Overexpression Of Shox2 And HCN4 Promotes The Differentiation Of Human Umbilical Cord Mesenchymal Stem Cells Into Pacemaker-like Cells

BackgroundCardiovascular disease has become the number one killer that threatens human health.Sick sinus syndrome（SSS）is one of the most common serious heart diseases and even increases the risk of death.Electronic pacemakers are currently the first-line treatment option for patients with sinus node dysfunction（SND）or SSS.Although electronic pacemakers are effective,current electronic devices have many limitations,such as lead or generator failure,lack of autonomous response,mutual interference with strong magnetic fields,and devicerelated infections.Biological pacemakers produced by somatic gene transfection,cell fusion or cell transplantation provide an alternative to electronic pacemakers.A somatic reprogramming strategy,including transfection of genetically encoded transcription factors,converts working cardiomyocytes into sinoatrial node pacemaker-like cells.Even as leadless pacemakers become smaller and less invasive,biological pacemakers are expected to expand the therapeutic potential of conduction system diseases.The If current,the pacing current,produced by the hyperpolarization-activated cyclic nucleotide-gated channel（HCN）gene,is the key to slow diastolic depolarization.HCN4 is the major isoform of HCN,and mutations in this channel lead to SND.In addition,the short stature homeobox-containing gene（Shox2）is equally important for the development and differentiation of sinoatrial node cells.Shox2 knockout mice died of SSS due to increased expression of Nkx2.5,Cx40 and Cx43 and downregulated expression of HCN4,Tbx3 and Isl1.Compared with adult bone marrow mesenchymal stem cells（BMSCs）,human umbilical cord mesenchymal stem cells（HUCMSCs）are easier to obtain,low cost,weak immunogenicity and good proliferation activity,and other advantages to provide a better foundation for subsequent research and clinical applications.For a long time,our research group has been committed to the research of stem cell regenerative medicine,and has the relevant research basis such as inducing the differentiation of BMSCs into pacemaker-like cells by transfecting pacemaker genes in vitro.To explore the feasibility and effect of transfecting Shox2 and HCN4 genes into HUCMSCs by lentiviral vector alone or in combination.Method1.HUCMSCs were cultured in vitro,and the morphology and proliferation characteristics of MSCs of different culture passages were observed under the microscope.2.Lentiviral vector construction:construct pLentis-Ubi-MCS-enhanced red fluorescent protein（ERFP）-HCN4 and pLentis-Ubi-MCS-enhanced green fluorescent protein（EGFP）Shox2 viral vectors,and pLentis-Ubi-MCS-ERFP and pLentis-Ubi-MCS-EGFP empty vector;the vector carries the puro resistance gene for the selection of stably transfected cell lines.3.Determine optimal viral multiplicity of infection（MOI）values and conditions for lentivirus infection of cells.The constructed lentiviral vectors were transfected into the 7th passage of well-grown HUCMSCs and divided into the following four groups:①control group（GFP/RFP group）②Shox2-MSCs group③HCN4-MSCs group④Co-transfection of Shox2-HCN4-MSCs group;Puromycin selection of stable strains.4.After transfection,the cells in each group were tested:①CCK8 was used to detect the proliferation of the cells in each group;②The fluorescence expression and morphological changes of the cells in each group were observed under a microscope;③The mRNA and protein expression levels of related genes were detected by fluorescence quantitative PCR and Western blotting respectively.,such as:Cx43,HCN4,Nkx2.5,etc.Result1.The HUCMSCs of P5 generation were in good condition after recovery,and showed typical vortex-like colony unit growth under the microscope after 24h of adherence.It can cover about 80-90%of the area of the bottom of the bottle in about 2 days,with a full shape and strong proliferation ability;the cells are cultured in vitro to P20 and still maintain good proliferation characteristics.2.The lentiviral overexpression vector was constructed successfully.In the experiment,gradient transfection was used,and the transfection efficiency of Shox2-MSCs group（GFP）and HCN4-MSCs group（RFP）was about 50-60%when MOI=250.With the extension of culture time,the fluorescence expression of cells in the experimental group continued to increase,the morphology also changed slightly,and the refractive index was better.When cotransfection was performed,the cells were observed to grow well under a microscope,and the co-transfection efficiency was about 40-50%.3.Cell proliferation ability test:The growth of cells in different groups was detected by CCK8 method.Compared with the control group,the growth rate of cells in Shox2-MSCs group and HCN4-MSCs group was significantly slowed down（p<0.05）,while the growth rate of cells in co-transfected Shox2HCN4-MSCs group slower（p<0.01）.4.Cell molecular level detection:（1）mRNA levels:The mRNA expressions of HCN4,Tbx18 and Tbx3 in the Shox2MSCs group and the Shox2-HCN4-MSCs group were significantly higher than those in the control group（p<0.05）,while the expressions of Nkx2.5 and Cx43 were significantly lower than those in the control group（p<0.01）;The mRNA expressions of Tbx3,Cx43 and Nkx2.5 were significantly different between the HCN4-MSCs group and the control group（p<0.05）;There was a statistical difference between the groups（p<0.05）;（2）Protein level:Compared with the control group,Cx45 protein level was up-regulated and Nkx2.5 and Cx43 protein levels were down-regulated in Shox2-MSCs group（p<0.01）,and Cx45 and Cx43 protein expressions were detected in HCN4-MSCs group.The Shox2HCN4-MSCs group was up（or）downregulated more significantly than the Shox2-MSCs group and the HCN4-MSCs group（p<0.001）.Conclusion1.The immortalized HUCMSCs were subcultured in vitro to 20 generations and still maintained typical morphological characteristics and good proliferation performance,which laid a seed cell foundation with good characteristics for subsequent research on the construction of stable and effective cardiac pacemaker-like cells.2.The successful transfection of Shox2/HCN4 into immortalized HUCMSCs by lentiviral recombinant vector alone or together provides a guarantee for the generation of stable pacemaker-like cell phenotypes in the next study.3.Transfection of Shox2 and HCN4 as a single factor or in combination can promote the differentiation of HUCMSCs into cardiac pacemaker-like cell phenotype,and the expressions of pacemaker-promoting genes HCN4,Shox2,Tbx3 and Cx45 were significantly up-regulated at the mRNA and protein levels.the expression levels of pacing inhibitory genes Nkx2.5 and Cx43 were significantly down-regulated.Compared with single gene transfection,the expression of the above pacing genes was further up-regulated in the co-transfection group,while the expressions of pacing inhibitory genes Nkx2.5 and Cx43 were further downregulated.Effect.It strongly supports the feasibility and effectiveness of our strategy to induce biological pacemaker-like cells by co-transfecting HUCMSCs with exogenous target genes.