The Derivation Of Corneal Endothelial Cell-like Cells From Skin Derived Precursors

The cornea is a transparent avascular tissue that consists of five layers from front to rear:epithelium,Bowman's membrane,stroma,and,Descemet's membrane and endothelium.Corneal endothelial cells(CECs)are composed of polygonal cells,mainly hexagonal cells.They form a single cell layer and connect to each other in mosaic structure covering the posterior surface of the cornea.The tight junctions as well as the ionic "pump" functions of CECs are important factors that maintain corneal dehydrated,transparency and normal thickness.Human CECs cannot regenerate in vivo,so the injured area can only be repaired by enlargement and migration of neighboring cells.When cell density is lower than 500-800/mm2,it is not sufficient to completely compensate the damage,resulting in corneal endothelial dysfunction which is characterized by corneal edema,bullous keratopathy,and loss of visual acuity.Penetrating corneal transplantation or corneal endothelial transplantation are now the most common and effective methods to treat corneal endothelial decompensation.However,severe lack of corneal materials cannot meet clinical demand.Therefore,looking for new functional corneal endothelial cells is very urgent.Differentiating stem cells into corneal endothelial cells can effectively solve the problems of insufficient corneal materials and difficult CECs culture in vitro.Restricted by ethics,the further research about embryonic stem cell is difficult to do,so more and more researchers focus on adult stem cells.Skin is the largest organ of mammal.Studies showed that skin regenerated depending mainly on the participation of skin stem cells.A variety of different types of stem cells had been found in skin,such as hair follicle stem cells,epidermal stem cells and dermal derived precursor cells,they are all adult stem cells.In addition to the application in the field of skin,skin stem cells have the potentialof differentiation to other type of cells because of high plasticity.Dermal stem cells derived from mesoderm,plasticity is higher,and differentiation to corneal endothelial cells may be a new research direction in the future.Skin-derived precursors(SKPs)from human and rodents dermis were discovered by Tomas,and they have been a research hotspot in recent years.As they are multipotent,in different conditions they can be differentiated into several functional cell types,like neurons and glial cells,bone and cartilage cells,smooth muscle cells,fat cells.SKPs can be easily isolated in large quantities and extraction method is simple.Thus,SKPs are ideal seed cells to be differentiated to CECs.Corneal transplantation is the major way to cure corneal endothelial dysfunction.Penetrating corneal transplantation is one important surgical method.However,it has many complications and the immune rejection ratio is high.Corneal endothelial transplantation includes:posterior lamellar keratop lasty(PLK),deep lamellar endothelial keratoplasty(DLEK),Descemet stripping endothelial keratoplasty(DSEK)and Descemet stripping automatic endothelial keratoplasty(DSAEK),Descemet membrane endothelial keratoplasty(DMEK).These techniques developed fast in recent years.Some researchers prepared corneal endothelial dysfunction models with ultrasonic emulsification or scraper,and then they injected cultivated CECs into anterior chamber with ROCK inhibitor in order to endothelial keratoplasty.It had succeeded in animals in recent years.These methods have common features,they have maintained normal epithelium and stroma,few complications,little surgical trauma,low immune rejection ratio,fast vision recovery.These provide new surgical choices for therapy of corneal endothelial dysfunction.Tissue-engineered cornea(TEC)combine scaffold and seeded cells to construct cornea substitute in vitro,which has biological activation.It has the potential to relieve pressure of lack of donor corneas.Scaffold is one important component,includes natural materials and synthetic materials.Synthetic materials include polylactic acid,polyglycolic acid and so on,but their cell adhere ability is not strong and mechanical strength could decrease.Natural materials mainly point to tissue derived from animal or human.This is the main source of materials for tissue-engineered cornea.Acellular human cornea matrix is the most ideal material for TEC.However,it also needs donor cornea,restricting its production.Recent year,acellular porcine cornea matrix(APCM)becomes the research hotspot.It has good tissue compatibility,low immunogenicity,and similar biomechanical characteristic to human cornea.In applications,it can support a lot of cells grow,and can be used to construct anterior,posterior,full-thickness tissue-engineered cornea.The animal experiments all had succeeded.Now there is related product in Chinese market for lamellar keratoplasty.Thus,APCM is the ideal scaffold material for tissue-engineered cornea.In conclusion,we first isolated SKPs to explore the feasibility of differentiating SKPs into CEC-like cells.By trying a lot of different methods,we had chosen the best methods and successfully induced SKPs to corneal endothelial cells which had similar morphology and markers to normal corneal endothelial cells.We also preliminarily explored the molecular mechanism of the induction,and found that SKPs differentiated into CEC-like cells probably by Wnt pathway.To detect the cell function in vivo,we transplanted CEC-like cells into rabbit corneal endothelial dysfunction models,and acquired good result.After transplantation in monkey corneal endothelial dysfunction models,results showed that CEC-like cells can exert excellent function similar to human CECs.Monkey corneas recovered transparency and could remain stable for a long time.We also explore the feasibility of tissue-engineered cornea construction using acellular porcine cornea matrix(APCM)and CEC-like cells.We found that skin-derived corneal endothelial cell-like cells grew well on APCM.Tissue-engineered corneas could be constructed with CEC-like cells.After transplantation,they could perform endothelial pump function partially.The renewable cell source,novel derivation method and good treatment effect may lead to potential clinical applications for corneal endothelial dysfunction or other corneal diseases in future.Purpose:Explore the feasibility of differentiating human skin derived precursors(SKPs)into corneal endothelial cell-like cells(CEC-like cells).Preliminarily explore the molecular mechanism of the differentiation.Methods:1.Isolation and cultivation of SKPs:Human skin was samples were collected from patients that underwent a double-eyelid operation.The epidermis was removed and incubated with Liberase.Then,the isolated dermal tissue was minced into small pieces and cells were dissociated.After filtered by cell mesh,cells were cultivated with SKP culture medium according to a certain density.2.Cultivation of B4G12 cells and collection of conditioned medium:The immortalized HCEC population B4G12 cells were cultured in human endothelial-SFM supplemented with human recombinant bFGF in culture flasks.Cells were passaged using trypsin.When the B4G12 cells grew to 70%-90%confluence,the medium was collected every 12 hours,and was stored in-80? refrigerator after filtering.3.Derivation of CEC-like cells from human SKPs:Human SKPs spheres were separated to single cells by trypsin,and then they were plated into 6-well plates coated with 1 chondroitin sulphate and laminin at a density of 1×105 cells/ml,5 × 105 cells/ml,or 1 × 106 cells/ml.(1)Methods of conditioned medium:Cultivate SKPs in B4G12 cell conditioned medium or its mixed medium with SKPs medium at a ratio of 3:1.The culture medium was changed every 2 days.(2)Method of coculture:SKPs were cocultured with B4G12 cell through transwell coculture system.B4G12 cells were cultured with SKPs through transwell.The culture medium was changed every 2 days.These methods were compared and the best was selected.4.Identification of CEC-like cells:CEC-like cells were identified by real time RT-PCR,immunofluorescence,and western blotting.Na+/K+ATPase,ZO-1,N-cadherin,CA2,Col4a2,Col8a2,Pitx2,FoxCl were detected by real time RT-PCR compared to SKPs.Expression of Na+/K+ATPase and ZO-1 were detected by immunofluorescence and western blotting.CEC-like cells were passaged and the expression of markers were detected by real time RT-PCR,immunofluorescence,western blotting.5.Preliminarily exploration of the differentiation molecular mechanism:expression of t-LRP6,p-LRP6,?-Catenin,Gapdh was detected in CEC-like cells and SKPs by western blotting.Results:1.After several experiments,we demonstrated that CEC-like cells could be derived from SKPs.The scheme that SKPs were cocultured with B4G12 cells at the density of 5×105 cells/ml is the best.During co-culturing with the B4G12 cells,the cell morphology began to change to a polygonal CEC-like shape on day 4.After 8 days,the polygonal CEC-like cells were the majority and formed a mosaic monolayer.They had similar morphology and characteristic to human CECs.When they were passaged,they grew well with similar morphology and characteristic to human CECs,and had strong proliferation ability.2.RT-PCR showed that CEC-like cells express significantly higher levels of several corneal endothelial markers and changed over time after derivation compared to SKPs The expression levels of differentiation transcription factors FoxC1 and Pitx2 were also significantly upregulated compared with SKPs.Immunofluorescent staining showed that the CEC-like cells expressed corneal endothelium major markers Na+/K+ATPase and ZO-1.Western blot analysis also showed that CEC-like cells express higher protein levels of Na+/K+ATPase alpha 1 and ZO-1 compared to SKPs.3.Western blot analysis showed that expression of three important molecules in Wnt pathway,active ?-Catenin,Phospho-LRP and total LRP,were significant higher in CEC-like cells than in SKPs.This indicated that CEC-like cells were differentiated through Wnt classical pathway.Upon stimulation with Wnt,LRP6 receptors increased and were phosphorylated,and free unphosphorylated ?-Catenin was accumulated leading to subsequent gene transcription and expression.Conclusions:SKPs could be differentiated into CEC-like cells through co-culturing with the B4G12 cells.This provides new sources of corneal endothelial cells.SKPs were differentiated into CEC-like cells probably through Wnt classical pathway.Purpose:Explore the function of skin-derived corneal endothelial cell-like cells in vivo and the feasibility to cure animal corneal endothelial dysfunction model.Methods:1.Rabbit corneal endothelial dysfunction model:rabbits were divided into two groups and the right eyes were selected for our experiment.The corneal endothelium was mechanically scraped with a lacrimal passage irrigator from the Descemet's membrane.HE staining and alizarin red staining were carried out to confirm the intact Descemet's membrane and no residual cells.2.Injection of cultivated CEC-like cells into the rabbit eyes:CEC-like cells were labeled by Dil reagent and suspended in 100?l culture medium.100?l aqueous humor was first extracted from the anterior chamber and then cells were injected into the anterior chamber.Each surgical eye was examined with a slit-lamp microscope,tenonometer,Visante OCT and confocal microscopy at certain times.Postoperative eye were removed.Alizarin red and trypan blue,immunofluorescent staining and HE staining were carried out to detect the CEC-like cells.3.Monkey corneal endothelial dysfunction model:monkeys were divided into two groups and the right eyes were selected for our experiment.The corneal endothelium was mechanically scraped with a lacrimal passage irrigator from the Descemet's membrane.4.Injection of cultivated CEC-like cells into the monkey eyes:CEC-like cells were labeled by Dil reagent and suspended in 50?l culture medium.50?l aqueous humor was first extracted from the anterior chamber and then cells were injected into the anterior chamber.Each surgical eye was examined by a slit-lamp microscope,tenonometer,Visante OCT,non-contact specular microscopy,B-mode ultrasound and fundus photography at certain times after surgery.Postoperative eye were removed HE staining and immunofluorescent staining were carried out to detect the CEC-like cells.Results:1.After CEC-like cells were transplanted into the rabbits corneal endothelial dysfunction models,the clarity of the CEC-like cell group corneas significantly improved,and the pupil and iris texture could be seen.After only about 7 days,the corneas became clearly transparent,while corneal opacity and stromal edema were still serious in the control group.Corneal thickness rapidly decreased after injection of CEC-like cells and polygonal cells covered the Descemet's membrane.However because of corneal opacity,this could not be clearly detected in control group.Histological examination showed Dil-labed CEC-like cells covered nearly full descemet's membrane and expressed Na+/K+ATPase in CEC-like cells injected group while almost no cells were detected on descemet's membranes in control group.2.CEC-like cells were transplanted into the monkey corneal endothelial dysfunction models,corneas became significantly clearly transparent that the pupil and iris texture could be seen,while the corneas of the control group were cloudy and thick.One month later,the corneas of CEC-like cell group became clearer and thinner,whereas corneal opacity and stromal edema became more and more serious in the control group.CEC-like cells were in the form of a polygonal monolayer.However because of the corneal opacity,this could not be clearly detected in the control group.Only mild inflammatory keratic or anterior chamber precipitates appeared after surgery and gradually disappeared a few weeks later.One year later,the cornea of the CEC-like cell group were still transparent while the corneal opacity and stromal edema were still serious in the control group.There were no new precipitates or vessels appeared during observation.B-mode ultrasound and fundus photography showed no pathological changes of the surgical eyes.Intraocular pressures were found to be in the normal range in all groups.Na+/K+ ATPase were also expressed in CEC-like cells,indicating the persistent pump function.HE staining showed that the cornea was almost normal and the CEC-like cells tightly connected to each other on the Descemet's membrane and there were little inflammatory cells in the cornea.In addition,no obvious tissue abnormalities were found in other parts of postoperative monkey.Conclusions:Skin-derived CEC-like cells have similar function with human CEC in vivo.We not only achieved good result in rabbit corneal endothelial transplantation but also acquired long-term therapeutic effect in monkey corneal endothelial dysfunction models.It provides new choice of therapy for corneal endothelial dysfunction.Purpose:Explore the feasib:ility of tissue-engineered cornea construction using acellular porcine cornea matrix(APCM)and CEC-like cells.Assess the animal transplantation effect.Methods:1.Preparation of APCM and APCM scaffold(AS):Fresh pig eyes were washed with phosphate buffer saline containing penicillin-streptomycin.The central corneas were excised using a trephine,and then treated with 1.5 M sterile sodium chloride solution and 5 U/mL DNase/RNase to remove the cells in porcine corneas.APCM was fabricated to.APCM lamella containing Bowman's membrane was cut to about 400?m-thickness to prepare AS and stored in-20? refrigerator.HE and DAPI staining were carried out to detect the morphology.2.Construction of tissue-engineered corneas(TECs):CEC-like cells were seeded on stromal side of AS at a certain density and cultivated for 7 days.HE and DAPI staining were carried out to detect the morphology.Alizarin red and trypan blue were carried out to detect the CEC-like cell density.3.Rabbit penetrating corneal transplantation:Before transplantation,the TECs were cut into 5 mm diameter grafts and penetrating keratoplasty was performed.The experimental group was transplanted with TEC,and control group was transplanted with AS.Each surgical eye was examined with a slit-lamp microscope,tenonometer,Visante OCT at certain times.Postoperative eye were removed.Immunofluorescent staining and HE staining were carried out to detect the CEC-like cells.Results:1.HE and DAPI staining showed that cells were removed completely from APCM.Collagen was well arranged and the bowman's membrane was intact.TECs were well constructed and CEC-like cells formed a monolayer which attached tightly to thestromal side.CEC-like cell density of TECs was similar to normal rabbit CECs.2.After transplantation,the corneas in experimental group transparency increased gradually and corneal thickness gradually decreased.Corneas in control group gradually became opaque and more and more thick.Epithelium gradually covered grafts in both groups.The grafts were completely re-epithelialized on 3 weeks and corneal fluorescein staining was negative.The intraocular pressures in two groups were all in normal range.There were no significant new vessels during observation.HE staining showed that,the CEC-like cells formed a monolayer well attached to the stroma in TEC group.Howerer,no CEC-like cells were detected in AS group.Epithelium covered grafs and a few host keratocytes immigrated into graft stroma in both groups.The Immunofluorescent staining of human nuclei confirmed that the endothelium in TEC group were seeded CEC-like cells,not the migration of host endothelium.CEC-like cells still expressed Na+/K+ ATPase,indicating the maintained pump function in vivo after transplantation.Conclusion:Skin-derived corneal endothelial cell-like cells grew well on APCM.Tissue-engineered corneas could be constructed with CEC-like cells.After transplantation,they could perform endothelial pump function partially.