1, N-cadherin, connexin 43, and Na+/K+-ATPase were localized at plasma membranes of cultured HCECs. mRNAs of the voltage-dependent anion channels (VDAC2 and VDAC3), sodium bicarbonate cotransporter member 4 (SLC4A4), and chloride channel proteins (CLCN2 and CLCN3) were detected by RT-PCR. During multiple passages, cultures without Asc-2P showed a decrease in growth and irregular cell morphology, whereas cultures with Asc-2P sustained cell growth and maintained the characteristic polygonal morphology. ELISA for 8-OHdG showed that the levels in mitochondrial DNA significantly decreased when HCECs were subcultured with Asc-2P. Combination of Asc-2P and bFGF on atelocollagen allows successful culture for HCECs. Asc-2P extends the lifespan of cultured HCECs, partly due to protection against oxidative DNA damage.

Proliferative capacity of corneal endothelial cells

The corneal endothelial monolayer helps maintain corneal transparency through its barrier and ionic “pump” functions. This transparency function can become compromised, resulting in a critical loss in endothelial cell density (ECD), corneal edema, bullous keratopathy, and loss of visual acuity. Although penetrating keratoplasty and various forms of endothelial keratoplasty are capable of restoring corneal clarity, they can also have complications requiring re-grafting or other treatments. With the increasing worldwide shortage of donor corneas to be used for keratoplasty, there is a greater need to find new therapies to restore corneal clarity that is lost due to endothelial dysfunction. As a result, researchers have been exploring alternative approaches that could result in the in vivo induction of transient corneal endothelial cell division or the in vitro expansion of healthy endothelial cells for corneal bioengineering as treatments to increase ECD and restore visual acuity. This review presents current information regarding the ability of human corneal endothelial cells (HCEC) to divide as a basis for the development of new therapies. Information will be presented on the positive and negative regulation of the cell cycle as background for the studies to be discussed. Results of studies exploring the proliferative capacity of HCEC will be presented and specific conditions that affect the ability of HCEC to divide will be discussed. Methods that have been tested to induce transient proliferation of HCEC will also be presented. This review will discuss the effect of donor age and endothelial topography on relative proliferative capacity of HCEC, as well as explore the role of nuclear oxidative DNA damage in decreasing the relative proliferative capacity of HCEC. Finally, potential new research directions will be discussed that could take advantage of and/or improve the proliferative capacity of these physiologically important cells in order to develop new treatments to restore corneal clarity.

Adhesion, Migration, and Proliferation of Cultured Human Corneal Endothelial Cells by Laminin-5

To investigate the expression of laminin-5 (LM5) and its receptors by human corneal endothelial cells (HCECs) and whether recombinant human LM5 influences adhesion, proliferation, and migration of cultured HCECs. The expression of LM5 and its receptors was examined in human donor corneas by immunohistochemistry, reverse transcription-polymerase chain reaction, and flow cytometry. HCECs cultured under serum-free conditions were used for analysis of the biological effects of LM5. Changes in HCEC adhesion and proliferation due to LM5 were evaluated by counting the number of cells. HCEC migration was assessed by quantifying the percentage of wound closure in the wound-healing assay with an image-processing and -analysis software program. Adult HCECs expressed the LM5 receptor α3β1 integrin, but not LM5 itself. Significantly more cells became adherent to recombinant LM5 (1.0 μg/mL)-coated dishes than to uncoated dishes in the cell adhesion assay. The proliferation of cultured HCECs was moderately promoted by LM5 (1.0 μg/mL) and soluble LM5 (20 ng/mL and 50 ng/mL) in the cell proliferation assay. A significantly higher percentage of wound closure was obtained with medium containing soluble LM5 than with control medium in the wound-healing assay. HCECs express the LM5 receptor α3β1 integrin. Recombinant LM5 promotes adhesion, migration, and moderate proliferation of cultured HCECs. It may be a critical factor in promoting HCEC culture and may contribute to the practical use of tissue-engineered HCECs.

Lipid-mediated gene transfer of acidic fibroblast growth factor into human corneal endothelial cells

The aim of this study was to optimize non-viral gene transfer conditions and investigate the effect of fibroblast growth factor-1 (FGF-1) gene transfer on human corneal endothelial cell (HCEC) proliferation. Five non-viral vectors (Lipofectin™, DMRIE-C™, DAC-30, Effectene™, FuGene™6) were used to transfect HCEC with plasmids coding for enhanced green fluorescent protein (EGFP) and FGF-1. Transfection efficiency and toxicity ( n=6) were quantified and optimized using the EGFP construct by FACS-analysis. Using optimal conditions HCEC were transfected with the FGF-1 plasmid and cell proliferation as well as expression of FGF-1 were determined at days 4 and 7 by counting and western blotting, respectively. Lipofectin (17±2·02%) transfected HCEC more successfully than DMRIE-C (11±1·46%), Effectene (9±0·62%), FuGene (9±0·93%) and DAC-30 (7±0·59%). Toxicity of the lipids ranged from 2 to 4%. Optimal HCEC proliferation was achieved with DAC-30/FGF-1 ( P<0·05), whereas all other vectors did not result in significantly increased cell proliferation. However, all of the transfected cells produced FGF-1 in different amounts as indicated by western blotting. Efficient and almost non-toxic transfer of the FGF-1 gene into HCEC can be successfully achieved by lipid-based techniques. Using optimal conditions significantly increased cell proliferation was independent on gene transfer efficiency. This may indicate that even a low transfection rate is sufficient to produce a concentration of FGF-1 that will have a stimulatory effect on HCECs.

Evaluation of potential organ culture media for eye banking using a human corneal endothelial cell growth assay

To evaluate the ability of different commercially available cell culture media to induce proliferation and morphological changes in primary cultures of human corneal endothelial cells (HCEC). This screening model was used in an attempt to establish a rational basis for the development of well-defined, serum-free preservation media for long-term organ culture of human donor corneas. A total of 11 different culture media enriched with 0%, 2%, 5%, and 10% fetal calf serum (FCS) were compared. The test media were divided into three groups: Group 1: Media based on minimal essential medium (MEM), currently used for long-term corneal organ culture in European eye banks; Group 2: F99-based media, enriched for growth of corneal endothelial cells at serum-reduced conditions; and Group 3: Media designed for growth of special cell types or for short-term corneal organ culture. The growth-promoting capacity of each test medium was quantified using an HCEC proliferation assay, whereas changes in cell morphology were evaluated by phase-contrast microscopy. The morphological characteristics of HCEC were best maintained in the group of F99-based media, which also induced the highest level of cell proliferation under serum-reduced conditions. Specifically, the medium F99-Sr (F99 enriched with ascorbic acid, insulin, bFGF, transferrin, selenium, and lipids) induced a two- to three-fold higher HCEC density at both 0% and 2% FCS when compared to all other test media, and it also maintained the most endothelial cell-like morphology. Also, at higher serum concentrations (5% and 10% FCS), the cell growth was most prominent in F99-Sr, as well as in the medium SFM that originally was designed for serum-free growth of vascular endothelial cells. This study suggests that the media F99-Sr and SFM should be further tested and refined as potential new storage solutions for long-term corneal organ culture at physiological temperatures.

The Role of Exogenous/Endogenous Basic Fibroblast Growth Factor (FGF2) and Transforming Growth Factor β (TGFβ-1) on Human Corneal Endothelial Cells Proliferation in Vitro

Adult human corneal endothelial cells (HCEC) have extremely low turnover rates but undergo rapid division in vitro when stimulated with soluble growth factors. We have investigated the role played by FGF2 and TGFβ-1 in the regulation of HCEC growth stimulation. HCEC from donors who were over 30 years old were cultured and experiments performed on cultures between the 2nd and the 6th passage in the presence of 5% NCS. Cell counts revealed a maximal stimulation of 2.1 × for FGF2 and 1.9 × for TGFβ-1 compared to control cultures. When both factors were added, a synergistic effect was noticed with a maximal stimulation of the proliferation rate of 4.5 × over controls. In addition, endogenous FGF2 produced by HCEC was quantitated in a sensitive EIA assay. After 5 days in culture, 106 cells contained 150 ng FGF2 and 35 ng was extracted from trypsin-digested ECM. Two molar NaCl washes of ECM released 15.6 ng FGF2, which induced a slight mitogenic activity (1.5 × over control) in HCEC cultures, which was partially inhibited by an anti-FGF2 antibody. Northern blot analysis of HCEC extracts revealed the presence of FGF receptors R1 and R2 mRNA. The bioactive FGFRs were demonstrated by the toxic effect of a mitotoxin FGF2-SAP. These results suggest that FGF2 could participate in the autocrine regulation of HCEC proliferation and survival. The synergy between exogenously added FGF2 and TGFβ demonstrates that a combination of different growth factors may be important to stimulate proliferation of these cells in vivo.

Activation of human corneal endothelial cell proliferation in malignant tumors of various locations and in diabetes mellitus

Activation of human corneal endothelial cell proliferation in malignant tumors of various locations and in diabetes mellitus