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.

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.

Central corneal epithelium self-healing after ring-shaped glycerin-cryopreserved lamellar keratoplasty in Terrien marginal degeneration

Dear Sir, I am Dr Yan-Long Bi, from the Department of Ophthalmology, Tongji University Affiliated to Tongji University School of Medicine, Shanghai, China. I write to present a case report of total limbal stem cells deficiency after treatment with ring-shaped lamellar keratoplasty secondary to Terrien marginal degeneration. During 3 years

Anti-inflammatory and anti-apoptotic effects of N-acetylcysteine in diabetic rat corneal epithelium

AIM:To characterize the anti-inflammatory and antiapoptotic effects of N-acetylcysteine(NAC)in streptozotocin(STZ)-induced diabetic rat corneal epithelium and human corneal epithelial cells(HCECs)exposed to a high-glucose environment.METHODS:HCECs were incubated in 0,5,50 mmol/L glucose medium,or 50 mmol/L glucose medium with NAC for 24h.Diabetes was induced in rats by intraperitoneal injection of 65 mg/kg STZ and some of these rats were topically administered NAC to corneas with 3 mice per group.We characterized receptor for advanced glycation end-products(RAGE)expression using immunofluorescence,and interleukin(IL)-1βand cleaved caspase-3(CCAP-3)expression using immunohistochemistry.Circulating tumor necrosis factor(TNF)-αconcentration was measured by ELISA and cleaved poly-ADP ribose polymerase(PARP)concentration was quantified by Western blotting.Apoptotic cells were detected using TUNEL assay and annexin V and propidium iodide staining.RESULTS:Diabetic rats had higher expression of RAGE(2.46±0.13 fold),IL-1β,and CCAP-3 in apoptotic cells of their corneas than control rats.The expression of RAGE(1.83±0.11 fold),IL-1β,and CCAP-3,and the number of apoptotic cells,were reduced by topical NAC treatment.HCECs incubated in 50 mmol/L glucose medium showed high concentrations of TNF-α(310±2.00 pg/mL)and cleaved PARP(7.43±0.56 fold),and more extensive apoptosis than cells in 50 mmol/L glucose medium.However,the addition of NAC reduced the concentrations of TNF-α(153.67±2.31 pg/mL)and cleaved PARP(5.55±0.31 fold)and the number of apoptotic cells.CONCLUSION:NAC inhibits inflammation and apoptosis in the corneas of diabetic rats and HCECs maintained in a high-glucose environment.

TREM-1 expression in rat corneal epithelium with Aspergillus fumigatus infection

AIM: To investigate the expression of triggering receptor expressed on myeloid cells-1(TREM-1) in the aberrant inflammation within the corneal epithelium at early period of fungal infection.METHODS: A total of 65 Wistar rats were randomly divided into control group, sham group and fungal keratitis(FK) group, in which the cornea was infected by Aspergillus fumigatus(A. fumigatus). After executed randomly at 8, 16, 24, 48 and 72 h after experimental model being established, the severity of keratomycosis in rats was scored visually with the aid of a dissecting microscope and slit lamp. Then corneas in three groups were collected to assess the expression of TREM-1through quantitative reverse transcription-polymerase chain reaction(RT-PCR), immunofluorescence technique and Western blot analysis. The correlation between FK inflammation and expression of TREM-1 was also analyzed.RESULTS: Corneal inflammation scores increased with time after fungal infection(F =49.74, P =0.000). The inflammation scores in FK group were obviously higher than those in sham group on the whole(F =137.78, P =0.000). Levels of TREM-1 in the infected rat corneal epithelium had elevated at 8h and peaked at 48h(P 【0.001,compared with control group). Western blot analysis also showed an obviously elevated TREM-1 level in rat corneal epithelium at 24 h and 48 h after fungal infection.Immunofluorescence technique showed that TREM-1mainly existed in corneal epithelium and infected corneal stoma of rat. TREM-1 protein expression was enhanced after fungal infection. Moreover, severity of FK inflammation was significantly related to TREM-1expression in FK(r =0.942, P =0.000).CONCLUSION: TREM-1 may contribute to amplify theinflammation in the cornea infected with A. fumigatus and play critical roles in the battle against A. fumigatus in the innate immune responses.

Expression of vitamin D receptor and cathelicidin in human corneal epithelium cells during fusarium solani infection

AIMTo observe the expression of vitamin D receptor (VDR) in human specimen and immortalized human corneal epithelium cells (HCEC) when challenged with fusarium solani. Moreover, we decided to discover the pathway of VDR expression. Also, we would like to detect the expression of cathelicidin antimicrobial peptide (CAMP) in the downstream pathway of VDR.METHODSImmunohistochemistry was used to examine the VDR expression in HCEC from healthy and fungal keratitis patients. Real time quantitative polymerase chain reaction (qPCR) was performed to observe the messenger ribonucleic acid (mRNA) change of VDR when immortalized HCEC were challenged with fusarium solani for different hours. CAMP was detected at both mRNA and protein levels.RESULTSWe found out that the VDR expression in fusarium solani keratitis patients' specimen was much more than that in healthy people. The mRNA and protein expression of VDR increased when we stimulated HCEC with fusarium solani antigen (P<0.01) and it could be inhibited by toll like receptor 2 (TLR2) monoclonal antibody. The CAMP expression was decreased because of fusarium solani antigen stimulation (P<0.01).CONCLUSIONThe VDR expression can be increased via TLR2/1-VDR pathway while the CAMP expression is decreased by the stimulation of fusarium solani antigen.

Reconstruction of Rabbit Corneal Layer Composed of Corneal Fibroblasts and Corneal Epithelium on the Lyophilized Amniotic Membrane

Many researchers have employed the cryopreserved amniotic membrane（CAM） and corneal epithelial cells in the treatment of a severely damaged burned cornea, with corneal epithelial cells cultured on an amniotic membrane （AM）. The lyophilized amniotic membrane（LAM） has a higher graft take and a longer shelf life; it is easier to store and safer because of gamma irradiation. Two Teflon rings（ Ahn＇s supporter） were made for culturing the cells on the LAM, and were then used to support the LAM. To reconstruct a corneal layer composed of corneal fibroblasts and epithelium, the corneal fibroblasts were first cultivated on the stromal side of LAM for five days, foUowed by epithelial cells culture on the epithelial side, by using the air-liquid interface culture. The reconstructed corneal layer composed of corneal fibroblasts and corneal epithelial cells has a much healthier basal layer of corneal epithelium than the reconstructed corneal epithelium, which was got by using only corneal epithelial cells, and resembles the epithelium of normal corneas, without the horny layer. Thus, the reconstruction of the corneal layer by using a LAM is considered to be a good in vitro model, not only for its application in toxicological test kits, but also for transplantation in patients with a severely damaged cornea.

EFFECT OF HUMAN AMNIOTIC MEMBRANE ON CORNEAL EPITHELIUM AND YAC-1 CELL

Objective To study the effect of the amniotic membrane on enhancing the proliferation ofcorneal epithelia and YAC-1 cell. Methods After the primary culture of the rabbit's corneal epithelia and YAC-1 cells, they were seeded on the upper surface or stromal matrix side of amniotic membrane respectively. The proliferation results were observed by MTT test. Results The amniotic membrane was found significantly enhancing the proliferation of corneal epithelia on the d1 ,d3 , and d5 after culture. The proliferation rate was 28.93% ,23.32% ,23.41 % (P<0 .05) respectively, but the d7 proliferation rate was 20.72% (P> 0.05). On the dl , d3 , d7 after culture , the YAC-1 cells proliferation rate was 34 .87% ,36 .28% ,33 .86% (P< 0.01) respectively. Conclusion Our results demonstrated that the amniotic membrane could enhance the proliferation of both corneal epithelia and YAC-1 cells significantly. Although amniotic membrane has been suggested as an ideal material for reconstruction of ocular surface, special attention should be paid during amniotic membrane transplantation for treating ocular surface lesion resulted from epibulbar tumors.

Differentiation of embryonic stem cells into corneal epithelium

Our project was to determine whether embryonic stem (ES) cells could be induced to differentiate into corneal epithelia by superficial corneoscleral limbal stroma. To achieve this goal, ES-GFP cell line D3 was pre-induced by retinoic acid (RA). The pre-induced cells were seeded on deepithelialized superficial corneoscleral slices (SCSS) to form a monolayer, and di-vided into three groups. Group 1 was cultured and passaged in vitro for direct detection. Group 2 was exposed to air-liquid interfaces for 10 days and implanted into the subcutaneous layer of nude mice for 2 weeks for further induction in vivo. Group 3 was cultured in vitro without any in-ducing factors for control. There were no teratomas found in nude mice which were implanted with differentiated ES cells after two weeks. The differentiated cells showed an appearance of epithelia both in vitro and in vivo. Expression of CK3, P63 and PCNA was detected by immuno-histochemical staining in the differentiated cells in group 1 and 2. Microvillis and zonula oc-cludens were observed on the surface of the differentiated cells under an electron microscope. In the control group, ES cells differentiated freely without any inducing factors. Most cells were shed and formed a neuronal dendrite-like structure, and a minority of cells appeared polymorphic. These results demonstrate that ES cells can differentiate into corneal epithelia on the surface of SCSS under the controlled condition. Differentiated ES cells could be used as epithelial seeding cells for the reconstruction of ocular surface and corneal tissue engineering in the future.

Putative epidermal stem cell convert into corneal epithelium-like cell under corneal tissue in vitro

Rhesus putative epidermal stem cells are being investigated for their potential use in regenerative corneal epithelium-like cells, which may provide a practical source of autologous seed cells for the construction of bioengineered corneas. The goal of this study was to investigate the potential of epi-dermal stem cells for trans-differentiation into corneal epithelium-like cells. Rhesus putative epidermal stem cells were isolated by type IV collagen attachment method. Flow cytometry analysis, immuno-histology and RT-PCR were conducted to identify the expression of specific markers (β1, α6 integrin, K15, K1/K10, K3/K12 and CD71) on the isolated rapid attaching cells. The isolated cells were cocultured with human corneal limbal stroma and corneal epithelial cells. After coculture, the expression of the same specific markers was evaluated in order to identify expression difference caused by the coculture conditions. K3/K12 expression was analyzed in coculture cells on day 2, 4, 6, 8 and 10. Putative epi-dermal stem cells in conditioned culture media were used as control. Putative epidermal stem cells were predominant in rapid attaching cells by type IV collagen attachment isolation. Before being co-cultured, the rhesus putative epidermal stem cells expressed K15, α6 and β1 integrin, but no CD71, K1/K10 and K3/K12. After coculture, these cells expressed K3/K12 (a marker of corneal epithelial cells), K15 and β 1 integrin, but no K1/K10. Cells being not coculture converted into terminally differentiated cells expressing K1/K10. These results indicate that rhesus putative epidermal stem cells can trans-differentiate into corneal epithelium-like cells and, therefore, may have potential therapeutic application as autologous seed cells for the construction of bioengineered corneas.

Netrin-1 promotes epithelium repair in corneal injury

●AIM:To explore netrin-1 functions on corneal epithelium in vitro and in vivo.●METHODS:In vitro the human corneal epithelial(HCE)cells were treated with serum free DMEM-F12 basic media containing 0,50,100,200,300,500,800,and 1000 ng/mL of netrin-1,respectively.The cells viability was detected by cell counting kit-8(CCK-8).The wound-healing assay was applied to assess the migration proficiency of HCE cells.Flow cytometry was used to analyze the cell-cycle distribution and apoptosis.In vivo,normal c57(6 wk)mice were demarcated with a trephine in the middle of the cornea to produce a 3-mm circular wound.Mice corneas were inflicted no epithelium with a 3-mm wound displayed,but remained the limbal epithelium intact.A blunt scalpel blade was used to remove the corneal epithelian cells,followed by topical netrin-1 application(200 ng/mL),and the group treated by PBS as control.The treated group was injected netrin-1 into the normal c57 mice inferior subconjunctival 4 h before trauma.Mouse corneal inflammation and neovascularization were observed under slit lamp microscope.The apoptosis of corneal cells was determined by TUNEL staining.●RESLUTS:A concentration of 200 ng/mL netrin-1 enhanced 25%of the HCE viability.The relative migration rates were 76.3%and 100%in control and netrin-1 treated group after cultured 72 h.Treated with netrin-1(200 ng/mL)decreased the apoptosis of HCE cells,as well as decreased their percentage from 19.3%±0.57%to 12.7%±0.42%of the total.The remaining wound area was 1.22 mm2 in control group but 0.22 mm2 in the netrin-1 treated group.Exogenous Netrin-1 inhibits apoptosis of corneal epithelial cells of c57 mice.TUNEL-positive cells at the epithelial layer of the corneas of the control and netrin-1 treated c57 mice at 24 h after wounding were 43.3%and 16.7%respectively.●CONCLUSION:Netrin-1 can reduce HCE apoptosis as well as promote its proliferation and migration.Topical application of netrin-1 promotes the injuryed cornea epithelial wound repair and inhibits apoptosis of corneal epithelial cells.These findings may offer potential therapies to repair the defects of corneal epithelial based on netrin-1.

Niche regulation of corneal epithelial stem cells at the limbus

Among all adult somatic stem cells, those of the corneal epithelium are unique in their exclusive location in a defined limbal structure termed Palisades of Vogt. As a result, surgical engraftment of limbal epithelial stem cells with or without ex vivo expansion has long been practiced to restore sights in patients inflicted with limbal stem cell deficiency. Neverthe- less, compared to other stem cell examples, relatively little is known about the limbal niche, which is believed to play a pivotal role in regulating self-renewal and fate decision oflimbal epithelial stem cells. This review summarizes relevant literature and formulates several key questions to guide future research into better understanding of the pathogenesis of limbal stem cell deficiency and further improvement of the tissue engineering of the corneal epithelium by focusing on the limbal niche.

SMILE与FS-LASIK术后角膜上皮重塑的临床研究

目的:比较飞秒激光小切口角膜基质透镜取出术(SMILE)与飞秒激光辅助准分子激光原位角膜磨镶术(FS-LASIK)术后角膜上皮厚度(CET)的变化特点。方法:收集2022-12/2023-11在乌鲁木齐爱尔眼科医院接受屈光手术患者187例187眼。按手术方式分为SMILE组110例110眼和FS-LASIK组77例77眼。应用眼前节光学相干断层扫描技术(OCT)分别于术前和术后1 wk,1、3、6 mo测量患者的CET。结果:比较术后6 mo时角膜中央区、旁中央区、中周区角膜上皮厚度变化量(△CET),SMILE的特点为中央区增厚最明显,中周区上皮最少;FS-LASIK的特点为旁中央区增厚最明显,中周区最少。术后1 wk,1、3、6 mo时SMILE与FS-LASIK组角膜0-7 mm范围的平均ΔCET与术前等效球镜均具有相关性。结论:SMILE和FS-LASIK术后6 mo内上皮增厚程度随时间变化有相似的趋势和不同的特点,二者△CET均与术前等效球镜正相关。

Fungus induces the release of IL- 8 in human corneal epithelial cells, via Dectin-1-mediated protein kinase C pathways

AIM: To identify whether Aspergillus fumigatus(A.fumigatus) hyphae antigens induced the release of interleukin-8(IL-8) in anti-fungal innate immunity of cultured human corneal epithelial cells(HCECs) and determine the involvement of intracellular signalling pathways. METHODS: HCECs were treated with A. fumigatus hyphae antigens with different concentrations and time.The cytoplasmic calcium of HCECs were assessed by fluorescence imaging. Western blot was used to detect the expression of Ca2 +-dependent protein kinase C(PKC). The IL-8 levels were determined by specific human IL-8 enzyme-linked immunosorbent assay(ELISA) and reverse transcriptase polymerase chain reaction(RT-PCR). Using a series of pharmacological inhibitors, we examined the upstream signalling pathway responsible for IL-8 expression in response to A.fumigatus hyphae antigens. RESULTS: Cells exposed to A. fumigatus hyphae antigens showed higher level of IL-8 m RNA expression and protein production. We demonstrated here that stimulation of HCECs with A. fumigatus hyphae triggers an intracellular Ca2 +flux and results in the activation of Ca2 +-dependent PKC(α, βⅠ and βⅡ) which can be attenuated by pre-treatment of cells with laminarin,suggesting that Dectin-1 signals pathway induced cytoplasmic calcium and influence the activation of PKC in HCECs. Inhibitors of Ca2 +-dependent PKC(Ro-31-8220 and Go6976) significantly abolished hyphae-induced expression of IL-8.CONCLUSION: Our findings suggest that A. fumigatushyphae-induced IL-8 expression was regulated by the activation of Dectin-1-mediated Ca2 +-dependent PKC in HCECs.

Efficacy of rhNGF-loaded amniotic membrane transplantation for rabbit corneal epithelial and nerve regeneration

AIM:To evaluate the efficacy of recombinant human nerve growth factor-loaded amniotic membrane(rh NGF-AM)on corneal epithelial and nerve regeneration in rabbit model.METHODS:Freshly prepared human amniotic membrane(AM)were immersed into PBS buffer containing 100 or 500μg/mL rh NGF for 15,30,and 60 min at 4℃.The in vitro release kinetics of rh NGF was measured with ELISA.For in vivo evaluation,the AM were immersed with 500μg/mL rh NGF for 30 min.Fifty-seven rabbits were selected to establish corneal epithelial defect model.In addition to the 19 rabbits in control group,38 rabbits received AM transplantation with or without rh NGF after the removal of central epithelium.Corneal epithelial defect area,sub-epithelial nerve fiber density,corneal sensitivity,rh NGF contents in resident AM and corneas were measured after the surgery.RESULTS:rh NGF was sustained release from the AM within 14 d in vitro,with the positive correlation with initial immersion concentration.The immersion of AM in 500μg/mL rh NGF for 30 min achieved the most stable release within 14 d.After transplantation in rabbit cornea,a high concentration of rh NGF in resident rh NGF-AM and cornea was maintained within 8 d.Corneal epithelial healing,nerve fiber regeneration and the recovery of corneal sensitivity were significantly accelerated after the rh NGF-AM transplantation when compared to simple AM transplantation(all P<0.05).CONCLUSION:Simple immersion of AM achieves the sustained release of rh NGF,and promotes corneal epithelial wound healing and nerve regeneration,as well as the recovery of corneal sensitivity in rabbit.

Dectin-1 agonist curdlan modulates innate immunity to Aspergillus fumigatus in human corneal epithelial cells

· AIM: To explore the immunomodulatory effects of curdlan on innate immune responses against Aspergillus fumigatus(A. fumigatus) in cultured human corneal epithelial cells(HCECs), and whether C-type lectin receptor Dectin-1 mediates the immunomodulatory effects of curdlan.·METHODS: The HCECs were stimulated by curdlan in different concentrations(50, 100, 200, 400 μg/m L) for various time. Then HCECs pretreated with or without laminarin(Dectin-1 blocker, 0.3 mg/m L) and curdlan were stimulated by A. fumigatus hyphae. The m RNA and protein production of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6) were determined by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The protein level of Dectin-1 was measured by Western blot.· RESULTS: Curdlan stimulated m RNA expression of TNF-α and IL-6 in a dose and time dependent manner in HCECs. Curdlan pretreatment before A. fumigatus hyphae stimulation significantly enhanced the expression of TNF-α and IL-6 at m RNA and protein levels compared with A. fumigatus hyphae stimulation group(P 【0.05).Both curdlan and A. fumigatus hyphae up-regulated Dectin-1 protein expression in HCECs, and Dectin-1expression was elevated to 1.5- to 2-fold by curdlan pretreatment followed hyphae stimulation. The Dectin-1blocker laminarin suppressed the m RNA expression and protein production of TNF-α and IL-6 induced by curdlan and hyphae(P 【0.05).· CONCLUSION: These findings demonstrated that curdlan pretreatment enhanced the inflammatory response induced by A. fumigatus hyphae in HCECs.Dectin-1 is essential for the immunomodulatory effectsof curdlan. Curdlan may have high clinical application values in fungal keratitis treatment.

Anti-inflammatory potential of human corneal stroma-derived stem cells determined by a novel in vitro corneal epithelial injury model

BACKGROUND An in vitro injury model mimicking a corneal surface injury was optimised using human corneal epithelial cells(hCEC).AIM To investigate whether corneal-stroma derived stem cells(CSSC) seeded on an amniotic membrane(AM) construct manifests an anti-inflammatory, healing response.METHODS Treatment of hCEC with ethanol and pro-inflammatory cytokines were compared in terms of viability loss, cytotoxicity, and pro-inflammatory cytokine release, in order to generate the in vitro injury. This resulted in an optimal injury of 20%(v/v) ethanol for 30 s with 1 ng/mL interleukin-1(IL-1) beta. Co-culture experiments were performed with CSSC alone and with CSSC-AM constructs.The effect of injury and co-culture on viability, cytotoxicity, IL-6 and IL-8 production, and IL1 B, TNF, IL6, and CXCL8 mRNA expression were assessed.RESULTS Co-culture with CSSC inhibited loss of hCEC viability caused by injury. Enzyme linked immunosorbent assay and polymerase chain reaction showed a significant reduction in the production of IL-6 and IL-8 pro-inflammatory cytokines, and reduction in pro-inflammatory cytokine mRNA expression during co-culture with CSSC alone and with the AM construct. These results confirmed the therapeutic potential of the CSSC and the possible use of AM as a cell carrier for application to the ocular surface.CONCLUSION CSSC were shown to have a potentially therapeutic anti-inflammatory effectwhen treating injured hCEC, demonstrating an important role in corneal regeneration and wound healing, leading to an improved knowledge of their potential use for research and therapeutic purposes.

Antagomir Dependent MicroRNA-205 Reduction Enhances Adhesion Ability of Human Corneal Epithelial Keratinocytes

Objective To investigate the effect of microRNA-205 reduction by antagomirs on adhesion ability of normal human corneal epithelial keratinocytes(NHCEKs).Methods Antagomir-205,complementary and inhibitory to microRNA-205,was used to suppress endogenous microRNA-205 in NHCEKs.The adhesion ability of treated NHCEKs was then assessed by cell adhesion assay.Immunoblot and immunohistochemistry were conducted to determine the level of two focal adhesion-related proteins,focal adhesion kinase(FAK) and paxillin(Pax).Phalloidin staining was performed to measure the level of filamentous actin in antagomir-treated NHCEKs.Results Antagomir-205 markedly reduced the level of microRNA-205 in NHCEKs and significantly enhanced adhesion ability of NHCEKs(P<0.01).Further protein analysis validated that inhibition of mi-croRNA-205 increased the number of phosphorylated FAK and phosphorylated Pax,and decreased filamen-tous actin.Conclusion Our findings suggest that microRNA-205 has down-regulating effect on cell motility in NHCEKs.

Limbal stem cells: Central concepts of corneal epithelial homeostasis

A strong cohort of evidence exists that supports the localisation of corneal stem cells at the limbus. The distinguishing characteristics of limbal cells as stem cells include slow cycling properties, high proliferative potential when required, clonogenicity, absence of differentiation marker expression coupled with positive expression of progenitor markers, multipotency, centripetal migration, requirement for a distinct niche environment and the ability of transplanted limbal cells to regenerate the entire corneal epithelium. The existence of limbal stem cells supports the prevailing theory of corneal homeostasis, known as the XYZ hypothesis where X represents proliferation and stratification of limbal basal cells, Y centripetal migration of basal cells and Z desquamation of superficial cells. To maintain the mass of cornea, the sum of X and Y must equal Z and very elegant cell tracking experiments provide strong evidence in support of this theory. However, several recent stud-ies have suggested the existence of oligopotent stem cells capable of corneal maintenance outside of the limbus. This review presents a summary of data which led to the current concepts of corneal epithelial homeostasis and discusses areas of controversy surrounding the existence of a secondary stem cell reservoir on the corneal

Evaluating alternative stem cell hypotheses for adult corneal epithelial maintenance

In this review we evaluate evidence for three different hypotheses that explain how the corneal epithelium is maintained. The limbal epithelial stem cell(LESC)hypothesis is most widely accepted. This proposes that stem cells in the basal layer of the limbal epithelium, at the periphery of the cornea, maintain themselves and also produce transient(or transit) amplifying cells(TACs). TACs then move centripetally to the centre of the cornea in the basal layer of the corneal epithelium and also replenish cells in the overlying suprabasal layers. The LESCs maintain the corneal epithelium during normal homeostasis and become more active to repair significant wounds. Second, the corneal epithelial stem cell(CESC) hypothesis postulates that, during normal homeostasis, stem cells distributed throughout the basal corneal epithelium, maintain the tissue. According to this hypothesis, LESCs are present in the limbus but are only active during wound healing. We also consider a third possibility, that the corneal epithelium is maintained during normal homeostasis by proliferation of basal corneal epithelial cells without any input from stem cells. After reviewing the published evidence, we conclude that the LESC and CESC hypotheses are consistent with more of the evidence than the third hypothesis, so we do not consider this further. The LESC and CESC hypotheses each have difficulty accounting for one main type of evidence so we evaluate the two key lines of evidence that discriminate between them. Finally, we discuss how lineage-tracing experiments have begun to resolve the debate in favour of the LESC hypothesis. Nevertheless, it also seems likely that some basal corneal epithelial cells can act as long-term progenitors if limbal stem cell function is compromised. Thus, this aspect of the CESC hypothesis may have a lasting impact on our understanding of corneal epithelial maintenance, even if it is eventually shown that stem cells are restricted to the limbus as proposed by the LESC hypothesis.