Stem cell therapy for myocardial infarction

Coronary heart disease and heart failure continue to be significant burdens to healthcare systems in the Western world and are predicted to become so in emerging economies. Despite mixed results in both experimental and clinical studies, stem cell therapy is a promising option for patients suffering from myocardial infarction or patients with chronic heart failure after myocardial infarction. However, many issues in the field of cellular cardiomyoplasty still need to be resolved. This thesis describes the experiments performed in a pre-clinical model in swine with reperfused myocardial infarction aiming at addressing several of these issues. Chapter two of this thesis shows that infarct size in swine can be measured accurately with multislice computed tomography, as compared to the “golden standard” histology. This study showed that myocardial viability can be assessed with multislice computed tomography. Furthermore, since we used magnetic resonance imaging in chapter three and four, we showed that for purposes of infarct size assessment multislice computed tomography compares well with magnetic resonance imaging, which is described in chapter three. Measurement of infarct size in patients with acute myocardial infarction is clinically relevant because infarct size is predictive of left ventricular function and geometric configuration and, hence, long-term clinical outcome. Information on infarct size obtained with multislice computed tomography would enhance the diagnostic armamentarium of physicians who lack access to cardiac magnetic resonance imaging or encounter patients who have contra indications to undergo magnetic resonance imaging. The review of umbilical cord blood derived cells in the fourth chapter of this thesis shows a great potential of these cells to regenerate damaged myocardium. These cells can easily be obtained in large numbers and are not harvested from diseased individuals, therefore they have a great differentiation and proliferation capacity. Moreover, they do not raise ethical difficulties question, as do embryonic stem cells. In chapter five, the effect of umbilical cord blood cells is assessed with magnetic resonance imaging in a porcine model of myocardial infarction. There was no positive effect on left ventricular function or infarct size four weeks after injection of intracoronary administration of the umbilical cord blood cells, which what not very surprising since only a few of the injected cells survived. Therefore, the immunogenic status of these cells is not fully understood yet. However, this study shows that cultured umbilical cord blood cells should be used with caution when applied intracoronary, since their large cell size result in occluded blood vessels, thereby causing micro infarctions. Hence, it cannot be excluded that a possible positive effect of the umbilical cord blood derived cells was obscured by the induction of micro infarctions caused by the mode of administration. Therefore intracoronary application is not suitable for these cells. Although intracoronary injection is an easy and less invasive technique to administer cells post myocardial infarction, intramyocardial injection was shown to result in positive effects by Kim et al. In contrast to the cells used in the initial experimental rodent studies, bone marrow derived mononuclear cells are the most common used cells in clinical trials. In chapter six the capacity of mononuclear cells and unselected bone marrow in a pre-clinical porcine model of reperfused myocardial infarction is evaluated. Our model closely mimics the clinical setting of myocardial infarction with regards to the route of administration and timing of stem cell therapy given in clinical trials. Four weeks after treatment with mononuclear cell injection a decrease in infarct size is observed as measured with magnetic resonance imaging. This was not observed after injection of unselected bone marrow. Histology showed that there was a trend towards more calcifications in the infarct area after the injection of unselected bone marrow. However, there was no beneficial effect of mononuclear cell or unselected bone marrow therapy on left ventricular function after myocardial infarction. Our results after mononuclear cell injection do not differ from those of the clinical trial performed in Leuven, Belgium by Janssens et al. All clinical trials used the method of intermittent balloon occlusion during intracoronary injection of cells through the wire lumen of the balloon catheter, based on the assumption that this would yield increased adhesion of the injected cells to the vascular wall during the no-flow period, thereby leading to a higher cell engraftment. However, in our previous studies we used a selective probing injection catheter without interruption of blood flow. Therefore, we tested these different injection techniques in chapter seven. We observed no differences in the number of bone marrow mononuclear cells engrafted in the myocardium when applied through these two catheters (probing or balloon catheter). Therefore, the lack of effect on left ventricular function of bone marrow derived mononuclear cells in our study (which was described in chapter six), cannot be explained by the injection technique used. Since cultured umbilical cord blood cells can cause micro infarctions when administered intracoronary in healthy myocardium (chapter five), we investigated in chapter eight whether cultured bone marrow derived mononuclear cells could cause micro infarctions four days after injection in healthy myocardium. We found that this was the case for cultured cells, but not for freshly isolated cells. Cultured cells are larger in size compared to freshly isolated cells, which result in the obstruction of the microcirculation. Although clinical trials suggest that intracoronary stem cell injection is safe, cultured stem cells should be used with caution when applied intracoronary. The labeling of injected cells with iron to be able to track them in vivo with magnetic resonance imaging is assessed in chapter nine. This study showed that due to the hemoglobin breakdown products containing iron which is present in hemorrhagic areas in the reperfused infarct, iron labeling of intramyocardially injected cells is not suitable to track the stem cells after injection. Future directions Future studies are required to investigate whether hemorrhage induced signal voids cause similar interference with cell detection with magnetic resonance imaging after intracoronary injection in reperfused myocardial infarctions or after injection in non-reperfused infarcts. Gadolinium may be a more suitable marker than iron to track cells in vivo with magnetic resonance imaging in reperfused myocardial infarctions. It should be further investigated what the effect of injection of cultured cells is, when applying a different mode of administration, for example intramyocardial cell injection. The effect of the stem cells used in our model seems to be disappointing compared to earlier study results in small rodents. It is possible that in our model bone marrow derived mononuclear cells will have a positive effect on left ventricular function in time. Therefore, in new experiments, swine should be monitored for a longer follow-up time, e.g. 2, 3, 6 and 12 months. For these experiments miniature swine should be used. Differentiation of bone marrow derived stem cells in vitro towards cardiomyocytes is an option to enhance the effect of stem cell therapy in large mammals. However, it should always be tested whether these cultured cells induce microinfarctions when applied intracoronary. Pre-differentiation into cardiomoyocytes might prevent the differentiation of injected cells into fibroblasts or inflammatory cells. The pre-differentiation might replace scar tissue by viable cardiomyocytes, and this might enhance the effect on infarct size on left ventricular function in vivo after injection. However, if the new cardiomyocytes are not able to survive in ischemic tissue, it should be investigated whether additional cytokine injections are needed to induce angiogenesis in the infarct tissue to enhance cell survival. Patients benefit from optimal pharmacological treatment after myocardial infarction. Over time, ejection fraction will increase and infarct size will decrease in patients suffering from myocardial infarction due to remodeling which is influenced by optimal pharmacological treatment. Therefore, in a pre-clinical porcine model of myocardial infarction the combination of optimal pharmacological treatment and stem cell therapy could be tested to evaluate the additional effect of stem cell therapy on the recovery of function, infarct size and the remodeling after myocardial infarction. However first, in animal models, it is necessary to determine the optimal cell number required to obtain optimal effects of the cells on infarct size and left ventricular function. It should be investigated how to access the cells (e.g. bone marrow aspiration versus cytokine mobilization), and whether they should be expanded ex vivo. Second, it should be determined which cell type will have maximal clinical effects. Third, the optimal delivery method will have to be determined. Finally, understanding the mechanism of cardiac regeneration in animals will shed a light on the optimal therapy for patients. The potential effect of stem cell therapy in patients should finally be assessed in large, randomized, placebo-controlled, double-blind clinical trials.

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