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©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
In vivo cardiac pacemaker function of differentiated human mesenchymal stem cells from adipose tissue transplanted into porcine hearts
Fabrice F Darche, Rasmus Rivinius, Ann-Kathrin Rahm, Eva Köllensperger, Uwe Leimer, Günter Germann, Miriam Reiss, Michael Koenen, Hugo A Katus, Dierk Thomas, Patrick A Schweizer
Fabrice F Darche, Rasmus Rivinius, Ann-Kathrin Rahm, Miriam Reiss, Michael Koenen, Hugo A Katus, Dierk Thomas, Patrick A Schweizer, Department of Cardiology, Medical University Hospital Heidelberg, Heidelberg D-69120, Germany
Fabrice F Darche, Rasmus Rivinius, Ann-Kathrin Rahm, Miriam Reiss, Hugo A Katus, Dierk Thomas, Patrick A Schweizer, DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, University of Heidelberg, Heidelberg D-69120, Germany
Fabrice F Darche, Rasmus Rivinius, Ann-Kathrin Rahm, Hugo A Katus, Dierk Thomas, Patrick A Schweizer, HCR (Heidelberg Center for Heart Rhythm Disorders), Department of Cardiology, Medical University Hospital Heidelberg, Heidelberg D-69120, Germany
Eva Köllensperger, Uwe Leimer, Günter Germann, Department of Plastic Surgery, ETHIANUM Klinik Heidelberg, Heidelberg D-69115, Germany
Michael Koenen, Department of Molecular Neurobiology, Max-Planck-Institute for Medical Research, Heidelberg D-69120, Germany
Author contributions: Darche FF and Schweizer PA designed and coordinated the study; Darche FF, Rivinius R, Rahm AK, Thomas D and Schweizer PA performed the experiments, acquired and analyzed data; Darche FF, Rivinius R, Rahm AK, Köllensperger E, Leimer U, Germann G, Reiss M, Koenen M, Katus HA, Thomas D and Schweizer PA interpreted the data; Darche FF and Schweizer PA wrote the manuscript; all authors approved the final version of the article; Thomas D and Schweizer PA contributed equally to this work.
Supported by Max-Planck-Society (TANDEM project to Koenen M and Schweizer PA); Ministry of Science, Research and the Arts Baden-Wuerttemberg (Sonderlinie Medizin to Thomas D); German Heart Foundation (Kaltenbach scholarship to Darche FF); German Cardiac Society (Otto-Hess scholarship to Rahm AK); Heidelberg Medical Faculty (Physician Scientist-Programm to Darche FF, Rivinius R and Rahm AK); German Cardiac Society (Research scholarship to Rivinius R); the German Society of Internal Medicine (Clinician-Scientist-Program to Rahm AK); and the German Centre for Cardiovascular Research (DZHK).
Institutional review board statement: Isolation and handling of haMSC were approved by the local ethical committee (University of Heidelberg ethical committee No. S-462/2010).
Institutional animal care and use committee statement: All experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996) and the European Community guidelines for the use of experimental animals. Protocols were approved by the local regulatory authority (AZ #359185.81/G-67/11, Regierungspräsidium Karlsruhe, Germany).
Conflict-of-interest statement: All authors have no any conflicts of interest.
Data sharing statement: No additional data.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
http://creativecommons.org/licenses/by-nc/4.0/ Corresponding author: Patrick A Schweizer, MD, Associate Professor, Senior Scientist, Department of Cardiology, University of Heidelberg, INF 410, Heidelberg D-69120, Germany.
patrick.schweizer@med.uni-heidelberg.de
Received: March 28, 2020
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
Processing time: 211 Days and 19 Hours
BACKGROUND
Mesenchymal stem cells (MSC) modified by gene transfer to express cardiac pacemaker channels such as HCN2 or HCN4 were shown to elicit pacemaker function after intracardiac transplantation in experimental animal models. Human MSC derived from adipose tissue (haMSC) differentiate into cells with pacemaker properties in vitro, but little is known about their behavior after intracardiac transplantation.
AIM
To investigate whether haMSC elicit biological pacemaker function in vivo after transplantation into pig hearts.
METHODS
haMSC under native conditions (nhaMSC) or after pre-conditioning by medium differentiation (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 atrioventricular (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 electronic pacemaker, assessment of escape recovery time, and pharmacological challenge to address catecholaminergic rate response. Finally, hearts were analyzed by histological and immunohistochemical investigations.
RESULTS
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). 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 HCN4 and other pacemaker-specific proteins involved in cardiac automaticity and impulse propagation.
CONCLUSION
dhaMSC transplanted into pig left ventricles sustainably induced rate-responsive ventricular pacemaker activity after in vivo engraftment for four weeks. The data suggest that pre-conditioned MSC may further differentiate along a pacemaker-related lineage after myocardial integration and may establish superior pacemaker properties in vivo.
Core Tip: Differentiated human mesenchymal stem cells from adipose tissue (dhaMSC) transplanted into the myocardium of domestic pigs are able to generate a biological pacemaker system in vivo. Electrocardiogram recordings and immunohistochemical analyses of pigs with transplanted dhaMSC indicated successful biological pacemaker generation. Pre-conditioning of cells was achieved by in vitro treatment of human mesenchymal stem cells in differentiation culture medium. Due to their reduced immunoreactivity, xenogeneic transplantation of human MSC into pig hearts was feasible without immunosuppression. dhaMSC maintained their immunoprivileged status over time.
