| Objective: To study and discuss the directional differentiation of human induced pluripotent stem cells(hi PSC)into human corneal endotheliad-like cells under defined conditions in vitro.Hi PSC differentiated into corneal endothelial cells by adding SB4315542,CHIR99021,fibroblast growth factor(FGF),XAV939,Blebbistatin,platelet-derived growth factor(PDGF),Laminin511 coating protein and other chemical components into the inducing differentiation medium.New Zealand white rabbits corneal endothelial dysfuncition model were established to verify the therapeutic function of hi PSC-derived endothelial-like cell in vivo,and to provide a new way and strategy for clinical treatment of corneal endothelial dysfunction.Methods: hi PSC was differentiated into human corneal endothelioid cells by a two-step induction scheme using a clear chemical composition medium.In the first step,SB4315542 and CHIR99021 were added to regulate TGF-β and Wnt/β-catenin signaling pathways,and hi PSC differentiation was induced to day 7.The expression of neural crest stem cell specific indicators SOX10 and β-catenin was detected by immunofluorescence staining,and the expression of neural crest stem cell specific indicators SOX9,SOX10,NGFR,HNK-1 andβ-catenin m RN was detected by QRT-PCR.The expression of NGFR(P75)and HNK-1proteins specific to neural crest stem cells was detected by flow cytometry.In the second step,XAV939,a novel Wnt/β-catenin signaling pathway inhibitor,Blebbistatin,a selective non-muscular myosin II inhibitor,PDGF and Laminin511 were used to induce neural ridge stem cells from day 7 to day 14.The expression of corneal endothelial cell specific indicator ZO-1 was detected by immunofluorescence staining,and the relative expression of CORneal endothelial cell specific COL4A1,COL8A1,COL8A2 and ZO-1 m RNA was detected by QRT-PCR.Adult New Zealand rabbits were divided into three groups: corneal endothelial decompensation model group,neural crest stem cells(NSCS)obtained from the 7th day of cell differentiation by anterior chamber injection,and corneal endothelioid cells(CORneal endothelial cells)obtained from the 14 th day of cell differentiation by anterior chamber injection.5 eyes in each group were observed under slit lamp and examined by anterior segment photography,optical coherence tomography(AS-OCT),central corneal thickness,histopathology and alizarin red staining.Results: During the differentiation process,the pluripotent stem cells were induced to gradually lose monoclonal morphology under light microscope,and the expression of SOX10 and β-catenin proteins was detected by immunofluorescence on the 7th day,and the purity of SOX10 expression could reach more than 80%.The expression purity of β-catenin was over90%.Qrt-pcr results showed that compared with undifferentiated hi PSC,m RNA expressions of SOX9,SOX10,NGFR,HNK-1 and β-catenin in neural crest cells harvested on day 7 of induced differentiation were significant,and the relative expression levels of SOX10 and HNK-1m RNA were the highest,and the differences were statistically significant.Flow cytometry showed that the high expression of HNK-1 and NGFR(P75)protein was 97.41%and 84.07%,respectively,and high purity neural crest cells were obtained.The neural crest cells continued to be induced and differentiated under optical microscope until day 14,and irregular single-layer hexagonal corneal endothelial cells with tightly connected structures were observed.Immunofluorescence showed that z O-1 was highly expressed in corneal endothelial cells.Qrt-pcr results showed that compared with undifferentiated hi PSC,the m RNA levels of COL4A1,COL8A1,COL8A2 and ZO-1 in corneal endothelial cells were significantly expressed,and the differences were statistically significant.In animal experiments,corneal edema was observed in the control group,and the degree of corneal edema and turbidity did not decrease with the extension of time during the observation period.The postoperative edema of the anterior chamber injection group was lighter than that of the control group,but the degree of corneal edema gradually increased with time.In the group of anterior chamber injection of corneal endothelial cells,the edema was significantly reduced 3 days after the operation,but the edema began to worsen 7 days after the operation.Corneal thickness was measured by anterior segment OCT,and the average corneal thickness in the corneal endothelial decompensation group was 1.606 mm,1.876 mm and 1.758 mm on day 1,3 and 7 after surgery,respectively.The mean corneal thickness of the anterior chamber injection group was 1.34 mm,1.537 mm,1.588 mm on day 1,3 and 7 after surgery,respectively.The mean corneal thickness of the anterior chamber injection group was 0.822 mm,0.787 mm and 1.08 mm,respectively,on day 1,3 and 7 after surgery.Corneal pathological examination of normal New Zealand rabbits showed that the epithelium,preelastic layer,stroma layer and endodermis were clearly visible,all layers were neatly arranged,and the corneal structure was complete.A large number of neatly arranged collagen tissues were found in the corneal stroma,and the endothelium was composed of monolayer cells.On the third day after operation,the sections of the patients in the anterior chamber injection group showed that the injected corneal endothelioid cells were all larger than the normal corneal endothelial cells.These cells appear to be loosely attached to the membrane of the corneal elastin.Human antinuclear staining of endothelial cells was positive,confirming that the cells adhered to the elastic layer were induced corneal endothelioid cells injected into the anterior chamber.Conclusion: i PSC can be directed to differentiate into CEC-like cell by using novel molecular compounds and chemically defined induction protocols.The CEC-like cells had corneal endothelial morphology and expressed corneal endothelial cell markers.Injection of CEC-like cells into the anterior chamber can relieve corneal edema in the short term.This study provides a new ideas for the directed differentiation of corneal endothelial cells. |
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