| Cardiovascular disease continues to be the leading cause of death.With the continual development of science and technology,implant valves,vascular stents,and alternative repaired substances can treat part of cardiovascular diseases.However,the cardiomyocyte,the foremost thick cell kind within the heart,lack a stable and reliable supply.Therefore,researchers have turned their attention to Pluripotent Stem Cells(PSCs)that have the potential to differentiate into different cell sorts.A variety of experiments has shown that somatic cell-derived cardiomyocytes have vital effects on repairing internal organ injury and restoring internal organ operation.The study on the differentiation of porcine pluripotent stem cells into cardiac muscle is predicted to supply a vital reference for the event of class cardiac muscle.With the development of xenotransplantation analysis,it’s additionally necessary to promote the availability of pig cardiomyocytes.In this study,porcine stem cells were used as the model to construct a culture system for the differentiation of stem cells into cardiomyocytes.The platform of porcine pluripotent stem cells established in the laboratory was used to screen and identify biological factors and chemical small molecules that promote the fate of differentiation of cardiomyocytes and explore the signaling pathway required for the development of porcine cardiomyocytes.The main experimental results are as follows:(1)The system of differentiation of porcine stem cells into cardiomyocytes was constructed,and small molecular substances such as FGF 10,RA,XAV939,Activin A,chir99021,and BMP4 were screened out to promote the fate of myocardial differentiation.Based on constructing the main gene expression of myocardium cells of pig species,this experiment uses pig extended pluripotent stem cells as materials and relies on different small molecules to construct the induction system of porcine pluripotent stem cells to differentiate into myocardium cells.By RT-PCR detection of specific marker genes in the process of myocardial induction,it was verified that(1)Wnt signal was activated before inhibition,(2)the ratio of 1:2 of BMP4 and Activin A,(3)the addition of RA in the second stage of induction and FGF10 in the third stage of induction can promote the induction of pig EPSCs into cardiomyocytes.At the last stage of induction,myocyte-specific genes NKX2-5 and TNNT2 were detected by RT-PCR,cell immunofluorescence staining,Western blot,etc.,to verify the acquisition of porcine EPSC-derived myocytes.(2)According to the transcriptome sequencing results of induced cells and original extended pluripotent stem cells,the signal pathways related to myocardial development were screened by comparing the state of induced cells.Transcriptome sequencing was performed on EPSC group,EPSC-derived cardiomyocytes(p EPSC-D8 group),and porcine heart tissue on the eighth day of induction,and it was found that EPSC group highly expressed OCT4,SOX2,NANOG,and other pluripotent related genes.Cardiac tissue and p EPSC-D8 showed high expression of ACTN,NKX2-5,TNNT2,TNNI,TBX5,TNNC,and other myocardial-specific genes,which could verify the similarity between the induced cells and porcine heart tissue.Through GO enrichment analysis of the induced differential genes in p EPSC-D8 and EPSC groups,it was found that most of the clusters were in the pathways related to nucleic acid and RNA synthesis,indicating that the induced cells were in the stage of vigorous metabolism and active growth,which confirmed the feasibility of the induced differentiation process.According to the GO enrichment analysis of the transcription group of heart tissue induced by the p EPSC-D8 group and piglets,it was screened that there were significant differences between the induced cells and heart tissue cluster genes in metabolic mode,mitochondria,ion pathways,etc.It was speculated that the induced cardiomyocytes were low in maturity,which may be the reason for the induced myocardium not having the ability to beat autonomously.By KEGG analysis of cardiac tissue and induced adhesion of p EPSC-D8 and EPSC groups,we concluded that MAPK,PI3K-Akt,and focal adhesion pathways play an important role in the development of pig hearts.In summary,this study established a stage induction system of porcine pluripotent stem cells into cardiomyocytes,and the induced cardiomyocytes were at the early stage of development,providing a reference for subsequent induction experiments... |
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