• 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 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.

• 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.

• Journal of Korean Ophthalmic Optics Society
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• v.8 no.1
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• pp.65-71
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• 2003

The corneal epithelium is constantly being shed. The mechanism of corneal desquamation is not fully understood. Apoptosis, programmed cell death, may play a role. Apoptosis can be induced by a number of factors and different mechanisms. The study was performed to examine the apoptotic index induced in human corneal epithelial cells maintained in tissue culture by various apoptotic inducers. Various inducers, recombinant human cytokines($INF{\gamma}$, $TNF{\alpha}$, FASAb), actinomycin D. camptothecin, cycloheximide, dexamethasone and etoposide, were purchased from commercial suppliers. Inducers at manufacturer-recommended concentration were added to the corneal epithelial cells for 48 hours. Cell viability was measured using MTT assay. The cells were then assessed for the level of apoptosis. Morphologic changes and quantification of apoptotic cells were determined and counted under fluorescence microscope after inducers-treated human corneal epithelial (HCE) cells for 48 hours with Hoechst 33342 staining. Annexin V-FITC/PI staining and DePsipher assay. The expression of Fas protein was studied by immunocytochemistry. All inducers induced apoptosis in HCE cells in a dose dependent manner. Actinomycin D. camptothecin and etoposide induced apoptosis at lower than manufacturer-recommended concentration, while cytokines, cycloheximide and dexamethasone induced apoptosis at higher concentrations at the end of 48 hours. All inducers elicited typical apoptotic morphologic changes (chromatin condensation, nucleus fragmentations non-orange-red colored mitochondria) and expresses Fas protein highly. Apoptotic index of HCE cells by these inducers was different from the other cell lines. RNA synthesis inhibitor and topoisomerase inhibitors induced apoptosis at lower concentration than manufacturer-recommended concentration. Cytokines, cycloheximide and dexamethasone were able to produce apoptosis at 10 times higher concentrations. RNA synthesis inhibitor and topoisomerase inhibitors are more sensitive than intracellular receptor-activators in apoptotic induction of HCE cells.

• Parasites, Hosts and Diseases
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• v.57 no.3
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• pp.217-223
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• 2019

Acanthamoeba castellanii has ubiquitous distribution and causes primary acanthamoebic keratitis (AK). AK is a common disease in contact lens wearers and results in permanent visual impairment or blindness. In this study, we observed the cytopathic effect, in vitro cytotoxicity, and secretion pattern of cytokines in human corneal epithelial cells (HCECs) induced by A. castellanii trophozoites and/or cysts. Morphological observation revealed that panked dendritic HCECs co-cultured with amoeba cysts had changed into round shape and gradually died. Such changes were more severe in co-culture with cyst than those of co-cultivation with trophozoites. In vitro cytotoxicity assay revealed the highest cytotoxicity to HCECs in the co-culture system with amoeba cysts. A. castellanii induced the expression of $IL-1{\alpha}$, IL-6, IL-8, and CXCL1 in HCECs. Secreted levels of $IL-1{\alpha}$, IL-6, and IL-8 in HCECs co-cultured with both trophozoites and cysts were increased at an early incubation time (3 and 6 hr). These results suggested that cytopathic changes and pro-inflammatory cytokines release of HCECs in response to A. castellanii, especially amoebic cysts, are an important mechanism for AK development.

• The Korean Journal of Vision Science
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• v.20 no.4
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• pp.531-541
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• 2018

Purpose : To evaluate the acute cytotoxic effect of polyhexamethylene biguanide (PHMB), epigallocatechin gallate (EGCG) and PHMB/EGCG mixture on the cultured human corneal epithelial cell (HCEpiC). Methods : HCEpiCs were cultured in the media of HCEpiC containing 0.00001~0.005% PHMB, 0.001~5% EGCG and 0.00005% PHMB/0.05% EGCG mixture respectively for 30, 60, 120 and 240 min. Cultured HCEpiCs were fixed and stained with Draq5 and cell viability and apoptosis were evaluated using confocal microscope and ImageXpress $Ultra^{TM}$. Results : Cultured HCEpiC did not show cytotoxic effect at below 0.00005% PHMB and below 0.05% EGCG concentration. In the media containing 0.00005% PHMB/0.05% EGCG, acute cytototoxic effect was not found, whereas damaged HCEpiCS were increased and survival cells were decreased in the media incubated for 240 min. Conclusion : The mixture of 0.00005% PHMB/0.05% EGCG showed non acute cytotoxic effect on the cultured HCEpiCs, however it is needed to investigate its chronic cytotoxic effect.

• Journal of Korean Ophthalmic Optics Society
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• v.5 no.1
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• pp.83-87
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• 2000

To investigate exhausted-medium-induced apoptosis in human corneal epithelial(HCE) cells, this study was performed DNA gel electrophoresis, M30 CytoDEATH staining and FAS-FAS ligand ELISA. SV-40 transfected cells were grown to confluency in culture for 7days. The supernatant was harvested and filtered with $0.22{\mu}m$ filter paper. Fresh HCE cells were exposed to the filtered exhausted medium for 1~2 days. Apoptotic cells were prepared for DNA extraction and run the agarose gel for DNA ladder pattern. M30 CytoDEATH was used a tool for easy and reliable determination of very early apoptosis in HCE cells. The control and exhausted medium were assayed for soluble FAS/FAS ligand protein by ELISA. HCE cells exposed to exhausted medium showed a typical DNA ladder pattern. Sporadic M30 CytoDEATH positive cells were detected among HCE cells exposed to exhausted medium. Soluble FAS/FAS ligand levels were not elevated in the exhausted medium compared to the fresh medium control. This study suggests that possible mechanism of exhausted medium induced apoptosis does not include the FAS-FAS ligand system.

• Biomolecules & Therapeutics
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• v.23 no.4
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• pp.379-385
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• 2015

The eye irritation potential of drug candidates or pharmaceutical ingredients should be evaluated if there is a possibility of ocular exposure. Traditionally, the ocular irritation has been evaluated by the rabbit Draize test. However, rabbit eyes are more sensitive to irritants than human eyes, therefore substantial level of false positives are unavoidable. To resolve this species difference, several three-dimensional human corneal epithelial (HCE) models have been developed as alternative eye irritation test methods. Recently, we introduced a new HCE model, MCTT HCE$^{TM}$ which is reconstructed with non-transformed human corneal cells from limbal tissues. Here, we examined if MCTT HCE$^{TM}$ can be employed to evaluate eye irritation potential of solid substances. Through optimization of washing method and exposure time, treatment time was established as 10 min and washing procedure was set up as 4 times of washing with 10 mL of PBS and shaking in 30 mL of PBS in a beaker. With the established eye irritation test protocol, 11 solid substances (5 non-irritants, 6 irritants) were evaluated which demonstrated an excellent predictive capacity (100% accuracy, 100% specificity and 100% sensitivity). We also compared the performance of our test method with rabbit Draize test results and in vitro cytotoxicity test with 2D human corneal epithelial cell lines.