The Roles Of AF9 In Reprogramming Bone Marrow Derived Mesenchymal Stem Cells Into Cardiac Progenitor Cells Via High-efficiency Protein Transduction

Object:Cardiovascular disease(CVD)is the top killer of public health in China.Regretfully,current treatments are only able to delay the progression of the disease but fail to regenerate lost myocardium.Recently,cardiac progenitor/stem cells(CPCs/CSCs)have been proven practicable and safe in several preclinical and clinical trials.However,the use of endogenous CPCs for heart repair requires heart biopsies,which may be dangerous for severely patients.Therefore,there is the need to develop new method to acquire exogenous CPCs.Due to their low immuogencity and immunomodulatory properties,bone marrow derived mesenchymal stem cells(BM-MSCs)have been considered as a potential candidate for both autologous and allogeneic stem cell therapy.From the perspective of lineage development,the shared mesodermal origin of BM-MSCs and CPCs suggests that cardiac reprogramming would help BM-MSCs overcome the weakness of cardiac competency and BM-MSCs derived CPCs might be an ideal cell type for human heart repair.Nevertheless,chromatin state constitutes the biggest epigenetic barrier during the cell fate transition.Searching for new chromatin remodeling factor is the key to promote cardiac repgramming.In this study,we investigated the roles and mechanisms of AF9 in reprogramming of bone marrow derived mecenchymal stem cells into cardiac progenitor cells via high-efficiency protein transduction.Methods:1.His-tagged AF9 fusion protein was expressed in E.coli expression strain carried in pSmartI-MLLT3 plasmid under optimized condition and purified by Ni-affinity chromatography.2.The proteins(GATA4,TBX5,NKX2.5 or AF9)were modified by nano-reagent(CN patent 201410823369.2)and incubated with BM-MSCs for 12 hours.Then,the cellular uptake of these proteins was detected by immnofluorescent staining.3.Quatitative PCR assay was carried out to detect mRNA expression of cardiac progenitor markers ISL1,FLK1,NKX2.5 and GATA4 after 8 days of reprogramming via GNT(GATA4,NKX2.5,TBX5)and GNTA(GATA4,NKX2.5,TBX5,AF9)respectively,whereby to evaluate the roles of the additon of reprogramming factor AF9 during cardiac progenitor reprogramming.Furthermore,the combination of GNTA was selected to in-depth investigation for converting human bone marrow mesenchymal stem cells into protein induced cardiac progenitor cell-like cells(piCPCs).Immnofluroscent staining and flow cytometry assay were conducted to examine that piCPCs induced with GNTA could positively express ISL1 and TBX5 and differentiate into three cardiac lineage cells.Then,dynamic changes of cardiac development related genes were analyzed by qPCR and Western Blot during reprogramming(from day 0,through day4 and day8,to day 16).4.To explore the mechanisms of AF9 during cardiac reprogramming,firstly,we used co-immunoprecipitation(Co-IP)and co-localization assays to assess the interaction between AF9 and GATA4.Then,the epigenetic expression patterns of histone methylation marks were observed at 4 different time points when reprogramming with GNTA.We performed lentiviral shRNA to knock down DOT1L in BM-MSCs,then analyzed how the reduction in DOT1L affected the level of H3K79me2 at MYH6 or MYH7 promoter regulated at day 8 reprogramming.Results:1.Recombinant protein AF9 was successfully expressed at 37? for 4h in E.coli BL21(DE3)strain and shown to be more than 90%pure by Ni-affinity chromatography.2.Intringly,after incubation for 12 hours,all the four proteins were able to reach inside of nucleus of BM-MSCs by nano-reagent transduction and over 95%cells were carried in positve signal when using immunofluorescent staining analysis.3.Compared to GNT group,the addition of AF9 enhanced the mRNA levels of GATA4(P<0.001)and ISL1(P<0.05).When treated with GNTA,BM-MSCs gradually had gathered into a mass and formed many rose-like colonies at day 8,that means,piCPCs were obtained aroud day 8 according to our reprogramming protocol.Meanwhile,the subset of piCPCs is mainly ISL1 and TBX5 positive cells(85.20±3.13%,n=3),which could differentiate into cardiomyocytes,smooth muscle cells and endothelial cells under specific condition.Further,qPCR and Western Blot assays also demonstrated cardiac progenitor markers significantly increased during the process of cardiac reprogramming and cardiogenic genes ISL1 and NKX2.5,known as earliest markers of cardiac lineage,reached to peak levels at day 8.4.As AF9 contributed to advance the reprogramming efficeny of triplet titranscription factors GNT,we then inquired the underlying interaction between AF9 and cardiac transcription factors.Co-IP result suggests that AF9 is linked to GATA4 and confocal microscopy showed the same that AF9 and GATA4 co-localize in the nuclei.H3K79me2,an epigenetic marker associated with transcriptional activation,is present at higher levels with the time of reprogramming.As expected,the level of H3K79me2 was indeed decreased at day 8 reprogramming in DOT1L deficency BM-MSCs,which indicated that DOTIL-mediated H3K79 methylation is crucial for cardiac progenitor cells reprogramming.Surprisingly,ChIP-qPCR datas also showed reduction in enrichment of H3K79me2 on MYH6 or MYH7 promoter at the same time-point when compared to scramble group.Conclusion:Taken together,this study demonstrated that AF9 is a potential chromatin modifier to enhance the reprogramming efficiency of BM-MSCs into cardiac progenitor cells.AF9 recruited DOT1L to the genes related to cardiac reprogramming and promoted their active transcription via deposition of H3K79me2 with the guide of GATA4/AF9 complex.Therefore,we highlight AF9 could accelerate the generation of exogenous CPCs,which would be widely used for biomedical applications,including cell therapy for heart repair,drugs development.