Experimental Study Of Exogenous Myocardial Repair By Mesenchymal Stem Cells And Reprogramming Factors

Optimization of Timing and Times for Administration of Atorvastatin-pretreated Mesenchymal Stem Cells in a Preclinical Model of Acute Myocardial InfarctionBackground:The therapeutic potential of mesenchymal stem cells(MSCs)for cardiac repair is hindered due to their low engraftment rate and low survival rate within the ischemic myocardium.Our previous studies showed that the combination of atorvastatin(ATV)and single injection of ATV-pretreated MSCs(ATV-MSCs)at 1 week post acute myocardial infarction(AMI)promoted MSC recruitment and survival with a rat acute myocardial infarction model.This study firstly aimed to investigate the dynamic changes of the recruitment factor,stromal cell-derived factor-1(SDF-1),along with the inflammation factor TNF-α and IL-6 expression through the whole AMI period to explore the optimal time window for MSCs transplantation.Furthermore,we intended to find whether the combinatorial therapy of intensive ATV with multiple injections of ATV-MSCs have greater efficacy at different stages of AMI to better define the optimal strategy for MSC therapy in AMI.Methods:In order to determine the optimal time window for MSC treatment,we firstly assessed SDF-1 dynamic expression and inflammation level in the myocardium.A total of 150 Sprague-Dawley(SD)rats were randomized into the sham operation(Sham),AMI control(AMI)and ATV treatment(ATV)groups for SDF-1 dynamic expression and the levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)over the 4-week period of AMI.Next 215 SD rats were randomized into 11 groups of Sham,AMI,single,dual,triple injections of ATV-MSCs with oral ATV at early(Earlyl,Early2,Early3),mid-term(Mid1,Mid2,Mid3)and late(Late1,Late2,Late3)stages of AMI for comparison.After AMI model establishment and different treatment in different groups,ATV-MSCs recruitment rate,cardiac function,infarct size,fibrotic region,angiogenesis,endogenous c-Kit+ cell mobilization and differentiation were evaluated and compared to find the best therapeutic approach.AMI model was created by left anterior descending artery ligation.Oral ATV(10mg/kg/day)were given by gastric gavage on the rat.ATV-MSCs were intravenously injected via tail vein.Results:As to the first part of the experiment of SDF-1 dynamic expression and inflammation level exploration,compared with AMI control,intensive ATV significantly augmented SDF-1 expression 1.5-2.6-fold in peri-infarcted region with inhibited inflammation over the 4-week period of AMI.ATV-MSCs implantation with ATV administration further enhanced MSC recruitment rate by 3.9%～24.0%,improved left ventricular ejection fraction(LVEF)by 20%～16.2%,and reduced infarct size in all groups 6 weeks post-AMI with most prominent improvement in Mid groups and still effective in Late groups.Mechanistically,ATV-MSCs remarkably suppressed inflammation and apoptosis while increased angiogenesis.Furthermore,triple injections of ATV-MSCs were much more effective than single administration during early and mid-term stages of AMI with the best effects in Mid 3 group.Conclusions:We conclude that with the usage of ATV,the selection of best time for stem cell transplantation is related to both of the SDF-1 expression and inflammation level.The optimal strategy for MSCs transplantation is multiple injections of ATV-MSCs at mid-term stage of AMI combined with intensive ATV administration.The translational potential of this strategy is clinically flexible and promising.Direct Cardiac Reprogramming by Transactivating Endogenous Transcriptional Factors Based on CRISPR-dCas9 SystemBackground:Acute myocardial infarction(AMI)and heart failure post-AMI have been the leading cause of deaths worldwide in the past decades.It is reported that AMI can wipe out nearly 25%of cardiomyocytes within several hours.Since adult cardiomyocytes are terminally differentiated somatic cells,it is of great importance to explore new treatments to achieve myocardial repair and regeneration after acute myocardial infarction.Direct reprogramming refers to the direct conversion of somatic cells into another type of mature somatic cells while skipping the stage of conversion to multipotent stem cells or progenitor cells.Conventionally,directly reprogramming cardiac fibroblasts into induced cardiomyocytes(iCMs)requires the ectopic expression of transcription factors.And the reprogramming efficiency is usually very low to achieve cardiac function recovery.Also,it remains largely unclear whether iCMs can be generate by endogenous chromatin trigger reprogramming.CRISPR-dCas9 activation system has been put into use to precisely targeted the desired genes to endogenously boost gene expression.Methods:In this study,we firstly designed candidate sgRNAs targeting a narrow promoter region close to the transcription start site(TSS),or a 200-bp region of distal enhancer of the cardiac reprogramming factors Mef2c(M),Gata4(G)and Tbx5(T),to directly target the endogenous transcription domains of M/G/T.Then we utilized the second generation of CRISPR activation,the SAM system,as a programmable and transcriptional regulator to effectively and precisely activate the endogenous expression level of reprogramming factors M/G/T.Finally,with the use of single transcriptional factor Gata4 sgRNA combo or all three transcriptional factors M/G/T sgRNA combo,we conducted in vitro cardiac reprogramming experiments to induce Mouse Embryonic Fibroblasts(MEFs)and fresh Cardiac Fibroblasts(fCFs)into iCMs.The reprogramming efficiency and function of iCMs were evaluated to determine whether sgRNAs with transactivators can replace the traditional exogenous overexpression of transcriptional factors.Results:Firstly,the sgRNA screening result showed that the sgRNAs targeting promoters of Gata4 and Tbx5 were sufficient to activate endogenous Gata4 and Tbx5 expression but not Mef2c.Instead,remodeling of the Mef2c enhancer could triggered activation endogenously.With the use of best Gata4-sgRNAs combination,along with SAM activators,we further concluded that targeted endogenous activation of Gata4 expression via sgRNAs in MEFs was sufficient to conduct direct cardiac reprogramming with retroviral mediated Mef2c and Tbx5 expression.And the reprograming efficiency of MEFs was similar to that of the exogenous retroviral M/G/T usage while fCFs could not achieved that high efficiency.In contrast,activate all three transcriptional factors endogenously via the corresponding sgRNAs was able to reprogram both MEFs and fCFs into iCMs though the efficiency in both cell types was relatively low.Conclusions:Our study exhibited the results of initial sgRNAs screening and found the effective sgRNAs that could significantly activate endogenous M,G,and T expression,thus induced MEFs and fCFs to iCM.Such endogenous gene activation mediated cardiac reprogramming provides a new strategy for converting fibroblasts into induced cardiomyocytes.It is likely that future efforts for design fibroblast-specific engineered nanoparticles carrying efficient reprogramming factor sgRNAs may be a more clinical translatable method of generating reprogrammed cardiomyocytes,thus to promote cardiac regeneration and improve cardiac function post AMI.