Autotransplantation Of Tbx18-MSCs In Treament Of Bradycardia

ObjectiveCurrently,cell transplantation and genetic technology are the two major aspects of researches concerning biological pacemakers(BPs).However,BPs based on single technology still cannot meet the requirements of practical clinical applications.In this study,cell transplantation and genetic therapy were combined to develop an innovative type of biological pacemaker.The proposed BP was transplanted into the epicardial of the right ventricle in a pig model of sinoatrial node damage to evaluate the pacing rate of the BP and response to alterations of the automatic nervous system.Methods1.Isolation,culture and identification of porcine mesenchymal stem cellsPorcine mesenchymal stem cells(MSCs)were obtained from bone marrow aspirated from anterior superior iliac spine of healthy young male pigs,followed by gradient isolation techniques,centrifugation and resuspension.The porcine MSCs were cultured in Dulbecco’s Modified Eagle Media(DMEM)supplemented with 20% fetal bovine serum.Trypsinization was performed after the cells became confluent.MSCs were subsequently subjected to flow cytometry for detection of stem cell biomarkers such as CD105,CD90,CD45 and CD35.2.Construction of biological pacemaker based on Lenti.Tbx18-transfected MSCsTbx18 overexpressing lentivirus was synthesized by HeYuan Biotech.Co.,reaching a titer of 2.09 ×108 TU/ml.Transfection was conducted using MSCs of passage 3.Various amount of Lenti.Tbx18 and Lenti.GFP was transfected into MSCs to ascertain optimal multiplicity of infection(MOI)by counting the efficiency of transfection.Western blotting was performed to assess the protein level of Tbx18.3.Transplantation of biological pacemaker in treatment of bradycardiaSinoatrial node damage model was developed through the right side of the 4th intercostal space of pigs under general anesthesia and mechanical ventilation.Pigs in the test group were injected with MSCs carrying Tbx18 and those in the control group were injected with MSCs transfected with Lenti.GFP and those in the blank group were injected with MSCs.After implantation of BPs,electrocardiogram(ECG)was regularly examined using PowerLab with LabChart software every week.In addition,isoproterenol was administered when evaluating autonomic response of the heart.RT-PCR were performed totest the biological safety of the pacemaker gene.After sacrifice of the experimental animals,myocardium around the injection site was procured for Western blotting and immunofluorescence analyses.Results1.Before adherence,MSCs grew in a dispersed and colony-like manner with a spherical shape and suspended in the culture medium.After 24 hours,a small quantity of cells was adherent and exhibited an oval or short spindle morphology.In about 5 days,the proliferation of porcine bone MSCs started to accelerate,with a long spindle shape.It took approximately 10-14 days for primary MSCs to approach 80-90% confluence.In our study,20% fetal bovine serum was required to maintain a well state of MSCs.Flow cytometry analysis indicated that the MSCs were CD105 and CD90 positive,and CD34 and CD45 negative.2.At 72 hours after transfection of Lenti.GFP and Lenti.Tbx18,green fluorescence could be visualized under fluorescent microscopy.Various MOI values leads to different transfecting efficiency.Along with the increment of MOI values,the ratio of green fluorescence-positive cells increased,with a stronger fluorescence intensity under fluorescence microscope.At a MOI of 60,more than 95% of the cells were transfected with Lenti.Tbx18,while the cells fell into a poor state when MOI was raised to 80 or 100.Therefore,a MOI of 60 was selected in subsequent experiments.Furthermore,the high expression of Tbx18 was confirmed by Western blottings.3.After damage of the atrial sinus node(ASN),the heart rate of the animal models decreased by 40-50% and the original P waves disappeared,which were replaced by newly developed and irregular P waves,suggesting successful establishment of ASN damage model.ECG was acquired after eliminating the support of temporary pacemaker 1 week postoperatively.The results revealed a significant elevation of heart rate in the Lenti.Tbx18 group at the third day,which lasted for three weeks.Afterwards,heart rates of BP implanted pigs slowly went down but were still faster than control group and blank group.The shape and direction of the QRS of the experimental group remained similar in comparison to the ECG monitored prior to surgeries.Administration of Isoprinosine upregulated the heart rate of animals in both group,revealing significantly statistical difference between the Lenti.Tbx18 and Lenti.GFP group.Immunofluorescence and immunoblotting analyses of the myocardial specimens around injection area demonstrated a high protein level of Tbx18.Conclusions1.The MSCs derived from porcine bone marrow can be successfully obtained and isolated.The MSCs can be easily cultured in 20% fetal bovine serum and possess a satisfied proliferating ability.2.Pacemaker protein Tbx18 can be safely,efficiently and stably overexpressed in porcine MSCs through a lentivirus vector in vitro.3.The proposed biological pacemaker based on Lenti.TBX18 transfected MSCs can pace the ventricle effectively after transplantation.Moreover,BP implanted porcine hearts are more sensitive in response to alterations of autonomous system.