Establishment of an untransfected human corneal epithelial cell line and its biocompatibility with denuded amniotic membrane

AIM: To establish an untransfected human corneal epithelial (HCEP) cell line and characterize its biocompatibility with denuded amniotic membrane (dAM). METHODS: The torn HCEP pieces were primarily cultured in DMEM/F12 media (pH 7.2) supplemented with 20% fetal bovine serum and other necessary factors, yielding an HCEP cell line which was its growth performance, chromosome morphology, tumorigenicity and expression of marker proteins analyzed. In addition, the biocompatibility of HCEP cells with dAM was evaluated through histological and immunocytochemistry analyses and with light, electron and slit-lamp microscopies. RESULTS: HCEP cells proliferated to confluence in 3 weeks, which have been subcultured to passage 160. A continuous untransfected HCEP cell line, designated as utHCEPC01, was established with a population doubling time of 45.42 hours as was determined at passage 100. The cells retained HCEP cell properties as were approved by chromosomal morphology and the expression of keratin 3. They, with no tumorigenicity, formed a multilayer epithelium-like structure on dAMs through proliferation and differentiation during air-liquid interface culture, maintained expression of marker proteins including keratin 3 and integrin p 1 and attached tightly to dAMs. The reconstructed HCEP was highly transparent and morphologically and structurally similar to the original. CONCLUSION: An untransfected and non-tumorigenic HCEP cell line was established in this study. The cells maintained expression of marker proteins. The cell line was biocompatible with dAM. It holds the potential of being used for in vitro reconstruction of tissue-engineered HCEP, promising for the treatment of diseases caused by corneal epithelial disorders.

The roles of surfactant protein D during Aspergillus fumigatus infection in human corneal epithelial cells

AIM: To investigate roles of surfactant protein D (SP-D) and relative cytokines in human corneal epithelial (HCE) cells Exposed to aspergillus fumigatus (AF) antigens. METHODS: HCE cells cultured 47 in vitro with AF antigens and sampled at 0, 0.5, 1 hour, 2, 4, 6 and 8 hours. The Expression of SP-D mRNA was evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR).The expression of SP-D protein was shown by ELISA and immunocytochemistry SP methods. The expression of NF-kappa B and relative downstream cytokines such as TNF-alpha, IL-1 beta, IL-8 and IL-10 in supernatant fluid were measured by ELISA. RESULTS: SP-D mRNA and protein were detected in untreated HCE cells. The expression of SP-D and the relative downstream cytokines rose after being stimulated with AF antigens. SP-D mRNA began to rise at 0.5 hour and the most significantly peak was in 2 hours. The protein of SP-D in supernatant fluid had the same trend with mRNA. Immunocytochemistry of SP-D showed positive expression and gradually increased to 6 hours, and then the expression began to decline. NF-kappa B was activated after treated by AF antigens and the changes had correlation with SP-D. TNF-alpha, IL-1 beta, IL-8 and IL-10 began to rise after given AF antigens 1 hour and were 1.82, 1.43, 1.12 and 1.28 times higher than the untreated HCE cells separately. The expression of TNF-alpha and IL-1 beta reached the peak at 2 hours, separately 2.80 and 2.86 times than the untreated. The expression of IL-8 and IL-10 gradually increased with a time-dependent manner. ' CONCLUSION: HCE cells exists SP-D and it may play a significant role in pathogenesis of keratomycosis. AF may induce human corneal epithelial cells to express inflammatory cytokines via SP-D and NF-kappa B pathway. SP-D possibly mediates the recognition to AF mycelium.

Acellular porcine corneal matrix as a carrier scaffold for cultivating human corneal epithelial cells and fibroblasts in vitro

AIM：To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts,and an acellular porcine cornea matrix（APCM） in vitro.·METHODS：The scaffold was prepared from fresh porcine corneas which were treated with 0.5%sodium dodecyl sulfate（SDS）solution and the complete removal of corneal cells was confirmed by hematoxylin-eosin（HE）staining and 4’,6-diamidino-2-phenylindole（DAPI）staining.Human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM,and then cell proliferative ability was evaluated by MTT assay.To construct a human corneal anterior lamellar replacement,corneal fibroblasts were injected into the APCM and cultured for 3d,followed by culturing corneal epithelial cells on the stroma construction surface for another 10d.The corneal replacement was analyzed by HE staining,and immunofluorescence staining.·R ESULTS：Histological examination indicated that there were no cells in the APCM by HE staining,and DAPI staining did not detect any residual DNA.The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells.At 10d,a continuous 3 to 5 layers of human corneal epithelial cells covering the surface of the APCM was observed,and the injected corneal fibroblasts distributed within the scaffold.The phenotype of the construction was similar to normal human corneas,with high expression of cytokeratin 12 in the epithelial cell layer and high expression of Vimentin in the stroma.·CONCLUSION：Corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix.This laid the foundation for the further transplantation in vitro.

Expression of dectin-1 during fungus infection in human corneal epithelial cells

AIM: To evaluate the expression of dendritic cell-associated C-type lectin-1(dectin-1) in human corneal epithelial(HCE) cells infected by fungus. · METHODS: A total of 20 cases of healthy donor corneas were group A,and 20 patients(20 eyes) suffered from fungal keratitis(FK) composed group B. Real-time qPCR and immunohistochemistry were applied to detect dectin-1 expression in corneal epithelium of both groups. HCE cells were cultured with aspergillus fumigatus(AF) antigens in vitro. The expression of dectin-1 mRNA was measured by real-time qPCR at the stimulation of 0,4,8 and 24h separately. Dectin-1 protein was detected by immunocytochemistry at 0 and 24h separately. ·RESULTS: Dectin-1 expressed in corneal epithelium of normal persons and FK patients. Vitro cellular experiment showed that the expression of dectin-1 mRNA in HCE cells began to increase after stimulation of AF antigens at 4h,and dectin-1 protein expression increased after stimulation at 24h. · CONCLUSION: Dectin-1 expressed in corneal epithelium of normal persons. AF antigens stimulation can elevate the expression of dectin-1 in HCE cells in vitro.

In vitro reconstruction and characterization of tissue-engineered human corneal epithelium with seeder cells from an untransfected human corneal epithelial cell line

AIM: To demonstrate the morphology and structure of in vitro reconstructed tissue-engineered human corneal epithelium (TE-HCEP) with seeder cells from an untransfected HCEP cell line. METHODS: The TE-HCEPs were reconstructed in vitro with seeder cells from an untransfected HCEP cell line, and scaffold carriers of denuded amniotic membrane (dAM) in air-liquid interface culture for 3, 5, 7 and 9 days, respectively. The specimens were examined with hematoxylin-eosin (HE) staining of paraffin-section, immunocytochemical staining, scanning and transmission electron microscopy. RESULTS: During in vitro reconstruction of TE-HCEP, HCEP cells formed a 3-4, 6-7 and 8-10 layers of an HCEP-like structure on dAMs in air-liquid interface culture for 3, 5 and 7 days, respectively. But the cells deceased to 5-6 layers and the structure of straified epithelium became loose at day 9. And the cells maintained positive expression of marker proteins (keratin 3 and keratin 12), cell-junction proteins (zonula occludens-1, E-cadherin, connexin 43 and integrin beta 1) and membrane transport protein of Na+-K+ ATPase. The HCEP cells in TE-HCEP were rich in microvilli on apical surface and established numerous cell-cell and cell-dAM junctions at day 5. CONCLUSION: The morphology and structure of the reconstructed TE-HCEP were similar to those of HCEP in vivo. The HCEP cells in the reconstructed TE-HCEP maintained the properties of HCEP cells, including abilities of forming intercellular and cell-extracellular matrix junctions and abilities of performing membrane transportation. The untransfected HCEP cells and dAMs could promisingly be used in reconstruction HCEP equivalent for clinical corneal epithelium transplantation.

Co-regulation of Dectin-1 and TLR2 in inflammatory response of human corneal epithelial cells induced by Aspergillus fumigates

AIM： To investigate the co-regulation of dendritic cell- associated C-type lectin-1 （Dectin-1）, Toll-like receptor 2 （TLR2）, and relative chemotactic factors in the Telomease- immortalized human corneal epithelial （THCE） cells after exposure to ,Aspergillus fumigatus （Af） hyphae. METHODS： The normal THCE cells were investigated as control. After cultured in vitro with Af hyphae, with or without laminarin and anti-TLR2 antibody for 4, 8, 16 and 24h, THCE cells were harvested. The expression of Dectin-1, TLR2, CXCL1 and CXCL8 mRNA were measured by real-time quantitative polymerase chain reaction at the stimulation of 4, 8 and 16h separately. The protein expression of Dectin-1 and TLR2 were analyzed at 8, 16, and 24h by Western blot. ~ RESULTS： The mRNA CXCL8 increased in THCE expression of CXCL1 and cells after stimulated by Af hyphae. The stimulatory effects on these inflammatory chemokines were shown in a dose-dependent manner and reached the peak at 8h. Af hyphae significantly stimulated the production of Dectin-1 and TLR2 in THCE cells at both mRNA and protein levels. The protein of Dectin-1 and TLR2 gradually increased till 16h. While pretreated with laminarin （a Dectin-1 inhibitor）, the expression of TLR2, CXCL1 and CXCL8 all decreased dramatically at the peak point, interestingly, when pretreated with TLR2 neutralizing antibody, the expression of Dectin -1, CXCL1 and CXCL8 also decreased dramatically at the peak point. CONCLUSION： These findings suggest that Dectin-1 and TLR2 co-regulated with each other after treated with inactive Af hyphae in the THCE cells, and they contribute together to the inflammatory responses by induction of chemokines CXCL1 and CXCL8.

Effects of different concentrations of tetramethylpyrazine,an active constituent of Chinese herb, on human corneal epithelial cell damaged by hydrogen peroxide

AIM: To discuss the effects of different concentrations of tetramethylpyrazine(TMP), an active constituent of Chinese herb, on damaged Shandong human corneal epithelial cell(SDHCEC) induced by hydrogen peroxide.METHODS: We detected the combined effects of TMP with concentrations ranging from 4 mg/m L to 0.03 mg/m L and 800 μM hydrogen peroxide on SDHCEC. The methyl thiazolyl tetrazolium(MTT) assay was processed at 3, 6and 12 h separately while the detection of cell apoptosis at 6h only by flow cytometry.RESULTS: The viability of SDHCEC with 0.5 mg/m L,0.25 mg/m L, 0.125 mg/m L and 0.06 mg/m L TMP joint with800 μM hydrogen peroxide at 3h and 6h was significantly higher than that with 800 μM hydrogen peroxide only, P 【0.05. However, except 0.25 mg/m L, TMP with other concentrations joint with 800 μM hydrogen peroxide at12 h could not significantly inhibit decreased SDHCEC viability induced by 800 μM hydrogen peroxide. At 12 h,TMP of 0.5 mg/m L, 0.25 mg/m L, 0.125 mg/m L and 0.06 mg/m L could significantly inhibit SDHCEC early apoptosis induced by 800 μM hydrogen peroxide, most remarkable at 0.25 mg/m L TMP, P 【0.05.CONCLUSION: Our results suggested that hydrogen peroxide can induce apoptosis related damage to SDHCEC. TMP can protect SDHCEC from the damage,and the protective effects may be associated with its anti-apoptosis mechanism.

The role of Dectin-1/Raf-1 signal cascade in innate immune of human corneal epithelial cells against Aspergillus fumigatus infection

AIM： To investigate the expression of the v-raf-1murine leukemia viral oncogene homolog 1（Raf-1） and its role in the innate immune response of human corneal epithelial cells（HCECs） infected by Aspergillus fumigatus.METHODS： HCECs were cultured in vitro.They were randomly divided into 4 groups,including control group,Aspergillus fumigatus group,GW5074（an inhibitor of Raf-1） group and Laminarin [an inhibitor of Dendriti-cell-associated C-type lectin 1（Dectin-1）] group.The protein expression level of total Raf-1 and p-Raf-1 was measured by Western blot.The expression of IL-6 and IL-8 m RNA in each group was detected by real-time polymerase chain reaction.RESULTS： In Aspergillus fumigatus group,total Raf-1 protein levels in HCECs remained unchanged at 5,15,30 and 45min after infection,while p-Raf-1 expression was significantly enhanced at 30 min after infection compared with control group.However,the expression of p-Raf-1 was apparently declined after treated with GW5074 or Laminarin compared with Aspergillus fumigatus group.The expression levels of IL-6,IL-8 m RNA were significantly increased after stimulation with fumigatus compared with control group.Pre-treated with GW5074 significantly inhibited Aspergillus fumigatus-induced upregulation of IL-8 and IL-6.CONCLUSION： Aspergillus fumigatus stimulation can elevate the expression of p-Raf-1 in HCECs in vitro.Dectin-1/Raf-1 signal pathway may play a role on regulating the expression of inflammatory cytokines,including IL-6 and IL-8.

Effects of epidermal growth factor on transforming growth factor-beta1-induced epithelial-mesenchymal transition and potential mechanism in human corneal epithelial cells

AIM: To evaluate the effects of epidermal growth factor(EGF) on transforming growth factor-beta1(TGF-β1)-induced epithelial-mesenchymal transition(EMT) in human corneal epithelial cells(HCECs). METHODS: HCECs were cultured and treated with TGF-β1 for establishing the model of EMT in vitro. Biological effect of EGF on TGF-β1-induced EMT was evaluated. Proteins and m RNAs expression changes of E-cadherin, N-cadherin and Fibronectin(EMT-relative markers) after TGF-β1 or TGF-β1 combined EGF treatment were detected by Western blot and RT-PCR, respectively. Viability and migration of HCECs were measured by CCK-8, transwell cell migration assay and cell scratch wound healing assay. Activation of Smad2, ERK, p38, JNK and Akt signaling pathways were evaluated by Western blot. Inhibitors of relevant signaling pathways were added to the HCECs to explore the key signal mechanism.RESULTS: With treatment of TGF-β1 only, three EMTrelative proteins and m RNA expression showed that EMT up-regulated in a concentration-dependent and time-dependent manner, with significantly decreasing cell viability(TGF-β1≥5 ng/m L, P<0.05) and increasing cell migration(TGF-β1≥5 ng/m L, P<0.01). The phosphorylation of Smad2 and p38 was a key process of TGF-β1-induced EMT. Meanwhile, EMT-relative proteins and m RNA expression showed that EGF inhibited TGF-β1-indued EMT, with significantly increasing cell viability(EGF≥10 ng/m L, P<0.01). It was noteworthy that EGF significantly enhanced cell migration although EMT was inhibited(EGF≥10 ng/m L, P<0.01), and the blockage of p38(by SB202190, a p38 inhibitor) was a potential mechanism of this phenomenon. CONCLUSION: EGF inhibits TGF-β1-induced EMT via suppressive p38, and promotes cells proliferation and migration in a non-EMT process by inhibiting p38 pathway.

Comparison of cytotoxicity and wound healing effect of carboxymethylcellulose and hyaluronic acid on human corneal epithelial cells

AIM: To investigate the cytotoxic effect on human corneal epithelial cells(HCECs) and the ability to faciliate corneal epithelial wound healing of carboxymethylcellulose(CMC) and hyaluronic acid(HA).METHODS: HCECs were exposed to 0.5% CMC(Refresh plus, Allergan, Irvine, California, USA) and 0.1% and 0.3%HA(Kynex , Alcon, Seoul, Korea, and Hyalein mini,Santen, Osaka, Japan) for the period of 30 min, and 4, 12,and 24 h. Methyl thiazolyl tetrazoiun(MTT)-based calorimetric assay was performed to assess the metabolic activity of cellular proliferation and lactate dehydrogenase(LDH) leakage assay to assess the cytotoxicity. apoptotic response was evaluated with flow cytometric analysis and fluorescence staining with Annexin V and propiodium iodide. Cellular morphology was evaluated by inverted phase-contrast light microscopy and electron microscopy. The wound widths were measured 24 h after confluent HCECs were scratch wounded.RESULTS: The inhibitory effect of human corneal epithelial proliferation and cytotoxicity showed the time-dependent response but no significant effect. Apoptosis developed in flow cytometry and apoptotic cells weredemonstrated in fluorescent micrograph. The damaged HCECs were detached from the bottom of the dish and showed the well-developed vacuole formations. Both CMC and HA stimulated reepithehlialization of HCECs scratched, which were more observed in CMC.CONCLUSION: CMC and HA, used in artificial tear formulation, could be utilized without any significant toxic effect on HCECs. Both significantly stimulated HCEC reepithelialization of corneal wounds.

Construction of eukaryotic plasmid expressing human TGFBI and its influence on human corneal epithelial cells

AIM: To detect the expression of transforming growth factor beta-induced gene (TGFBI) protein in human corneal tissue and overexpress it in the human corneal epithelial cells in order to discuss the function of TGFBI in the pathogenesis of corneal dystrophy. METHODS: Immunohistochemistry (IHC) was used to detect the expression of TGFBI in the human cornea tissue. TGFBI cDNA was obtained by reverse transcription-PCR from human corneal total RNA extracted from cornea transplant donor and cloned into pCMV-N-HA vector. The recombinant pCMV-N-HA-TGFBI plasmid transfected human corneal epithelial cells. Forty-eight hours later, mRNA and proteins were harvested from cells for real-time PCR analysis and western blot assay respectively. RESULTS: IHC indicated TGFBI mainly exist below the human corneal epithelium layer. Transfection of recombinant pCMV-N-HA-TGFBI into human corneal epithelial cells resulted in effective expression of TGFBI, as shown by increased mRNA level detected by real-time KR as well as increased protein level detected by Western blot. Meanwhile the result of real-time KR and Western blot shown the expression of MMP1,MMP3(matrix rnetalloproteinases MMP) increased while the expressin of TIMP1 (tissue inhibitors of matrix metalloproteinases TIMP) decreased. CONCLUSION: TGFBI mainly exists below the corneal epithelial layer, recombinant eukaryotic expression vector harboring human TGFBI cDNA was obtained and efficiently overexpressed in human corneal epithelial cells. Meanwhile the TGFBI overexpression in human corneal epithelial cells result in MMPI., MMP3 increasing and TIMP1 decreasing. The result might be helpful for studying the function and role of TGFBI in pathogenesis of corneal dystrophy.

Early expression of mannose-binding lectin 2 during Aspergillus fumigatus infection in human corneal epithelial cells

AIM: To evaluate the early expression of mannose-binding lectin 2(MBL2) in human corneal epithelial cells(HCECs) infected by Aspergillus fumigatus(AF).METHODS: HCECs cultured in vitro with AF antigens and sampled at 0, 0.5, 1, 2, 4, 6 and 8h. The expression of MBL2 m RNA was evaluated by semiquantitative reverse transcription-polymerase chain reaction(RT-PCR). The expression of MBL2 protein in supernatant fluid was shown by enzyme linked immunosorbent assay(ELISA). MBL2 protein in HCECs was detected by immunocytochemistry at 0 and 24 h.RESULTS: MBL2 m RNA and protein are expressed in normal HCECs. The expression of MBL2 m RNA and protein in supernatant fluid begin to increase after being stimulated with AF antigens. The most significantly peak of MBL2 m RNA is in 2h. The protein of MBL2 in supernatant fluid decrease gradually after 0.5h. The protein in HCECs expression increase after stimulation of24 h.· CONCLUSION: MBL2 receptor expressed in normal HCECs in vitro. The stimulation by AF antigens can increase the early expression of it.

Cytoprotective effect of amniotic membrane extracts on human corneal epithelial cells exposed to benzalkonium chloride in vitro

Background:The goal was to explore the protective effect and potential mechanism of amniotic membrane extracts(AME)on the ocular surface exposed to benzalkonium chloride(BAC).Methods:The human corneal epithelial cell(HCEC)line SD-HCEC1s was cultured in 5 groups:normal control(NC),NC+AME,BAC,BAC+NC,and BAC+AME.Cell viability analysis,flow cytometry analysis,real-time polymerase chain reaction(PCR),and western blot were employed to measure changes in cell function.Matrix metalloproteinases(MMPs)and inflammatory cytokines were assayed by enzyme-linked immunosorbent assay(ELISA)and activity assays.Results:Real-time PCR and western blot analysis demonstrated that the expressional level of caspase-8 was increased while the levels of Muc1,Muc4,and Muc16 were decreased after treatment with 0.02%BAC for 1 h.When the SD-HCEC1s were withdrawn from the BAC and switched to media containing 10%AME for 2 days,the expression level of capsase-8 was decreased while the levels of Muc1,Muc4,and Muc16 were increased.Real-time PCR and ELISA demonstrated that the mRNA and protein levels of MMP-1,MMP-3,MMP-13,CXCL1,interleukin(IL)-1β,IL-6,and tumor necrosis factor-alpha(TNF-α)were significantly increased after treatment with 0.02%BAC,whereas those of MMP-8 were decreased.When the 0.02%BAC was withdrawn and the SD-HCEC1s were cultured in 10%AME,the mRNA and protein levels of MMP-1,MMP-3,MMP-13,CXCL1,IL-1β,IL-6,and TNF-αwere decreased,while those of MMP-8 were increased.MMP-8 activity assays confirmed that IL-1βand TNF-αdownregulated the protein levels of MMP-8.Conclusions:AME protects SD-HCEC1s when stressed in BAC via upregulation of MMP-8 and downregulation of IL-1βand TNF-α.AME may have the potential functions to be employed as a topical adjunctive therapy in eyes chronically exposed to BAC.

Nerve Growth Factor Modulate Proliferation of Cultured Rabbit Corneal Endothelial Cells and Epithelial Cells

Summary： In order to investigate the effect of nerve growth factor （NGF） on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF.MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570 nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner. 50 U/mI. and 500 U/mI. NGF got more increase of proliferation than that of 5 U/mL NGF did. Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.

Differentiation of human amniotic epithelial cells into corneal epithelial-like cells in vitro

·AIM: To explore the feasibility that human amniotic epithelial cells (hAECs) have the potential to differentiate into corneal epithelial -like cells under the microenvironment replicated by spontaneously immortalized human corneal epithelial cells (S-ihCECs). ·METHODS: hAECs were isolated by enzyme digestion, and flow cytometry was used to analysis the expression of CD29/90/166/73/34 and HLA -DR. Recovered and cultured S -ihCECs, immunocytochemistry was used to detect the expression of CK3/12. The proliferation of S - ihCECs handled by different concentrations of mitomycin was detected by CCK -8. The proliferation of hAECs cultured by S-ihCECs culture media collected at different time was analyzed by CCK -8. After filtered out the optimal conditions, we collected S-ihCECs culture media for 5 days, then prepared conditioned medium to incubate hAECs, inverted phase contrast microscope and scanning electron microscope were used to observe the change of morphology in hAECs. Quantitative real -time reverse transcription -polymerase chain reaction (QRT - PCR) was carried out to evaluate the expression of Oct - 4, NANOG, PAX6, and CK12 in the differentiation period. Immunocytochemistry and western bloting were used to detect the expression of CK3/12. ·RESULTS: The culture media collected every 12h, from 20μg/mL mitomycin pretreatment S -ihCECs could significantly promote the proliferation of hAECs. In the period of differentiation, the morphology of differentiated hAECs was obviously different compared with the control group, and the distinctive CK3/12 for corneal epithelial cells was detected.·CONCLUSION: This study showed that hAECs can differentiate into corneal epithelial -like cells by replication of the corneal epithelial microenvironment, using the culture media collected from S -ihCECs, and it is possible that S -ihCECs culture media could be used in corneal tissue engineering. ·

Induced pluripotent stem cells as a potential therapeutic source for corneal epithelial stem cells

Corneal blindness caused by limbal stem cell deficiency(LSCD) is one of the most common debilitating eye disorders. Thus far, the most effective treatment for LSCD is corneal transplantation, which is often hindered by the shortage of donors. Pluripotent stem cell technology including embryonic stem cells(ESCs) and induced pluripotent stem cells(iPSCs) have opened new avenues for treating this disease. iPSCs-derived corneal epithelial cells provide an autologous and unlimited source of cells for the treatment of LSCD. On the other hand, iPSCs of LSCD patients can be used for iPSCs-corneal disease model and new drug discovery. However, prior to clinical trial, the efficacy and safety of these cells in patients with LSCD should be proved. Here we focused on the current status of iPSCs-derived corneal epithelial cells used for cell therapy as well as for corneal disease modeling. The challenges and potential of iPSCs-derived corneal epithelial cells as a choice for clinical treatment in corneal disease were also discussed.

Herpes simplex virus-1 infection or Simian virus 40-mediated immortalization of corneal cells causes permanent translocation of NLRP3 to the nuclei

AIM: To investigate into the potential involvement of pyrin containing 3 gene(NLRP3), a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of corneas against viruses.METHODS: The herpes viral keratitis model was utilized in BALB/c mice with inoculation of herpes simplex virus-1(HSV-1). Corneal tissues removed during therapy of patients with viral keratitis as well as a Simian vacuolating virus 40(SV40)-immortalized human corneal epithelial cell line were also examined.Immunohistochemistry was used to detect NLRP3 in these subjects, focusing on their distribution in tissue or cells. Western blot was used to measure the level of NLRP3 and another two related molecules in NLPR3 inflammasome, namely caspase-1 and IL-1β.RESULTS: The NLRP3 activation induced by HSV-1infection in corneas was accompanied with redistribution of NLRP3 from the cytoplasm to the nucleus in both murine and human corneal epithelial cells. Furthermore,in the SV40-immortalized human corneal epithelial cells,NLRP3 was exclusively located in the nucleus, and treatment of the cells with high concentration of extracellular potassium(known as an inhibitor of NLRP3activation) effectively drove NLRP3 back to the cytoplasm as reflected by both immunohistochemistry and Western blot.· CONCLUSION: It is proposed that herpes virus infection activates and causes redistribution of NLRP3 to nuclei. Whether this NLRP3 translocation occurs with other viral infections and in other cell types merit further study.

Transplantation of tissue-engineered human corneal epithelium in limbal stem cell deficiency rabbit models

AIM: To evaluate the biological functions of tissue-engineered human corneal epithelium (TE-HCEP) by corneal transplantation in limbal stem cell deficiency (LSCD) rabbit models. METHODS: TE-HCEPs were reconstructed with DiI-labeled untransfected HCEP cells and denuded amniotic membrane (dAM) in air-liquid interface culture, and their morphology and structure were characterized by hematoxylin-eosin (HE) staining of paraffin-sections, immunohistochemistry and electron microscopy. LSCD models were established by mechanical and alcohol treatment of the left eyes of New Zealand white rabbits, and their eyes were transplanted with TE-HCEPs with dAM surface outside by lamellar keratoplasty (LKP). Corneal transparency, neovascularization, thickness, and epithelial integrality of both traumatic and post transplantation eyes were checked once a week by slit-lamp corneal microscopy, a corneal pachymeter, and periodic acid-Schiff (PAS) staining. At day 120 post surgery, the rabbits in each group were sacrificed and their corneas were examined by DiI label observation, HE staining, immunohistochemistry and electron microscopy. RESULTS: After cultured for 5 days on dAM, HCEP cells, maintaining keratin 3 expression, reconstructed a 6-7 layer TE-HCEP with normal morphology and structure. The traumatic rabbit corneas, entirely opaque, conjunctivalized and with invaded blood vessels, were used as LSCD models for TE-HCEP transplantation. After transplantation, obvious edema was not found in TE-HCEP-transplanted corneas which became more and more transparent, the invaded blood vessels reduced gradually throughout the monitoring period. The corneas decreased to normal thickness on day 25, while those of dAM eyes were over 575 mu m in thickness during the monitoring period. A 45 layer of epithelium consisting of TE-HCEP originated cells attached tightly to the anterior surface of stroma was reconstructed 120 days after TE-HCEP transplantation, which was similar to the normal control eye in morphology and structure. In contrast, intense corneal edema, turbid, invaded blood vessels were found in dAM eyes, and no multilayer epithelium was found but only a few scattered conjunctiva-like cells appeared. CONCLUSION: The TE-HCEP, with similar morphology and structure to those of innate HCEP, could reconstruct a multilayer corneal epithelium with normal functions in restoring corneal transparency and thickness of LSCD rabbits after transplantation. It may be a promising HCEP equivalent for clinical therapy of corneal epithelial disorders.

Profile of biological characterizations and clinical application of corneal stem/progenitor cells

Corneal stem/progenitor cells are typical adult stem/progenitor cells.The human cornea covers the front of the eyeball,which protects the eye from the outside environment while allowing vision.The location and function demand the cornea to maintain its transparency and to continuously renew its epithelial surface by replacing injured or aged cells through a rapid turnover process in which corneal stem/progenitor cells play an important role.Corneal stem/progenitor cells include mainly corneal epithelial stem cells,corneal endothelial cell progenitors and corneal stromal stem cells.Since the discovery of corneal epithelial stem cells(also known as limbal stem cells)in 1971,an increasing number of markers for corneal stem/progenitor cells have been proposed,but there is no consensus regarding the definitive markers for them.Therefore,the identification,isolation and cultivation of these cells remain challenging without a unified approach.In this review,we systematically introduce the profile of biological characterizations,such as anatomy,characteristics,isolation,cultivation and molecular markers,and clinical applications of the three categories of corneal stem/progenitor cells.

Nucleotide oligomerization domain 2 contributes to the innate immune response in THCE cells stimulated by Aspergillus fumigatus conidia

AIM: To investigate the expression of nucleotide oligomerization domain 2 (NOD2) in the immortalized human corneal epithelial cell line (THCE), and its role in the innate immune response triggered by inactive Aspergillus fumigatus (Af) conidia. METHODS: The normal THCE cells were investigated as controls. After incubation with inactive Af conidia for 0.5, 2, 4, 6, and 8 hours, THCE cells were harvested, mRNA expression of NOD2 and receptor interacting protein 2 (RIP2) was detected by RT-PCR. Intracellular proteins including NOD2, NF-kappa B and proinflammatory cytokines such as TNF-alpha, IL-8, IL-6 in the cell supernatant were analyzed by ELISA. RESULTS: Our data indicate that NOD2 expressed in the normal THCE cells. After triggered by the inactive Af conidia, the expression of NOD2, RIP2 mRNA and the secretion of NOD2, NF-kappa B, TNF-alpha, IL-8, IL-6 both increased in a time-depended manner, and reached the peak point at 4, 6, 6, 4, 6, 6, 4 hours, respectively. And after pretreated with NOD2 neutralizing antibody, the expression of RIP2, NF-kappa B, TNF-alpha, IL-8 both decreased dramatically at the peak point, while the secretion of IL-6 changed little. CONCLUSION: The results of this study suggest that NOD2 exists and expresses in the THCE cells, and contributes to the innate immune responses triggered by inactive Afconidia by induction of proinflammatory cytokines such as TNF-alpha and IL-8 through the NF-kappa B pathway.