After two decades of intensive research and attempts of clinical translation, stem cell based therapies for cardiac diseases are not getting closer to clinical success. This review tries to unravel the obstacles and focuses on underlying mechanisms as the target for regenerative therapies. At present, the principal outcome in clinical therapy does not reflect experimental evidence. It seems that the scientific obstacle is a lack of integration of knowledge from tissue repair and disease mechanisms. Recent insights from clinical trials delineate mechanisms of stem cell dysfunction and gene defects in repair mechanisms as cause of atherosclerosis and heart disease. These findings require a redirection of current practice of stem cell therapy and a reset using more detailed analysis of stem cell function interfering with disease mechanisms. To accelerate scientific development the authors suggest intensifying unified computational data analysis and shared data knowledge by using open-access data platforms.

Cellular inflammation following acute myocardial infarction has gained increasing importance as a target mechanism for therapeutic approaches. We sought to investigate the effect of syngeneic cardiac induced cells (CiC) on myocardial inflammation using 18F-FDG PET (Positron emission tomography)-based imaging and the resulting effect on cardiac pump function using cardiac magnetic resonance (CMR) imaging in a mouse model of myocardial infarction. Mice underwent permanent left anterior descending coronary artery (LAD) ligation inducing an acute inflammatory response. The therapy group received an intramyocardial injection of 10⁶ CiC into the border zone of the infarction. Five days after myocardial infarction, 18F-FDG PET was performed under anaesthesia with ketamine and xylazine (KX) to image the inflammatory response in the heart. Flow cytometry of the mononuclear cells in the heart was performed to analyze the inflammatory response. The effect of CiC therapy on cardiac function was determined after three weeks by CMR. The 18F-FDG PET imaging of the heart five days after myocardial infarction (MI) revealed high focal tracer accumulation in the border zone of the infarcted myocardium, whereas no difference was observed in the tracer uptake between infarct and remote myocardium. The CiC transplantation induced a shift in 18F-FDG uptake pattern, leading to significantly higher 18F-FDG uptake in the whole heart, as well as the remote area of the heart. Correspondingly, high numbers of CD11⁺ cells could be measured by flow cytometry in this region. The CiC transplantation significantly improved the left ventricular ejection function (LVEF) three weeks after myocardial infarction. The CiC transplantation after myocardial infarction leads to an improvement in pump function through modulation of the cellular inflammatory response five days after myocardial infarction. By combining CiC transplantation and the cardiac glucose uptake suppression protocol with KX in a mouse model, we show for the first time, that imaging of cellular inflammation after myocardial infarction using 18F-FDG PET can be used as an early prognostic tool for assessing the efficacy of cardiac stem cell therapies.

Background: The immune response is a crucial factor for mediating the benefit of cardiac cell therapies. Our previous research showed that cardiomyocyte transplantation alters the cardiac immune response and, when combined with short-term pharmacological CCR2 inhibition, resulted in diminished functional benefit. However, the specific role of innate immune cells, especially CCR2 macrophages on the outcome of cardiomyocyte transplantation, is unclear. Methods: We compared the cellular, molecular, and functional outcome following cardiomyocyte transplantation in wildtype and T cell- and B cell-deficient Rag2^del mice. The cardiac inflammatory response was assessed using flow cytometry. Gene expression profile was assessed using single-cell and bulk RNA sequencing. Cardiac function and morphology were determined using magnetic resonance tomography and immunohistochemistry respectively. Results: Compared to wildtype mice, Rag2^del mice show an increased innate immune response at steady state and disparate macrophage response after MI. Subsequent single-cell analyses after MI showed differences in macrophage development and a lower prevalence of CCR2 expressing macrophages. Cardiomyocyte transplantation increased NK cells and monocytes, while reducing CCR2⁻MHC-II^lo macrophages. Consequently, it led to increased mRNA levels of genes involved in extracellular remodelling, poor graft survival, and no functional improvement. Using machine learning-based feature selection, Mfge8 and Ccl7 were identified as the primary targets underlying these effects in the heart. Conclusions: Our results demonstrate that the improved functional outcome following cardiomyocyte transplantation is dependent on a specific CCR2 macrophage response. This work highlights the need to study the role of the immune response for cardiomyocyte cell therapy for successful clinical translation.

Angiogenesis plays a central role in the healing process following acute myocardial infarction. The PET tracer [⁶⁸Ga]-NODAGA-RGD, which is a ligand for the α_vβ₃ integrin, has been investigated for imaging angiogenesis in the process of healing myocardium in both animal and clinical studies. It’s value as a prognostic marker of functional outcome remains unclear. Therefore, the aim of this work was to establish [⁶⁸Ga]-NODAGA-RGD for imaging angiogenesis in the murine infarct model and evaluate the tracer as a predictor for cardiac remodeling in the context of cardiac stem cell therapy. [⁶⁸Ga]-NODAGA-RGD PET performed seven days after left anterior descending coronary artery (LAD) occlusion in 129S6 mice showed intense tracer accumulation within the infarct region. The specificity was shown in a sub-group of animals by application of the competitive inhibitor cilengitide prior to tracer injection in a subgroup of animals. Myocardial infarction (MI) significantly reduced cardiac function and resulted in pronounced left ventricular remodeling after three weeks, as measured by cardiac MRI in a separate group. Cardiac induced cells (CiC) that were derived from mESC injected intramyocardially in the therapy group significantly improved left ventricular ejection fraction (LVEF). Surprisingly, CiC transplantation resulted in significantly lower tracer accumulation seven days after MI induction. Accordingly, we successfully established the PET tracer [⁶⁸Ga]-NODAGA-RGD for the assessment of α_vβ₃ integrin expression in the healing process after MI in the mouse model. Yet, our results indicate that the mere extent of angiogenesis following MI does not serve as a sufficient prognostic marker for functional outcome.

Background: Ventricular arrhythmias (VA) are a common cause of sudden death after myocardial infarction (MI). Therefore, developing new therapeutic methods for the prevention and treatment of VA is of prime importance. Methods: Human bone marrow derived CD271⁺ mesenchymal stem cells (MSC) were tested for their antiarrhythmic effect. This was done through the development of a novel mouse model using an immunocompromised Rag2^−/− γc^−/− mouse strain subjected to myocardial “infarction-reinfarction”. The mice underwent a first ischemia-reperfusion through the left anterior descending (LAD) artery closure for 45 min with a subsequent second permanent LAD ligation after seven days from the first infarct. Results: This mouse model induced various types of VA detected with continuous electrocardiogram (ECG) monitoring via implanted telemetry device. The immediate intramyocardial delivery of CD271⁺ MSC after the first MI significantly reduced VA induced after the second MI. Conclusions: In addition to the clinical relevance, more closely reflecting patients who suffer from severe ischemic heart disease and related arrhythmias, our new mouse model bearing reinfarction warrants the time required for stem cell engraftment and for the first time enables us to analyze and verify significant antiarrhythmic effects of human CD271⁺ stem cells in vivo.

Cellular inflammation is an integral part of the healing process following acute myocardial infarction and has been under intense investigation for both therapeutic and prognostic approaches. Monocytes and macrophages are metabolically highly active and show increased uptake rates of glucose and its analog, ¹⁸F-FDG. Yet, the specific allocation of the radioactivity to the inflammatory cells via positron emission tomography (PET) imaging requires the suppression of glucose metabolism in viable myocardium. In mice, the most important model organism in basic research, this can be achieved by the application of ketamine/xylazine (KX) for anesthesia instead of isoflurane. Yet, while the consensus exists that glucose metabolism is effectively suppressed, a strategy for reproducible image analysis is grossly lacking and causes uncertainty concerning data interpretation. We introduce a simple strategy for systematic image analysis, which is a prerequisite to evaluate therapies targeting myocardial inflammation. Mice underwent permanent occlusion of the left anterior descending artery (LAD), inducing an acute myocardial infarction (MI). Five days after MI induction, 10MBq ¹⁸F-FDG was injected intravenously and a static PET/CT scan under ketamine/xylazine anesthesia was performed. For image reconstruction, we used an algorithm based on three-dimensional ordered subsets expectation maximization (3D-OSEM) followed by three-dimensional ordinary Poisson maximum a priori (MAP) reconstruction. Using this approach, high focal tracer uptake was typically located in the border zone of the infarct by visual inspection. To precisely demarcate the border zone for reproducible volume of interest (VOI) positioning, our protocol relies on positioning VOIs around the whole left ventricle, the inferobasal wall and the anterolateral wall guided by anatomical landmarks. This strategy enables comparable data in mouse studies, which is an important prerequisite for using a PET-based assessment of myocardial inflammation as a prognostic tool in therapeutic applications.