The Mechanism Study Of Diabetic Retinopathy Based On IPSC-ECs And IPSC-RPE Cells Derived From Patients

Objective:To establish induced pluripotent stem cells(iPSCs)line derived from blood cells of patients with diabetic retinopathy(DR)by reprogramming technology,and then construct iPSCs-derived vascular endothelial cells(DR-iPSC-ECs)and retinal pigment epithelial cells(DR-iPSC-RPE)model.The characteristics of changes in differentiated cell morphology,molecular biology,and function will be explored to study the pathogenesis of DR.Methods:1.The peripheral blood of patients with clinically diagnosed DR was collected,and peripheral blood mononuclear cells(PBMCs)were separated via gradient centrifugation.Then PBMCs were carried out for primary culture.PBMCs were transfected with Sendai virus vector to induce reprogramming into iPSCs,and then the obtained iPSCs were identified in terms of cell morphology,stem cell marker gene expression,differentiation potential,and safety.2.In vitro directed differentiation technique was used to make bloodderived iPSCs of DR patients and normal healthy people into ECs.The ECs derived from iPSCs of DR patients were considered as DR-iEC group,and the ECs derived from normal human iPSCs were Control-iEC group.The expression of specific genes and protein markers of ECs were detected by RT-qPCR and immunofluorescence staining.iEC RNA was extracted for high-throughput sequencing to test the differential gene expression between the DR-iEC group and Control-iEC group.3.The iPSCs of DR patients and normal people were induced to differentiate into RPE cells.The gene and protein expression differences in the RPE cells of the DR-iRPE group and the Control-iRPE group were detected by RNA-sequence and quantitative proteomics.Results:1.Two i PS cell lines derived from DR patients were successfully established.These two cell lines have the following characteristics:(1)Both iPSCs cell lines had typical ESCs-like clonal morphology.(2)Immunofluorescence staining showed that such cells were both positive with OCT4 and SSEA4.(3)Immunofluorescence staining confirmed that both iPSCs cell lines could differentiate into three germ layer cells with positive expressions of AFP(endoderm),α-SMA(mesoderm),and PAX6(ectoderm)respectively.(4)RT-PCR also showed that both iPSCs cell lines had the differentiating ability for the positive expression of SOX17,GATA4(endoderm),MIXL1,TBXT(mesoderm),MAP2,PAX6(ectoderm).2.The iPSCs derived from DR patients and normal people were differentiated into ECs.Immunofluorescence staining showed that the differentiated ECs were positive with ECs specific markers CD31,CD144 and v WF.The tube-forming experiment displayed that the tube-forming ability in the DR-iEC group was better than that in the Control-iEC group.3.The iPSCs derived from DR patients and normal people were differentiated into RPE in vitro.Immunofluorescence staining showed that the RPE derived from iPSCs could positively express ZO-1 and Mi TF,and the expression level of ZO-1 in the DR-iRPE group was lower than the normal group.The morphology of RPE cells in the DR-iRPE group was irregular.Compared with the Control-iRPE,the RPE barrier function of the DR group decreased.4.Compared with the Control-iEC group,the qPCR assay exhibited that the expressions of IL18 and NLRP12 were up-regulated in the DR-iEC group,which was consistent with the RNA-seq results.Under high glucose conditions,the apoptosis-related genes of TLR4 and CASP3 in the DR-iEC group were significantly up-regulated compared with the normal glucose condition,while the expressions of TLR4 and CASP3 in the Control-iEC group were not significantly different from those under the normal glucose condition.5.RNA-seq results revealed that compared with the Control-iRPE group,aging-related genes and EMT-related genes were up-regulated,and cilia assembly-related genes were down-regulated in the DR-iRPE group.The gene expressions of IL6,FOS,and VEGFA were up-regulated,and the expression of CFAP53 was down-regulated,and the difference was statistically significant.ELISA results showed that the VEGF secreted by RPE cells in the DR-iRPE group was higher than that in the Control-iRPE group.6.The integrated analysis of RNA-seq and proteomics showed that the differential genes/proteins of RPE cells in the DR-iRPE group and the Control-iRPE group had a certain correlation.Compared with the ControliRPE group,the cytokine response-related genes(CD38,IFIT3,TRIM22,etc.)in the DR-iRPE group were significantly up-regulated.The expressions of apoptosis-related genes(BBC3,TLR3,PRODH,etc.)were up-regulated,and the oxidative stress-related genes(CYP1B1,PNPT1,etc.)were up-regulated,and TXNRD2 was down-regulated,aging-related genes(HMGA2 and PNPT1)were up-regulated,and CDKN2 A was downregulated.Conclusion:1.Two iPSC cell lines derived from blood cells of DR patients are successfully generated by non-integrating Sendai virus transfection.2.iECs and iRPE cells can be effectively obtained from iPSCs in DR patients and normal persons by in vitro directed differentiation technique,which provides a good cell model for further study on the pathogenesis of DR.3.The integrated analysis of RNA-seq and proteomics uncovers that the differential expressions of genes and proteins in the DR-iRPE group and the Control-iRPE group are closely related to oxidative stress,apoptosis,aging,etc.,which are of great significance for drug screening and new treatment exploration in the future.