Genetically Modified Porcine Mesenchymal Stem Cells by Lentiviral Tbx18 Create a Biological Pacemaker

Abstract

Background Tbx18 is a vital transcription factor involved in embryonic sinoatrial node (SAN) formation process but is gradually vanished after birth. Myocardial injection of lentiviral Tbx18 converts cardiomyocytes into pacemaker-like cells morphologically and functionally. In this in vitro and in vivo study, genetical modification of porcine bone mesenchymal stem cells (BMSCs) by recapturing the Tbx18 expression creates a biological pacemaker which was examined. Methods The isolated porcine BMSCs were transfected with lentiviral Tbx18, and the induced pacemaker-like cells were analyzed using real-time polymerase chain reaction and western blotting to investigate the efficiency of transformation. Then, the induced pacemaker-like cells were implanted into the right ventricle of the SAN dysfunction porcine model after the differentiation process. Biological pacemaker activity and ectopic pacing region were tested by an electrocardiograph (ECG) monitor. Results The isolated porcine BMSCs expressed specific surface markers of stem cells; meanwhile, the expression of myocardial markers was upregulated significantly after lentiviral Tbx18 transfection. The porcine SAN dysfunction model was constructed by electrocoagulation using a surgical electrotome. The results showed that the mean heart beat (HR) of BMSCs-Tbx18 was significantly higher than that of BMSCs-GFP. An ectopic pacing region was affirmed into the right ventricle by ECG after implantation of BMSCs-Tbx18. Conclusion It was verified that Lenti-Tbx18 is capable of transducing porcine BMSCs into pacemaker-like cells. Genetically modified porcine BMSCs by lentiviral Tbx18 could create a biological pacemaker. However, further researches in large-scale animals are required to rule out unexpected complications prior to application in clinical practice.

Comparative Study of Transcriptome in the Hearts Isolated from Mice, Rats, and Humans

“…Transcription factors strongly expressed in SA have been studied for nearly 20 years as genes controlling cardiac automaticity. Not only SHOX2 [ 41 , 42 ] and HOXA2 [ 43 ] but also TBX18 [ 44 , 45 , 46 , 47 ], ISL1 [ 48 , 49 ], TBX3 [ 46 , 50 , 51 ], and TBX5 [ 52 ] have been reported. In our analysis, there were 81 SA-specific genes common to mice and humans, including seven transcription factors such as SHOX2 and HOXA2 , and TBX18 and ISL1 ( Supplementary File S1 ).…”

“…Although transcription factors that characterize the SA function have been intensively investigated for a couple of decades, these studies technically had to take into account the differences among animal species. For examples, TBX18 [ 44 , 45 , 46 , 47 ], TBX3 [ 46 , 50 , 51 ], and TBX5 [ 52 , 57 , 58 ] have been repeatedly reported as important transcription factors characterizing SA. Tbx18 is identified by in situ hybridizations from the superior vena cava to 75% of the SA head, and Tbx5 is expressed throughout the SA at 14.5 days of mouse embryonic development.…”

Recombinant Adeno-Associated Viral Vector-Mediated Gene Transfer of hTBX18 Generates Pacemaker Cells from Ventricular Cardiomyocytes

“…Previous work had identified that SAN development is tightly regulated by the transcription factors Shox2, TBX3, TBX5 and TBX18 [ 17 ]. Using these transcription factors, studies focused on overexpression to either increase the efficiency of the differentiation process of stem cells—either embryonic or induced-pluripotent-derived—into the pacemaker-like phenotype [ 33 , 34 , 35 , 36 , 37 , 44 , 45 , 46 ] or used a viral vector-mediated approach to demonstrate the conversion of cardiomyocytes into pacemaker-like cells [ 1 , 27 , 32 , 33 ]. Although all were successful to some degree, TBX18 was shown to be upstream of all these factors [ 25 , 26 ] and demonstrated the greatest potential in achieving functional pacemaker cells in either a stem cell or gene therapy context.…”

“…Newer attempts at using hTBX18 have attempted to use both gene- and gene-cell hybrid-based approaches to generate SAN-like pacemaker cells, both in vivo or in vitro, before implantation [ 32 , 33 , 34 , 35 , 36 , 37 ]. Further studies are required to assess the engraftment potential, pacing efficacy and any proarrhythmic effects of these strategies [ 32 ].…”

Effect of Shenfu Injection on Differentiation of Bone Marrow Mesenchymal Stem Cells into Pacemaker-Like Cells and Improvement of Pacing Function of Sinoatrial Node

“…Recently, for the purpose of circumventing the limitations and negative impacts of electronic pacemakers, stem cell-based biological pacemakers have been widely investigated so as to initiate the spontaneous activity of formerly quiescent cardiomyocytes [ 3 – 5 ]. Recent investigations have mainly focused on the induction of pacemaker-like cells through genetic reprogramming of stem cells [ 6 ]. The hyperpolarization-activated nucleotide-gated channel (HCN4) is the predominant isoform in the murine sinus atrial node (SAN) at both RNA and protein levels [ 7 ].…”