TBX18 Gene Induced Adipose-derived Stem Cells To Differentiate Into Pacemaker-like Cells

Part Ⅰ Culture and identification of ADSCsObjective:To study the biological characteristics of rat and canine ADSCs and find the optimal culture conditions of ADSCs.Methods:Rat and canine ADSCs derived from inguen were separate and culture by enzyme digestion method. The morphology of ADSCs was observed by light microscope. Flow cytometry was used to detect surface antigen of ADSCs. CCK-8 was used to detect proliferation of ADSCs. Rat ADSCs were induced to adipogenic and osteogenic differentiation. Oil red O staining and alkaline phosphatase staining was used to detect the adipogenic and osteogenic differentiation of ADSCs. RT-PCR was used to detect the expression of transcription factor Oct-4 mRNA and Sox-2 mRNA of ADSCs at high and low glucose culture conditions and different generations.Results:Rat ADSCs were short fusiform or triangle. Canine ADSCs were long fusiform. The expressions of CD29, CD44 and CD73 were positive. The expressions of CD34 and CD14 were negative. The expressions of surface markers between rat and canine ADSCs had no significant difference. Rat ADSCs and Canine ADSCs have the ability of proliferation in vitro, and there is no significant difference in population doubling time between Rat ADSCs and Canine ADSCs. Rat ADSCs can induce to adipogenic and osteogenic differentiation. Oil red O staining and alkaline phosphatase staining are positive. The expressions of Oct-4mRNA and Sox-2mRNA of rat ADSCs at the low glucose culture condition were significantly higher than those in high glucose culture condition.The expression of Oct-4mRNA of P3 rat ADSCs were significantly higher than those of P10 and P20 ADSCs. The expression of Sox-2mRNA significantly decreased at P20.Conclusion:ADSCs with multiple differentiation potential were successfully isolated and cultured. It is more favorable to maintain the differentiation potential of ADSCs at the low glucose culture conditions. The differentiation potential of ADSCs gradually decreased with the increased culture time.Part Ⅱ Construct recombined adenovirus vector carring human TBX18 gene and transfect into ADSCsObjective:To observe the transfection efficiency of ADSCs transfected with recombinant adenovirus vector of TBX18.Methods:Construct the adenovirus vector of human TBX18. The adenovirus vector carrying TBX18 gene and green fluorescent protein (GFP) was transfected into rat ADSCs, and the control group was transfected with GFP. Different multiplicity of infection (MOI) values were 0,10,20,50,80,100 and 1000 respectively. Cell morphology and green fluorescence expression were observed at 24-48 hours after transfection. Transfection efficiency was detected by flow cytometry at 48 hours after transfection. CCK-8 was used to detect the proliferation of transfected and untransfected ADSCs. RT-PCR as used to detect expression of TBX18 at 48 hours and 7 days after ADSCs transfection.Results:The recombinant adenovirus vector pAd-MCMV-GFP-TBX18 (Ad-TBX18) was successfully constructed, and the control group was Ad-GFP. After transfection, the expression of green fluorescence gradually increased with the increase of MOI value. When MOI is 80 or above 80, some green fluorescent cells died and the cytoplasm appeared vacuoles. Green fluorescent expression was very strong when MOI is 1000, but most of the cells died. Flow cytometry showed that the transfection efficiency was more than 70% when MOI reached 50 or more than 50. The transfection efficiency of TBX18 was 75.7+5.6% when MOI reached 50. RT-PCR showed that the expression of TBX18mRNA was significantly higher than that control group at 48 hours and 7 days after ADSCs transfection.Conclusion:The recombinant adenovirus vector carrying human TBX18 gene was successfully constructed. The optimal MOI value was 50. TBX18 could be stably expressed in ADSCs after transfection.Part Ⅲ TBX18 induced ADSCs to differentiate into pacemaker-like cells in cardiac microenvironmentObjective:To study the effect of TBX18 on the differentiation of rat ADSCs in cardiac microenvironment.Methods:ADSCs transfected with Ad-TBX18 and Ad-GFP co-cultured with neonatal rat cardiomyocytes at ratio of 1:5 or 1:10. The beating rates of cells were observed by microscope in the co-culture system. The expression of HCN4mRNA, CX43mRNA and CX45mRNA in the co-culture system was detected by RT-PCR. WB was used to detect the expression of HCN4 of the co-culture system and neonatal rat cardiomyocytes. Immunofluorescence was used to detect the expression of CTNI, HCN4, CX43 and CX45 after co-culturing for 7d. Whole cell patch clamp technique was used to detect If current of green fluorescent cells after co-culturing for 5-7d. Before the patch clamp was performed,2-APB was used to block the electrical conduction of the cells.Results:After co-culturing for 7 days, we observed synthetic cell body formed by transfected ADSCs and neonatal rat cardiomyocytes. They generated synchronous beat.The maximum beating rates in GFP group and Tbx18 group were 75bpm vs 60 bpm at 5 days,60bpm vs 90bpm at 6 days and 60bpm vs 88bpm at 7 days after co-culturing. The beating frequency of single green fluorescent cell was 30-60 pbm after co-culturing for 5-7 days. The myocardial specific marker cTnI was detected in two groups by immunofluorescence after co-culturing for 7 days. The positive rates of cTnI expression between two groups had no significant difference (28.6+5.8% vs 26.6+5.9%). HCN4mRNA expression was significantly higher in Tbx18 group than that in GFP group. HCN4 protein level was significantly higher than that of the control group and neonatal rat cardiomyocytes. Immunofluorescence assay showed that HCN4 expression was positive in ADSCs transfected with TBX18 and the positive rate was 13±5%. HCN4 expression was not detected in GFP group. RT-PCR analysis showed that the expression of CX45mRNA in Tbxl8 group was significantly higher than GFP group. The expression of CX43mRNA in Tbxl8 group was significantly lower than GFP group. The expression of CX43 and CX45 in the TBX18 was detected by immunofluorescence assay. ADSCs transfected with TBX18 were detected If current that can be completely blocked by specific blocker CsCl. ADSCs transfected with GFP did not detect the If current. The maximum current density of the If current is 5.43+1.36pA/pF.Conclusion:TBX18 can induce ADSCs to differentiate into pacemaker-like cells and increase the differentiation efficiency of pacemaker-like cells in cardiac microenvironment.Part IV ADSCs transfected with TBX18 waere injected into myocardium of canine with complete atrioventricular blockObjective:ADSCs transfected with TBX18 were injected into myocardium and observe pacemaker activity in canine with complete atrioventricular block.Methods:6 canines were implanted ⅤⅥ pacemaker and treated with radiofrequency ablation of atrioventricular node. Ad-TBX18 was transfected into canine ADSCs. The cell suspension was made after 48 hours. Every dog was inject 1×107 cells.3 canines were given ADSCs transfected with Ad-TBX18 by a manageable injection catheter. The injection site was myocardium of right high interventricular septal.1 canine was openned chest to inject ADSCs transfected with Ad-TBX18. The injection site was epicardial myocardium of basal ventricular septum.2 canines were only performed WI pacemaker implantation and atrioventricular node ablation. We observed beating rates at 7 days and 14 days after operation.Results:ADSCs were successfully transfected with Ad-TBX18. Pacemaker implantation was performed and the model of complete atrioventricular block was successfully constructed in 6 canines.1 canine openned chest and died 1 day after operation. There was no significant difference in average heart rate between TBX18-ADSCs group and control group at 1 week and 2 weeks after operation. There was no significant change in the heart rates in other 5 dogs immediately after ablation, 1 week and 2 weeks after the operation. The heart rates were 40-50bpm. The ablation site was found by autopsy. The injection site was not found. No green fluorescent cells were found in the frozen sections of the right ventricle.Conclusion:There is no significant increase in heart rate in canines with complete atrioventricular block after injecting transfected ADSCs. To further explore the conditions of ability of differentiate into pacemaker like cells of ADSCs transfected with TBX18, and to confirm its pacemaker activity in vivo with subsequent studies.