A Preliminary Study On Induction Of Mouse ES/iPS Cells Differentiation To Corneal Endothelium Ex Vivo

PurposeEmbryonic stem cells(ES-cells)and indued pluripotent stem cells(iPS-cellls)have extensive selfrenewal capacity and are competent to differentiate into all types of cells in vivo and in vitro,bringing huge hope for future regenerative medicine.To explore the possibility of mouse ES and iPS cell to differentiate into corneal endothelial cells in the given condition mimicking the embryonic corneal endothelium development in vitro.Considering the neural crest(NC)origination of corneal endothelium,generating NC-like cell in the first stage is necessary.So we inquiried possibility and compared efficiency of different methods such as RA-treartment in EB differentiation and b FGF+BMP4 induction in monolayer adherent culture)yeilding NC cells combined with sequential induction via coculture to develop a high efficient diffferentiation system.Methods1.Rrabbit's Descemet membrane and lens capsue membrane peeled down for corneal endothelial cell(CEC)and lens epithelial cell(LEC)isolation and expansion,normal phenotype was observed.LEC-CM was collected to investigate the regulatory effect on CEC.MTT and ICC of Ki67 were explored to proliferation ability comparison,West blot to specific marker expression comparison.2.ES and iPS cells were cultured on feeder with lif to maintain its undifferentiation status.Its pluripotent was proved by associated marker detection and teratoma forming.Removal of feeder and lif made embryoid body formingin suspension culture.Various concentrantion RA used for NC induction,m RNA expression profile according to time phase was compared and.various coating methods were carried using Fibronectin(Fn)?Laminin(Ln)?gelatin(g)?poly-l-lysine(PLL)?aquosus humor as extra cell strom for EB migration and differentiation,then efficiency was observed and compared.Migration of NC cell on gelatin coating was then analysized by ICC/IF.3.Passage2-3 CEC or LEC of rabbit was applied to various coculturemodel:(1)collectionofconditioned medium,(2)microencapsulation in coculture,(3)coculture in transwell-insert,(4)Feeder after MMC treatement,(5)active cell layer.ES/iPS cells derived NC cells were sequentialy induced in various coculture conditions.Methods were compared and the best was confirmed.CEC specific marker was detected using ICC/IF.4.The NC inductiion was optimized by two factors conbimed(b FGF+BMP4)in monolayer adherent culture,and sequential induction to CEC differentiation was still done using LEC-CM.ICC and realtime PCR were explored for phenotype change analysis as previous study.Results1.Rabbit CEC?LEC can be cultivated ex vivo with good proliferation and nomal phenotype.LEC-CM had little effect on CEC proliferatin,but CEC cultured in LEC-CM expressed higher ZO-1?N-cadherin and Vimentin.2.ES/iPS in routine culture with Lif and Feeder maintained pluripotent differentiation potential,expressing Oct4,SSEA-1and strong AP activity,forming teratoma in vivo,as well as forming embryoid body in suspension culture without lif and Feeder.Protocol of 0.5-1u M RA treatment at EBd4 for 4 days gave rise to NC-like cells whose m RNA expression profile detected by realtime PCR confirmed NC differentiation.When EBs were seeded onto gelatin coated plate,cells migrating showed expression of NC marker P75,AP-2?,SOX10 in ICC/IF.3.In CEC and LEC CM sequential induction,CEC-like cell composed clone well formed giving appearance of polygonal shape and CEC specific marker expression in ICC/IF such as AQP1?ZO-1?Na~+-K~+-ATPase?N-cad?VE-cad at day 7.While others coculture failed to producing so vigorous growth or directional differentiation.LEC-CM proved more efficient induction effect as m RNA profile accounted.4.ES-cells was effiencially induced to NC cell differentiation by two factors combined(b FGF+BMP4)and senquential induction in LEC-CMproduced higher yielding of CEC–like cell with CEC markers detected at day 6.Conclusions1.LEC-CM had little effect on CEC proliferation,while maintained CEC phenotype.2.RA drived mouse ES and iPS cells differentiation to NC-like cell.Gelatin coating for longtime provided opitimum condition for NC cell migration and sequential differentiation.3.CM as coculture was an easier?feasible and more controllable model.CEC-CM and LEC-CM both promoted ES/iPS cells derived NC-cells to CEC-differentiation in sequential induction.4.On the basis of establishing ES cell differentiation system to NC by monolayer adherent culture with two factors,LEC-CM for sequential induction to CEC gained higher efficiency in shorter period.