Studies On The Transcriptional Regulatory Mechanisms Of Tetralogy Of Fallot Through Induced Pluripotent Stem Cell Model

[Background and Objective]Congenital heart disease(CHD)is the most common birth defect disease in humans,it's the leading cause of death in non-infectious diseases in infants.Tetralogy of Fallot(TOF)is the most common complex and cyanosisc ongenital heart disease with a high mortality rate,and untreated patients have a mortality rate of 70-75%.However,due to the current lack of disease models,the etiology,cytology and molecular etiology of TOF remain unclear.Therefore,we need a new tool or disease model to systematically study TOF,so that we can better explore the pathophysiological mechanisms in the process of its occurrence and development.Induced pluripotent stem cells(iPSCs)is a kind of cells similar to embryonic stem cells(ESCs)obtained by transfecting reprogramming factors into somatic cells.Based on its well-defined genetic background,iPSCs provide a new tool for studying the developmental biology process and genetic diseases.In the research of cardiovascular disease,base on its good ability to retain the genetic background of samples and mimic the development process of the heart,iPSCs provide a unique cellular platform for exploring cardiac abnormal caused by genetic variation.Based on the above background,we established firstly iPSCs lines from TOF or healthy donors to explore the pathogenesis of TOF.Then,iPSCs were induced to differentiate into cardiomyocytes,and meanwhile functional differences of iPSCs between TOF and healthy donors at the cellular level were compared.Finally,the molecular pathogenesis of TOF at the transcriptome level was explored based on the cardiomyocytes model from iPSCs.[Methods:]The first part:The skin tissues from TOF or health donors were collected,and skin fibroblasts were obtained by tissue adherent method.The classic "OCT3/4,SOX2,Klf4 and c-Myc" four reprogramming factors were transfected into primary cultured skin fibroblasts by Sendai virus.The pluripotency genes and proteins of iPSCs lines were detected by alkaline phosphatase staining,immunofluorescence staining,flow cytometry and qPCR.The karyotypes of these cells were analyzed by G band karyotype analysis.Finally,the differentiation potential of iPSCs lines were tested by teratoma formation assay in NOD-SCID mice.The second part:Using the small molecules CHIR99021 and IWP-2 to intervened the Wnt pathway,combined with vitamin C and BMP4 to induce iPSCs lines to differentiate into cardiomyocytes by monolayer culture-based method.The differentiated cardiomyocytes were purified by lactic acid metabolism method.The specific genes and proteins of the induced cardiomyocytes were detected by immunofluorescence,flow cytometry and qPCR.The ultrastructure of cardiomyocytes was detected by transmission electron microscopy.Then,the proliferative abilities of different groups of iPSCs derived cardiomyocytes were detected by CCK8 and Ki67 staining.The migration abilities of the cardiomyocytes were detected by scratch assay.The third part:The cardiomyocytes induced from iPSCs were obtained from TOF or healthy donors(The samples were collected from each iPSCs line which was induced from three independent differentiated and purified procedures).After amplification of the cells transcriptome,samples were sequenced by high-throughput sequencing technology,and the transcriptome sequencing data were analyzed to find the differential expression genes between the TOF group and the healthy control.Further bioinformatics analysis was used to detect key differential expression genes.Then,qPCR was used to verify key differential genes.Furthermore,siRNA was used to knock down the expression of the key differential gene BMP10 in AC 16 cardiomyocytes.The cell proliferation was detected by CCK8 assay and EdU staining in cardiomyocytes with BMP10 gene knockdown successfully.The migration of cardiomyocytes was detected by scratch assay.The above experiments were used to clarify the function of BMP10 in cardiomyocytes.Finally,western blot and qPCR were used to explore the possible invovled in pathway and molecules regulated by BMP10.[Results]The first part:The skin fibroblasts were obtained successfully from three TOF or two healthy donors by using the tissue adherent method,and five fibroblast cell lines had good proliferative abilities.Through using Sendai virus carrying the classical"OSKM" reprogramming factors,we successfully induced the above five fibroblast lines into iPSCs with a similar morphology to ESCs.In the process of culture,we found that two TOF and one healthy control cell lines had good clone morphology and proliferation abilities,while the other 2 cell lines showed poor proliferation abilities and obvious self-differentiation.Therefore,we selected three good cells for identification and study.The alkaline phosphatase staining results of three iPSCs lines were positive.The genes or proteins associated with pluripotency such as OCT4 and Nanog were highly expressed,which were detected by immunofluorescence,flow cytometry and qPCR.The karyotype is normal after 20 passage.Teratoma formation assay was used to confirm that all three cell lines had good differentiation abilities to all three germ layers.The second part:Through the intervention of Wnt pathway combined with vitamin C and BMP4,the three iPSCs lines successfully differentiated into cardiomyocytes which could beat spontaneously.Moreover,the results from flow cytometry showed that the above inductive method possesssed a high efficiency and the positive rate(CTNT+)of three cell lines were higher than 80%.In addition,there was no difference on induction efficiency between the TOF group and healthy control group.The positive rate of three differentiated cardiomyocytes was successfully purified to nearly 95%by using the method of lactic acid metabolism.The differentiated cardiomyocytes showed high expression of myocardial specific protein CTNT and ?-actinin by immunofluorescence.The results of qPCR showed that myocardial specific gene expression including GATA4,NKX2.5,MEF2C,MLC-2v,Hand1,CTNT were also significantly increased.There was no significant difference in cardiomyocytes volume,beating rate,ion channel gene expression and ultrastructure between the TOF group and the healthy control.The data from CCK8 and Ki67 staining showed that the proliferative abilities of cardiomyocytes derived from TOF group were lower than those of health control and the TOF-1 group was significantly reduced.Scratch assay confirmed that the migration abilities of cardiomyocytes derived from the TOF group was also significantly reduced.The third part:A total of 1388 differentially expressed genes were screened between the TOF group and the healthy control group.Compared with the healthy control group,there were 1006 up-regulated genes and 382 down-regulated genes in TOF group.Through GO enrichment analysis of differentially expressed genes,we found that down-regulated genes were significantly enriched in terms associated with cardiac development.Further bioinformatics analysis found that MLY2,CSRP3,BMP10,TBX5,TBX20,WNT2,ISL1,CXCL12,TNF,SNAI1,S1PR1 and SERPINE1 genes are located at the center of the local gene clusters.The qPCR verified that the gene expression level of MLY2,CSRP3,BMP10,TBX5,TBX20,CXCL12,TNF and SERPINE1 were significantly lower in the TOF group than those in the healthy control group.Finally,we successfully knocked down the BMP 10 gene in AC 16 cardiomyocytes through siRNA,and found that the proliferative ability of cardiomyocytes significantly decreased,while the migration ability was not remarkablely changed.Further downstream regulatory mechanism analysis revealed that the phosphorylation levels of smadl/5 and Erk1/2 were down-regulated,and the expression levels of downstream transcription factors TBX5 and TBX20 were down-regulated after BMP10 knockdown.Similarly,the expression levels of p-Smad1/5,p-Erk,TBX5,and TBX20 were also down-regulated in the iPSCs-TOF groups.[Conclusions]1.This study established iPSCs cell lines from TOF and healthy donors successfully,which can be used as a disease model to research the pathogenesis of TOF at the cellular level.2.There was no difference in cardiomyocyte differentiative efficiency between iPSCs from TOF group and those from heath control group.However,the proliferation and migration abilities of cardiomyocytes derived from TOF group were decreased.Therefore,we speculated that the abnormality of cardiomyocyte proliferation and migration abilities may be associated with TOF during cardiac development.3.After analyzing and verifying the transcriptome data,we found that down-regulatory genes MLY2,CSRP3,BMP10,TBX5,TBX20,CXCL12,TNF and SERPINE1 may be associated with the occurrence of TOF through affecting the proliferation and migration of cardiomyocytes.4.The further mechanism study demonstrated that the abnormal expression of BMP10 could affect the proliferation of cardiomyocytes by regulating the downstream Smad1/5 and MAPK signaling pathways,thus participated in the occurrence of TOF.Moreover,during the development of TOF,TBX5 and TBX20 may be the downstream regulatory transcription factors of BMP10.