Corneal Endothelial Cell Biology And Biomimetic Promotion For IPS Cell Differentiation To Corneal Endothelial Cell

Part1The influence of aqueous humor on the growth of bovinecorneal endothelial cells in vitroObjective: The aim of this study was to evaluate the role of aqueous humor on bovinecorneal endothelial cells (B-CEC) in vitro.Methods: Aqueous humor of1.2ml was collected from the anterior chamber ofbovine and sterilized, and the liquid supernatant was obtained. The B-CEC wereisolated from bovine cornea and then cultured in low glucose Dulbecco ModifiedEagle Medium (DMEM) with10%fetal bovine serum (FBS) in vitro. Aqueous humorwere added into the medium with the final concentration of2.5%,5.0%,10.0%,15.0%and20.0%, respectively, and no aqueous humor was added in the control group. Cellcounting kit-8(CCK-8) assay was used to detect the absorbency value of B-CEC forthe evaluation of cell proliferation. Progression of the cell cycle was analyzed by flowcytometry (FCM). When cells reached confluence,1ml plastic pipette tip was used toscratch the cell single layer, and the cells were incubated consequently in10%FBSculture medium with or without aqueous humor for24hours. Healing area of the cellsingle layer was tested. The cells were incubated at a density of6×105cells/ml andcultured using medium with or without10.0%aqueous human for5days, and thenumber of the cells was analyzed by DAPI fluorescence stain.Results: The confluent B-CEC showed a slabstone-like and hexagonal appearance.CCK-8assay revealed that the absorbance values of B-CEC was significantlydifferent among the various culture groups (F=4.051, P=0.007), and the absorbancevalue in different concentrations of aqueous human culture groups were significantlyhigher than that in the control group (P<0.01) and those in10%aqueous humanshowed the highest result. FCM showed that the percentage of the cells in S and G2phases was (34.80±3.13)%in the10.0%aqueous humors group and (23.06±1.13)%inthe control group, showing a significant difference (t=-5.729, P=0.005). The scratchtest showed that would area of the cell signal layer was (0.116±0.019) mm2in the10.0%aqueous humors group and (0.358±0.049) mm2in the control group, showing asignificant difference (t=13.842, P=0.000). The density of cells in the10.0%aqueous humor group was (1439±110)/field, which was more than (1162±45)/field in thecontrol group (t=-11.020, P=0.000).Conclusion: Aqueous humor at the concentration of10.0%can promote the growthand proliferation of B-CEC. The result suggests that10.0%aqueous humor can beused as a promoting agent in the culture of B-CEC. Part2The stimulatory effect of ROCK inhibitor on bovinecorneal endothelial cellsObjective: To investigate the effect of a specific inhibitor of Rho-associated proteinkinase (ROCK), Y-27632, on cultured bovine corneal endothelial cells (B-CEC).Methods: Primary cultures of B-CEC were established from explants of theendothelial cell layer. B-CEC cultured with1%serum conditions media were used foranalysis of the proliferation effects of Y-27632. Progression of the cell cycle andapoptosis were analyzed by flow cytometry. Apoptotic cells which stained withAnnexinV-FITC/propidium iodide (PI) were also observed using fluorescencemicroscope. The phase-contrast microscopy was used to observe the morphologicalcharacteristics of B-CEC induced by Y-27632. Immunofluorescence analysis for theexpression of β-actin, AQP1and Na+/K+-ATPase in B-CEC was carried out aftertreated with Y-27632. The abilities of early adhesion and cell-cell interactions aftertrypsin enzyme digestion with Y-27632treatment were evaluated. Wound-healingscratch test and hanging drop aggregation assay were performed to determine changesin cellular migration.Results: The proliferation of cultured B-CEC was moderately promoted by10μMY-27632for cell growth assay. Y-27632induced morphological changes in thecultured B-CEC and normal cell morphology could recover after Y-27632removed.In addition, Y-27632significantly enhanced both the adhesion and migration ofB-CEC from the assays of early adherent experiment, trypsin enzyme digestion andscratch test. Furthermore, the hanging drop aggregation assay showed that Y-27632 promoted B-CEC to form cellular network and sheet proliferation along liquid-airinterface and move up to the surface of the lid of dish.Conclusion: Our results demonstrate that the use of Y-27632as an additive is a goodstrategy to achieve proliferation, adhesion and migration of cultured B-CEC. Y-27632will be favorable to future CEC engraftment therapy and topical application for CECdeficiency. Part3The biomimetic promotion for iPS cell differentiation tocorneal endothelial cellsObjective: in order to promote the differentiation of induced pluripotent stem (iPS)cells into corneal endothelial cell (CEC)-Like cells and obtain new way for gettingCEC derived from iPS cells, the applications of a variety of conditions whichreplicated corneal developmental context and microenvironments in vivo wereexplored.Methods: Morphology of the iPS cell was observed by phase-contrast microscopy.The procedure of iPS cell differentiation to corneal endothelial cells as follow:(1)The co-culture of iPS cells with B-CEC was carried out and their morphologicalchanges were observed under phase-contrast microscopy and scanning electronmicroscopy (SEM).(2) In order to supply a biomimetic environments and replicatecorneal developmental context, GSK-3β inhibitor VI, retinoic acid (RA), b-FGF,TGF-β2, Aqueous humor, bovine corneal endothelial extracellular matrix (B-ECM)and co-culture were used to promote iPS cell differentiation to corneal endothelialcells. Immunofluorescent stains of iPS markers (SSEA4、Oct4、TRA-1-60and SOX2),neural stem cell marker (Nestin), and CEC markers [zonula occludins-1(ZO1),N-Cadherin, Na+/K+-ATPase and AQP1] were applied and visualized underfluorescence microscopy. Results: After co-culture induction, most of the iPS cells performance out of the state.SEM evaluation indicated the rough surfaces of B-ECM, which were composed of aseries of filament fiber arrangement. GSK-3β inhibitor VI and RA were key factorsfor differentiation to corneal endothelium, which promoted the induction from iPScells to neural cells. After differentiating induction, CEC-like cells derived from iPScells showed polygonal shape and expressed the neural stem cell maker of Nestin.Aqueous humor, TGF-β2and B-ECM were important for further differentiation ofCEC-like cells from iPS cells. The expressions of N-Cadherin, ZO-1andNa+K+-ATPase markers, which characterize corneal endothelial cells, were positive insome differentiated cells.Conclusion: When GSK-3β inhibitor VI, retinoic acid, b-FGF, TGF-β2, aqueoushumor were used and co-culture with B-ECM and CEC, iPS cell differentiation tocorneal endothelial cells was induced. The entire context of a cell’s microenvironmentis important for iPS cell differentiation. The cues from the replication of CECdevelopmental process and biomimetic environments of the properties of cornealnative tissue can promote the differentiation of iPS cells into CEC-Like cells. Ourstudy supply a new way for getting CEC derived from iPS cells by biomimeticparadigm.