Published online Oct 26, 2020. doi: 10.4252/wjsc.v12.i10.1133
Peer-review started: March 28, 2020
First decision: April 22, 2020
Revised: May 3, 2020
Accepted: August 24, 2020
Article in press: August 24, 2020
Published online: October 26, 2020
Loss or dysfunction of sinoatrial nodal cells leads to sick sinus syndrome, often requiring the implantation of an electronic pacemaker. With respect to technical limitations of electronic pacemaker devices, research efforts are aimed at developing biological alternatives that might adapt more adequately to physiological requirements of the patients. In the search for a novel approach to overcome electronic pacemakers, multiple approaches have successfully utilized biological pacemakers in vitro and/or in large animal models using multiple gene or cell therapeutic strategies.
Gene and cell therapy are two different possible approaches that can be pursued to establish a biological pacemaker. As gene therapy strategies are hampered by non-sustained effects of gene transfer and the risk of neoplasia, stem cell therapy offers a promising alternative approach to biological pacemaking, aiming at the replacement of lost or dysfunctional pacemaker cells. Human mesenchymal stem cells (MSC) are attractive candidates as they are easily obtained from adipose tissue or bone marrow, can be expanded to high numbers in vitro, show the ability to differentiate into several mesenchymal cell lineages, and dispose of immunotolerant properties discarding the need for immunosuppression. However, the efficacy of MSC differentiation into cardiac myocytes is limited as transdifferentiation requires potentially teratogenic substances such as 5-azacytidine or amphotericin, hampering its use with respect to a future clinical application. To establish biological pacemaking, a hybrid model combining gene and stem cell therapy by introducing HCN2 into MSC has been proposed. According to this approach, MSC primarily served as a "vector shuttle" to guide the depolarizing gene HCN2 to the heart. Recently, we reported the differentiation of human MSC (hMSC) derived from adipose tissue into cells with cardiac pacemaker characteristics. Pacemaker cell differentiation was achieved by cell culture using a customized cell medium, omitting the need for genetic manipulation.
The main objective of this study was to show whether in vitro differentiated hMSC derived from adipose tissue (dhaMSC) may elicit biological pacemaker function after transplantation into porcine myocardium. To this end, we performed intramyocardial implantation of dhaMSC in pigs with experimental total atrioventricular (AV)-block to elucidate the capacity of dhaMSC to integrate within the myocardium and to generate escape pacemaker activity.
In vitro differentiation of native hMSC derived from adipose tissue (nhaMSC) was realized by treating nhaMSC in differentiation medium, which mainly consisted of RPMI 1640, B27 supplement and human recombinant BMP4. nhaMSC or dhaMSC (n = 6 pigs each, 5 × 106 cells/animal) were injected into the porcine left ventricular free wall. Animals receiving PBS injection served as controls (n = 6). Four weeks later, total AV block was induced by radiofrequency catheter ablation, and electronic pacemaker devices were implanted for backup stimulation and heart rate monitoring. Ventricular rate and rhythm of pigs were evaluated during a follow-up of 15 d post-ablation by 12-lead-ECG with heart rate assessment, 24-h continuous rate monitoring recorded by an electronic pacemaker, assessment of escape recovery time, and a pharmacological challenge to address catecholaminergic rate response. Pace-mapping experiments were undertaken on the exposed heart by stimulation at the site of cell injection using an epicardial electrode connected to an external pacemaker choosing a pacing rate 20 bpm higher than the prevalent escape rate. Twelve-lead ECG monitoring was performed, and the electrical activation front of spontaneous and paced rhythms was evaluated with respect to similarity and origin. Finally, hearts were analyzed by histological and immunohistochemical investigations.
In vivo transplantation of dhaMSC into the left ventricular free wall of pigs elicited spontaneous and regular rhythms that were pace-mapped to ventricular injection sites (mean heart rate 72.2 ± 3.6 bpm; n = 6) after experimental total AV block. Ventricular rhythms were stably detected over a 15-d period and were sensitive to catecholaminergic stimulation (mean maximum heart rate 131.0 ± 6.2 bpm; n = 6; P < 0.001). Pigs, which received nhaMSC or PBS presented significantly lower ventricular rates (mean heart rates 47.2 ± 2.5 bpm and 37.4 ± 3.2 bpm, respectively; n = 6 each; P < 0.001) and exhibited little sensitivity towards catecholaminergic stimulation (mean maximum heart rates 76.4 ± 3.1 bpm and 60.5 ± 3.1 bpm, respectively; n = 6 each; P < 0.05). To evaluate the nature and origin of escape rhythms after experimental total AV block, we performed pace-mapping. For pigs with transplanted dhaMSC, a complete 12/12 matching of escape and pace-mapped beats was observed in the 12-lead ECG. By contrast, pace-maps and escape rhythms of pigs with transplanted nhaMSC or treated with PBS differed, indicating an origin of spontaneous rhythms other than the site of injection. Histological and immunohistochemical evaluation of hearts treated with dhaMSC revealed local clusters of transplanted cells at the injection sites that lacked macrophage or lymphocyte infiltrations or tumor formation. Intense fluorescence signals at these sites indicated membrane expression of the pacemaker-specific proteins HCN1 and HCN4, the calcium channel Cav1.2, and the connexins Cx31.9 and Cx45 involved in cardiac automaticity and impulse propagation.
In our proof-of-concept study we were able to show that pre-conditioned MSC, differentiated towards a nodal-type lineage in vitro, sustainably and catecholamine-responsively pace the hearts of pigs at physiological rates after engraftment within the ventricular myocardium and further differentiate towards a cardiac pacemaker phenotype in vivo as shown by the presence of HCN4 staining. In contrast, immunohistochemical analysis indicated that transplanted non-pre-conditioned nhaMSC did not express relevant pacemaker-specific markers leading to the assumption that prior in vitro differentiation (“pre-conditioning”) of MSC is a prerequisite for further in vivo pacemaker differentiation. Based on this approach, a cell-based biological pacemaker may become feasible using adult stem cells and bypassing viral transduction of pacemaker genes or transcription factors.
To establish a biological pacemaker system in a human setting, high ethical and safety standards have to be set. The use of immunotolerant, viral-free differentiated MSC is an important step forwards to meet these requirements. Considering the results of our proof-of-concept study, we advocate larger animal trials with long-term follow-up to validate our findings and to eventually pave the way for human translation.