Background: Insulin-like growth factor 1 (IGF-1) and hepatocyte growth factor (HGF) are among the most promising growth factors for promoting cardiorepair. Here, we evaluated the combination of cell- and gene-based therapy using mesenchymal stem cells (MSC) genetically modified to overexpress IGF-1 or HGF to treat acute myocardial infarction (AMI) in a porcine model. Methods: Pig MSC from adipose tissue (paMSC) were genetically modified for evaluation of different therapeutic strategies to improve AMI treatment. Three groups of infarcted Large White pigs were compared (I, control, non-transplanted; II, transplanted with paMSC-GFP (green fluorescent protein); III, transplanted with paMSC-IGF-1/HGF). Cardiac function was evaluated non-invasively using magnetic resonance imaging (MRI) for 1 month. After euthanasia and sampling of the animal, infarcted areas were studied by histology and immunohistochemistry. Results: Intramyocardial transplant in a porcine infarct model demonstrated the safety of paMSC in short-term treatments. Treatment with paMSC-IGF-1/HGF (1:1) compared with the other groups showed a clear reduction in inflammation in some sections analyzed and promoted angiogenic processes in ischemic tissue. Although cardiac function parameters were not significantly improved, cell retention and IGF-1 overexpression was confirmed within the myocardium. Conclusions: The simultaneous administration of IGF-1- and HGF-overexpressing paMSC appears not to promote a synergistic effect or effective repair. The combined enhancement of neovascularization and fibrosis in paMSC-IGF-1/HGF-treated animals nonetheless suggests that sustained exposure to high IGF-1 + HGF levels promotes beneficial as well as deleterious effects that do not improve overall cardiac regeneration.

The intrapericardial delivery has been defined as an efficient method for pharmacological agent delivery. Here we hypothesize that intrapericardial administration of cardiosphere-derived cells (CDCs) may have an immunomodulatory effect providing an optimal microenvironment for promoting cardiac repair. To our knowledge, this is the first report studying the effects of CDCs for myocardial repair using the intrapericardial delivery route. CDCs lines were isolated, expanded and characterized by flow cytometry and PCR. Their differentiation ability was determined using specific culture media and differential staining. 300,000 CDCs/kg were injected into the pericardial space of a swine myocardial infarcted model. Magnetic resonance imaging, biochemical analysis of pericardial fluid and plasma, cytokine measurements and flow cytometry analysis were performed. Our results showed that, phenotype and differentiation behavior of porcine CDCs were equivalent to previously described CDCs. Moreover, the intrapericardial administration of CDCs fulfilled the safety aspects as non-adverse effects were reported. Finally, the phenotypes of resident lymphocytes and TH1 cytokines in the pericardial fluid were significantly altered after CDCs administration. The pericardial fluid could be considered as a safe and optimal vehicle for CDCs administration. The observed changes in the studied immunological parameters could exert a modulation in the inflammatory environment of infarcted hearts, indirectly benefiting the endogenous cardiac repair.

Acute myocardial infarction triggers a strong inflammatory response in the affected cardiac tissue. New therapeutic tools based on stem cell therapy may modulate the unbalanced inflammation in the damaged cardiac tissue, contributing to the resolution of this pathological condition. The main goal of this study was to analyze the immunomodulatory effects of cardiosphere-derived cells (CDCs) and their extracellular vesicles (EV-CDCs), delivered by intrapericardial administration in a clinically relevant animal model, during the initial pro-inflammatory phase of an induced myocardial infarction. This effect was assessed in peripheral blood and pericardial fluid leukocytes from infarcted animals. Additionally, cardiac functional parameters, troponin I, hematological and biochemical components were also analyzed to characterize myocardial infarction-induced changes, as well as the safety aspects of these procedures. Our preclinical study demonstrated a successful myocardial infarction induction in all animals, without any reported adverse effect related to the intrapericardial administration of CDCs or EV-CDCs. Significant changes were observed in biochemical and immunological parameters after myocardial infarction. The analysis of peripheral blood leukocytes revealed an increase of M2 monocytes in the EV-CDCs group, while no differences were reported in other lymphocyte subsets. Moreover, arginase-1 (M2-differentiation marker) was significantly increased in pericardial fluids 24 h after EV-CDCs administration. In summary, we demonstrate that, in our experimental conditions, intrapericardially administered EV-CDCs have an immunomodulatory effect on monocyte polarization, showing a beneficial effect for counteracting an unbalanced inflammatory reaction in the acute phase of myocardial infarction. These M2 monocytes have been defined as “pro-regenerative cells” with a pro-angiogenic and anti-inflammatory activity.

The optimal timing of cardiac stem cells administration is still unclear. We assessed the safety of same-day and delayed (one week) delivery and the possible influence of the timing on the therapeutic outcomes of allogeneic porcine cardiac stem cells administration after acute myocardial infarction in a closed-chest ischemia-reperfusion model. Female swine surviving 90 min occlusion of the mid left anterior descending coronary artery received an intracoronary injection of 25x106 porcine cardiac stem cells either two hours (n = 5, D0) or 7 days (n = 6, D7) after reperfusion. Controls received intracoronary injection of vehicle on day 7 (n = 6, CON). Safety was defined in terms of absence of major cardiac events, changes to the ECG during injection, post-administration coronary flow assessed using the TIMI scale and cardiac troponin I determination after the intervention. Cardiac Magnetic Resonance was performed for morphological and functional assessment prior to infarction, before injection (D7 and CON groups only), at one and 10 weeks. Samples were taken from the infarct and transition areas for pathological examination. No major adverse cardiac events were seen during injection in any group. Animals receiving the therapy on the same day of infarction (D0 group) showed mild transient ST changes during injection (n = 4) and, in one case, slightly compromised coronary flow (TIMI 2). Cardiac function parameters and infarct sizes were not significantly different between groups, with a trend towards higher ejection fraction in the treated groups. Ventricular volumes indexed to body surface area increased over time in control animals, and decreased by the end of the study in animals receiving the therapy, significantly so when comparing End Diastolic Volume between CON and D7 groups (CON: 121.70 ml/m2 ± 26.09 ml/m2, D7: 98.71 ml/m2 ± 8.30 ml/m2, p = 0.037). The treated groups showed less organization of the collagenous scar, and a significantly (p = 0.019) higher amount of larger, more mature vessels at the infarct border. The intracoronary injection of 25x106 allogeneic cardiac stem cells is generally safe, both early and 7 days after experimental infarction, and alleviates myocardial dysfunction, with a greater limitation of left ventricular remodeling when performed at one week.