Cell viability and extracellular matrix synthesis in a co-culture system of corneal stromal cells and adipose-derived mesenchymal stem cells

AIM：To investigate the impact of adipose-derived mesenchymal stem cells（ADSCs） on cell viability and extracellular matrix（ECM） synthesis of corneal stromal cells（CSCs）. METHODS：ADSCs and CSCs were obtained from the corneas of New Zealand white rabbits and indirectly cocultured in vitro. The proliferative capacity of CSCs in the different groups was assessed by CCK-8 assays. Annexin V-fluorescein isothiocyanate（FITC）/proliferation indices（PI） assays were used to detect the apoptosis of CSCs. The expression levels of matrix metalloproteinase（MMP）, such as MMP1, MMP2, MMP9, and collagens were also evaluated by Western blot. RESULTS：ADSCs significantly promoted proliferation and invasion of CSCs in the indirect co-culture assays. The co-cultural group displayed much higher ability of proliferation, especially under the co-culture conditions of ADSCs for 3d, compared with that CSCs cultured alone. The PI of CSCs in the co-culture system were increased approximately 3-8-fold compared with the control group. A significant change was observed in the proportions of cells at apoptosis（early and late） between the negative control group（6.34% and 2.06%） and the ADCSs-treated group（4.69% and 1.59%）. The expression levels of MMPs were down regulated in the co-culture models. Compared with the control group, the decrease intensities of MMP-1, MMP-2 and MMP-9 in CSCs/ADSCs group were observed, 3.90-fold, 1.09-fold and 3.03-fold, respectively. However, the increase intensities of collagen type（I, II, III, IV, and V） in CSCs were observed in CSCs/ADSCs group, 3.47-fold,4.30-fold, 2.35-fold, 2.55-fold and 2.43-fold, respectively, compared to that in the control group. The expressions of aldehyde dehydrogenase and fibronectin in CSCs were upregulated in the co-culture models.CONCLUSION：ADSCs play a promotive role in CSCs＇ growth and invasion, which may be partially associated with MMPs decrease and collagens increase, resulting in a positive participation in the plasticity and ECM synthesis of CSCs. This provided a new insight into the extensive role of ADSCs in CSCs and a potential molecular target for corneal therapy.

Cytotoxicity of pilocarpine to human corneal stromal cells and its underlying cytotoxic mechanisms

AIM： To examine the cytotoxic effect of pilocarpine, an anti-glaucoma drug, on human corneal stromal（HCS）cells and its underlying cytotoxic mechanisms using an in vitro model of non-transfected HCS cells.· METHODS： After HCS cells were treated with pilocarpine at a concentration from 0.15625 g/L to 20.0 g/L,their morphology and viability were detected by light microscopy and MTT assay. The membrane permeability,DNA fragmentation and ultrastructure were examined by acridine orange（AO）/ethidium bromide（EB） double-staining. DNA electrophoresis and transmission electron microscopy（TEM）, cell cycle, phosphatidylserine（PS）orientation and mitochondrial transmembrane potential（MTP） were assayed by flow cytometry（FCM）. And the activation of caspases was checked by ELISA.· RESULTS： Morphology observations and viability assay showed that pilocarpine at concentrations above0.625 g/L induced dose- and time-dependent morphological abnormality and viability decline of HCS cells. AO/EB double-staining, DNA electrophoresis and TEM noted that pilocarpine at concentrations above 0.625 g/L induced dose- and/or time-dependent membrane permeability elevation, DNA fragmentation, and apoptotic body formation of the cells. Moreover, FCM and ELISA assays revealed that 2.5 g/L pilocarpine also induced S phase arrest, PS externalization, MTP disruption, and caspase-8,-9 and-3 activation of the cells.· CONCLUSION： Pilocarpine at concentrations above0.625 g/L（1/32 of its clinical therapeutic dosage） has a dose- and time-dependent cytotoxicity to HCS cells by inducing apoptosis in these cells, which is most probably regulated by a death receptor-mediated mitochondrion-dependent signaling pathway.

Local anesthetic lidocaine induces apoptosis in human corneal stromal cells in vitro

AIM:To demonstrate the apoptosis-inducing effect of iidocalne on human corneal stromal(HCS)cells fn vitm,and provide experimental basis for safety anesthetic usage In clinic of ophthalmology.METHODS:In vitro cultured HCS cells were treated with lidocaine at different doses and times,and their morphology was monitored successively with inverted phase contrast microscopy.The membrane permeability of them was detected by acridine orange/ethidium bromide(AO/EB)double staining.The DNA fragmentation of them was examined by agarose gel electrophoresis,and their ultrastructure was observed by transmission electron microscopy(TEM),respectively.RESULTS:Exposure to lidocaine at doses from0.3125g/L to 20g/L induced morphological changes of HCS cells such as cytoplasmic vacuolation,cellular shrinkage,and turning round,and elevated membrane permeability of these cells in AO/EB staining.The change of morphology and membrane permeability was doseand time-dependent,while lidocaine at dose below0.15625g/L could not induce these changes.Furthermore,lidocaine induced DNA fragmentation and ultrastructural changes such as cytoplasmic vacuolation,structural disorganization,chromatin condensation,and apoptotic body appearance of the cells.CONCLUSION:Lidocaine has significant cytotoxicity on human corneal stromal cells in vitro in a dose-and time-dependent manner by inducing apoptosis of these cells.The established experimental model and findingsbased on this model here help provide new insight into the apoptosis-inducing effect of local anesthetics in eye clinic.

Effects of corneal stromal cell-and bone marrow-derived endothelial progenitor cell-conditioned media on the proliferation of corneal endothelial cells

AIM： To explore the effects of conditioned media on the proliferation of corneal endothelial cells （CECa） and to compare the efficiency of different conditioned media （CM）. METHODS： Rat CECs, corneal stromal cells （CSCs）, bone marrow -derived endothelial progenitor cells （BEPCs）, and bone marrow-derived mesenchymal stem cells （BMSCs） were isolated and cultured in vitra CM was collected from CSCs, BEPCs, and BMSCSo CECs were cultivated in different culture media. Cell morphology was recorded, and gene and protein expression were analyzed.~ RESULTS： After grown in CM for 5d, CECs in each experimental group remained polygonal, in a cobblestone- like monolayer arrangement. Immunocytofluorescence revealed positive expression of Na＋/K＋-ATP, aquaporin 1 （AQP1）, and zonula occludens 1 （ZO-1）. Based on quantitative polymerase chain reaction （qPCR） analysis, Na ＋/K ＋-ATP expression in CSC-CM was notably upregulated by 1.3-fold （＋0.036） （P〈0.05, n=3）. The expression levels of ZO-1, neuron specific enolase （NSE）, Vimentin, paired homebox 6 （PAX6）, and procollagen type VII （COL8A1） were notably upregulated in each experimental group. Each CM had a positive effect on CEC proliferation, and CSC-CM had the strongest effect on proliferation.~ CONCLUSION： CSC-CM, BEPC-CM, and BMSC-CM not only stimulated the proliferation of CECs, but also maintained the characteristic differentiated phenotypes necessary for endothelial functions. CSC-CM had the most notable effect on CEC proliferation. KEYWORDS： conditioned medium; corneal endothelial cell; corneal stromal cell; bone marrow-derived endothelial progenitor cell; proliferation

Establishment of an untransfected human corneal stromal cell line and its biocompatibility to acellular porcine corneal stroma

AIM: To establish an untransfected human corneal stromal (HCS) cell line and characterize its biocompatibility to acellular porcine corneal stoma (aPCS). METHODS: Primary culture was initiated with a pure population of HCS cells in DMEM/F12 media (pH 7.2) containing 20% fetal bovine serum and various necessary growth factors. The established cell line was characterized by growth property, chromosome analysis, tumorigenicity assay, expression of marker proteins and functional proteins. Furthermore, the biocompatibility of HCS cells with aPCS was examined through histological and immunocytochemistry analyses and with light, electron microscopies. RESULTS: HCS cells proliferated to confluence 2 weeks later in primary culture and have been subcultured to passage 140 so far. A continuous untransfected HCS cell line with a population doubling time of 41.44 hours at passage 80 has been determined. Results of chromosome analysis, morphology, combined with the results of expression of marker protein and functional proteins suggested that the cells retained HCS cell properties. Furthermore, HCS cells have no tumorigenicity, and with excellent biocompatibility to aPCS. CONCLUSION: An untransfected and non-tumorigenic HCS cell line has been established, and the cells maintained positive expression of marker proteins and functional proteins. The cell line, with excellent biocompatibility to aPCS, might be used for in vitroreconstruction of tissue-engineered HCS.

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.

Effects of Adipose-derived Mesenchymal Stem Cell Exosomes on Corneal Stromal Fibroblast Viability and Extracellular Matrix Synthesis

Background： Corneal stromal cells （CSCs） are components of the corneal endothelial microenvironment that can be induced to form a functional tissue-engineered corneal endothelium. Adipose-derived mesenchymal stem cells （ADSCs） have been reported as an important component of regenerative medicine and cell therapy for corneal stromal damage. We have demonstrated that the treatment with ADSCs leads to phenotypic changes in CSCs in vitro. However, the underlying mechanisms of such ADSC-induced changes in CSCs remain unclear. Methods： ADSCs and CSCs were isolated from New Zealand white rabbits and cultured in vitro. An Exosome Isolation Kit, Western blotting, and nanoparticle tracking analysis （NTA） were used to isolate and confirm the exosomes from ADSC culture medium. Meanwhile, the optimal exosome concentration and treatment time were selected. Cell Counting Kit-8 and annexin V-fluorescein isothiocyanate/ propidium iodide assays were used to assess the effect of ADSC-derived exosomes on the proliferation and apoptosis of CSCs. To evaluate the effects ofADSC-derived exosomes on CSC invasion activity, Western blotting was used to detect the expression of matrix metalloproteinases （MMPs） and collagens. Results： ADSCs and CSCs were successfully isolated from New Zealand rabbits. The optimal concentration and treatment time of exosomes for the following study were 100 μg/ml and 96 h, respectively. NTA revealed that the ADSC-derived exosomes appeared as nanoparticles （40-200 nm）, and Western blotting confirmed positive expression of CD9, CDSI, flotillin-1, and HSP70 versus ADSC cytoplasmic proteins （all P〈 0.01 ）. ADSC-derived exosomes （50μg/ml and 100μg/ml） significantly promoted proliferation and inhibited apoptosis （mainly early apoptosis） of CSCs versus non-exosome-treated CSCs （all P 〈 0.05）. Interestingly, MMPs were downregulated and extracellular matrix （ECM）-related proteins including collagens and fibronectin were upregulated in the exosome-treated CSCs versus non-exosome-treated CSCs （MMPI： t = 80.103, P 〈 0.01; MMP2： t = 114.778, P 〈 0.01; MMP3： t = 56.208, P 〈 0.01; and MMP9： t = 60.617, P〈 0.01; collagen I： t = -82.742, P〈 0.01; collagen II： t = -72.818, P〈 0.01; collagen III： t = -104.452, P〈 0.01; collagen IV： t = - 133.426, P 〈 0.01, and collagen V： t - -294.019, P 〈 0.01 ; and fibronectin： t = -92.491, P 〈 0.01, respectively）. Conclusion： The findings indicate that ADSCs might play an important role in CSC viability regulation and ECM remodeling, partially through the secretion of exosomes.

The anti-scarring effect of corneal stromal stem cell therapy is mediated by transforming growth factorβ3

Background:Corneal stromal stem cells(CSSC)reduce corneal inflammation,prevent fibrotic scarring,and regenerate transparent stromal tissue in injured corneas.These effects rely on factors produced by CSSC to block the fibrotic gene expression.This study investigated the mechanism of the scar-free regeneration effect.Methods:Primary human CSSC(hCSSC)from donor corneal rims were cultivated to passage 3 and co-cultured with mouse macrophage RAW264.7 cells induced to M1 pro-inflammatory phenotype by treatment with interferonγand lipopolysaccharides,or to M2 anti-inflammatory phenotype by interleukin-4,in a Transwell system.The timecourse expression of human transforming growth factorβ3(hTGFβ3)and hTGFβ1 were examined by immunofluorescence and qPCR.TGFβ3 knockdown for>70%in hCSSC[hCSSC-TGFβ3(si)]was achieved by small interfering RNA transfection.Naïve CSSC and hCSSC-TGFβ3(si)were transplanted in a fibrin gel to mouse corneas,respectively,after wounding by stromal ablation.Corneal clarity and the expression of mouse inflammatory and fibrosis genes were examined.Results:hTGFβ3 was upregulated by hCSSC when co-cultured with RAW cells under M1 condition.Transplantation of hCSSC to wounded mouse corneas showed significant upregulation of hTGFβ3 at days 1 and 3 post-injury,along with the reduced expression of mouse inflammatory genes(CD80,C-X-C motif chemokine ligand 5,lipocalin 2,plasminogen activator urokinase receptor,pro-platelet basic protein,and secreted phosphoprotein 1).By day 14,hCSSC treatment significantly reduced the expression of fibrotic and scar tissue genes(fibronectin,hyaluronan synthase 2,Secreted protein acidic and cysteine rich,tenascin C,collagen 3a1 andα-smooth muscle actin),and the injured corneas remained clear.However,hCSSC-TGFβ3(si)lost these anti-inflammatory and anti-scarring functions,and the wounded corneas showed intense scarring.Conclusion:This study has demonstrated that the corneal regenerative effect of hCSSC is mediated by TGFβ3,inducing a scar-free tissue response.