• Journal of Embryo Transfer
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• v.33 no.2
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• pp.85-97
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• 2018

The trans-differentiation potential of mesenchymal stem cells (MSCs) is employed, but there is little understanding of the cell source-dependent trans-differentiation potential of MSCs into corneal epithelial cells. In the present study, we induced trans-differentiation of MSCs derived from umbilical cord matrix (UCM-MSCs) and from dental tissue (D-MSCs), and we comparatively evaluated the in vitro trans-differentiation properties of both MSCs into corneal epithelial-like cells. Specific cell surface markers of MSC (CD44, CD73, CD90, and CD105) were detected in both UCM-MSCs and D-MSCs, but MHCII and CD119 were significantly lower (P < 0.05) in UCM-MSCs than in D-MSCs. In UCM-MSCs, not only expression levels of Oct3/4 and Nanog but also proliferation ability were significantly higher (P < 0.05) than in D-MSCs. In vitro differentiation abilities into adipocytes and osteocytes were confirmed for both MSCs. UCM-MSCs and D-MSCs were successfully trans-differentiated into corneal epithelial cells, and expression of lineage-specific markers (Cytokeratin-3, -8, and -12) were confirmed in both MSCs using immunofluorescence staining and qRT-PCR analysis. In particular, the differentiation capacity of UCM-MSCs into corneal epithelial cells was significantly higher (P < 0.05) than that of D-MSCs. In conclusion, UCM-MSCs have higher differentiation potential into corneal epithelial-like cells and have lower expression of CD119 and MHC class II than D-MSCs, which makes them a better source for the treatment of corneal opacity.

• Journal of Life Science
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• v.32 no.9
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• pp.712-720
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• 2022

The purpose of this study was to investigate the efficacy of rat corneal-derived epithelial cells as an in vitro model to evaluate the harmfulness of the cornea caused by particulate matter 2.5 (PM_2.5). To establish an experimental model for the effect of PM_2.5 on corneal epithelial cells, it was confirmed that primary cultured cells isolated from rat eyes were corneal epithelial cells through pan-cytokeratin staining. Our results showed that PM_2.5 treatment reduced cell viability of primary rat corneal epithelial (RCE) cells, which was associated with the induction of apoptosis. PM_2.5 treatment also increased the generation of reactive oxygen species due to mitochondrial dysfunction. In addition, the production of nitric oxide and inflammatory cytokines was increased in PM_2.5-treated RCE cells. Furthermore, through heatmap analysis showing various expression profiling between PM_2.5-exposed and unexposed RCE cells, we proposed five genes, including BLNK, IL-1RA, Itga2b, ABCb1a and Ptgs2, as potential targets for clinical treatment of PM-related ocular diseases. These findings indicate that the primary RCE cell line is a useful in vitro model system for the study of PM_2.5-mediated pathological mechanisms and that PM2.5-induced oxidative and inflammatory responses are key factors in PM2.5-induced ocular surface disorders.

• Journal of Korean Ophthalmic Optics Society
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• v.4 no.1
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• pp.1-6
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• 1999

The corneal epithelium is constantly shed and apoptosis may play an important role in this turn-over. We sought to define that serum-free medium was able to induce apoptosis of corneal epithelial cells. SV-40 transfected human corneal epithelial(HCE) cells were grown to 70% confluency in culture. Serum-free medium was added to cells and the cells incubated for 1, 2, 3, or 6 days. Apoptosis of cells at different times was assessed by staining cells with Giemsa or Hoechst 33342 and measuring DNA fragmentation using the TUNEL assay. HCE cells exposed to serum-free medium demonstrated a high incidence of apoptosis, which increased over time to $50{\pm}4%$ after 3 days. They also stained positively with TUNEL assay. Serum-free medium caused time dependent apoptosis of HCE cells. Thus, serum-like nutrient might be important in corneal epithelial cell homeostasis.

• Journal of Veterinary Science
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• v.23 no.4
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• pp.62.1-62.10
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• 2022

Background: The corneal and limbal morphology relevant to corneal epithelial maintenance in ten different species was examined using histological methods. Objectives: The presence of a Bowman's layer, limbal epithelial cell, and superficial stromal morphology was examined in the following species to evaluate the differences in corneal thickness and epithelium: Java sparrows, frogs, macaws, spoonbills, red pandas, penguins, horses, Dobermans, orangutans, and humans. Methods: Corneal sections (4 ㎛) were obtained from ten ocular globes from three different animal classes: Aves, Amphibia, and Mammalia. All sections were stained with hematoxylin and eosin and periodic acid-Schiff reaction. After microscopy, all stained slides were photographed and analyzed. Results: Significant morphological differences in the corneal and limbal epithelia and their underlying stroma between species were observed. The number of corneal epithelial cell layers and the overall corneal epithelial thickness varied significantly among the species. The presence of a Bowman's layer was only observed in primates (orangutans and humans). Presumed supranuclear melanin caps were noted in four species (orangutans, macaws, red pandas, and horses) in the limbal basal epithelial layer (putative site of corneal epithelial stem cells). The melanin granules covered the apex of the cell nucleus. Conclusions: Supranuclear melanin capping has been described as a process within the epidermis to reduce the concentration of ultraviolet-induced DNA photoproducts. Similarly, there may be a relationship between limbal stem cell melanin capping as a protective mechanism against ultra-violet radiation.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.3
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• pp.262-269
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• 2005

Many researchers have employed cryopreserved amniotic membrane (CAM) in the treatment of a severely damaged cornea, using corneal epithelial cells cultured on an amniotic membrane (AM). In this study, two Teflon rings were made for culturing the cells on the LAM and CAM, and were then used to support the AM, which is referred to in this paper as an Ahn's AM supporter. The primary corneal epithelial cells were obtained from the limbus, using an ex-plantation method. The corneal epithelium could be reconstructed by culturing the third­passage corneal epithelial cells on the AM. A lyophilized amniotic membrane (LAM) has a higher rate of graft take, a longer shelf life, is easier to store, and safer, due to gamma irradiation, than a (AM. The corneal epithelium reconstructed on the LAM and (AM, supported by the two­Teflon rings, was similar to normal corneal epithelium. However, the advantages of the LAM over that of the (AM make the former more useful. The reconstruction model of the corneal epithelium, using AM, is considered as a good in vitro model for transplantation of cornel epithelium into patients with a severely damaged cornea.

• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.289-292
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• 2002

The corneal tissue consists of three layers : epithelium, stroma, and endothelium. Central cornea is a highly differentiated tissue whereas the limbus contains the epithelial stem cell. In the present study. we report the engineering of the three-dimensional reconstructed cornea derived from rabbit limbal epithelial and stromal cells. The differentiation degree of corneal stem cells were assessed in serum concentration and inoculation density of stromal cells. Optimal condition differentiation of corneal stem cells is achieved when 5% FBS was supplemented to culture medium and $1-2{\times}10^5$ cells/ml inoculation density of stromal cells.

• Parasites, Hosts and Diseases
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• v.54 no.6
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• pp.697-702
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• 2016

Acanthamoeba keratitis has been increasing in recent years. Main risk factors are contact lens wear and their cleaning solutions. Most contact lens wearers use multipurpose disinfecting solutions (MPDS) for cleansing and disinfecting microorganisms because of its convenience. We determined amoebicidal effects of MPDS made in Korea and their cytotoxicity on human corneal epithelium cells. Fifteen commercial MPDS (A to O) were tested for their amoebicidal effects on Acanthamoeba castellanii trophozoites and cysts by using a most probable number (MPN) technique. Among them, 7 kinds of MPDS showed little or no amoebicidal effects for 24 hr exposure. Solutions A, B, G, H, L, and O showed positive amoebicidal effects, and solutions M and N killed almost all trophozoites and cysts after 24 hr exposure. However, 50%-N solution showed 56% cytotoxicity on human corneal epithelial cells within 4 hr exposure, and 50%-O solution also showed 62% cytotoxicity on human cells within 4 hr exposure. Solution A did not show any cytotoxicity on human cells. These results revealed that most MPDS made in Korea were ineffective to kill Acanthamoeba. The solutions having amoebicidal activity also showed high levels of cytotoxicity on human corneal epithelial cells. New formulations for improved MPDS that are amoebicidal but safe for host cells are needed to prevent Acanthamoeba keratitis.

• The Korean Journal of Physiology and Pharmacology
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• v.18 no.2
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• pp.89-94
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• 2014

DA-6034, a eupatilin derivative of flavonoid, has shown potent effects on the protection of gastric mucosa and induced the increases in fluid and glycoprotein secretion in human and rat corneal and conjunctival cells, suggesting that it might be considered as a drug for the treatment of dry eye. However, whether DA-6034 induces $Ca^{2+}$ signaling and its underlying mechanism in epithelial cells are not known. In the present study, we investigated the mechanism for actions of DA-6034 in $Ca^{2+}$ signaling pathways of the epithelial cells (conjunctival and corneal cells) from human donor eyes and mouse salivary gland epithelial cells. DA-6034 activated $Ca^{2+}$-activated $Cl^-$ channels (CaCCs) and increased intracellular calcium concentrations ($[Ca^{2+}]_i$) in primary cultured human conjunctival cells. DA-6034 also increased $[Ca^{2+}]_i$ in mouse salivary gland cells and human corneal epithelial cells. $[Ca^{2+}]_i$ increase of DA-6034 was dependent on the $Ca^{2+}$ entry from extracellular and $Ca^{2+}$ release from internal $Ca^{2+}$ stores. Interestingly, these effects of DA-6034 were related to ryanodine receptors (RyRs) but not phospholipase C/inositol 1,4,5-triphosphate ($IP_3$) pathway and lysosomal $Ca^{2+}$ stores. These results suggest that DA-6034 induces $Ca^{2+}$ signaling via extracellular $Ca^{2+}$ entry and RyRs-sensitive $Ca^{2+}$ release from internal $Ca^{2+}$ stores in epithelial cells.

• The Korean Journal of Physiology and Pharmacology
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• v.21 no.2
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• pp.189-195
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• 2017

This study aimed to compare the cellular toxicities of three clinically used dry eye treatments; 3% diquafosol tetrasodium and hyaluronic acid at 0.3 and 0.18%. A methyl thiazolyltetrazoiun (MTT)-based calorimetric assay was used to assess cellular proliferation and a lactate dehydrogenase (LDH) leakage assay to assess cytotoxicity, using Human corneal epithelial cells (HCECs) exposed to 3% diquafosol tetrasodium, 0.3% hyaluronic acid (HA), or 0.18% HA or 1, 6 or 24 h. Cellular morphology was evaluated by inverted phase-contrast light microscopy and electron microscopy, and wound widths were measured 24 h after confluent HCECs were scratched. Diquafosol had a significant, time-dependent, inhibitory effect on HCEC proliferation and cytotoxicity. HCECs treated with diquafosol detached more from the bottoms of dishes and damaged cells showed degenerative changes, such as, reduced numbers of microvilli, vacuole formation, and chromatin of the nuclear remnant condensed along the nuclear periphery. All significantly stimulated reepithelialization of HCECs scratched, which were less observed in diquafosol. Therefore, epithelial toxicity should be considered after long-term usage of diquafosol and in overdose cases, especially in dry eye patients with pre-existing punctated epithelial erosion.

• Korean Journal of Optics and Photonics
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• v.34 no.2
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• pp.45-52
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• 2023

In this study, we have investigated the effect of photobiomodulation (PBM) on corneal wound healing, using a low-power light-emitting diode (LED) at different wavelengths. We found that LEDs with wavelengths ranging from 623 to 940 nm had no significant cytotoxic effects on corneal epithelial cells. The effect of PBM on promoting cell migration was analyzed by scratch assay, and it was found that PBM at 623 nm significantly increased cell migration and promoted wound healing. Furthermore, the expression of genes related to cell migration and wound healing was analyzed, and it was found that PBM at 623 nm upregulated the expression of the genes FGF-1 and MMP2, which are known to promote cell proliferation and extracellular matrix degradation. These findings suggest that PBM with low-powered light at specific wavelengths, particularly 623 nm, could be utilized to treat corneal injury.