In the current study, we demonstrated that modulation of the microenvironment, using antioxidants, results in an increase in stem cell survival early after transplantation to the myocardium in small animals. Furthermore, we show that noninvasive reporter gene imaging can be used to study stem cell biology in cardiac disease. Studies like the one described here will play a major role in the understanding of the interaction between stem cells and their microenvironment in the living subject.
In the current study, we provide evidence that increased oxidative stress is involved in the decreased stem cell viability observed under severe hypoxic conditions. Furthermore, our data suggest that the increase in oxidative stress seen in hypoxia is due to an increase in the production of ROS, and that the beneficial effect seen with antioxidants (as used in this study) is mainly through the metabolism of ROS, rather than the modulation of pro-oxidant enzymes. From the present study, we are not able to discern if other pro-oxidant enzymes could play a role in the increase of oxidative stress seen in hypoxia. Overall, the beneficial effect of oxidative stress blockade likely provides a more favorable microenvironment for stem cell engraftment and survival in the heart, as previously shown in the hindlimb [17
]. Another factor to be taken into consideration is that the oxidative stress levels and how different cells adjust to different pathophysiological states may vary across different cell types (e.g., mesenchymal stem cells or rat cardiomyoblasts—used in this study). Thus, extrapolating these results to other cell populations or pathophysiological states should be done with caution. In the current study design, the benefit observed is not a long-term effect, as other mechanisms are likely responsible for the cell death in medium and long term (e.g., prolonged inflammation, immune response), which should be the target in future studies. Conceivably, strategies that target medium and late cell death may lead to novel therapeutic strategies for a more effective stem cell therapy approach. Due to the study design, from our study, we are not able to separate the effects of cell pretreatment from the potential beneficial effects provided by antioxidants in the drinking water. However, due to the early and relatively short-term duration of the beneficial effects of antioxidant blockade on cell survival, it is unlikely that the latter contributed significantly to the early increase in cell survival observed in our study.
In the present study, we chose to use the firefly luciferase reporter gene system, imaged using a cooled CCD camera, because it allows fast and high-throughput imaging of cell survival. This is the first report, to our knowledge, where reporter gene technology has been successfully used to report increased survival of transplanted cells in the myocardium, through exogenous and systemic modulation of the microenvironment. While cell survival in our study was similar to that observed in other studies [6
], some reports have reported higher rates of cell survival after transplantation [7
], and there are many potential reasons for that. As mentioned before, different cell types may have different responses to comparable challenge and thus, different survival. One of the most likely explanations is that a combination of immune response, ischemia, and apoptosis account for the relatively short survival of the transplanted cells [8
]. While use of antioxidants improved cell engraftment and survival, providing evidence to the deleterious role of increased oxidative stress in early stem cell death, it was not sufficient to counteract the deleterious stimuli faced by transplanted cells. In other words, it is unlikely that antioxidant intervention by itself will be sufficient to result in a meaningful increase in stem cell survival after transplantation. However, a combination of antioxidant blockade with other interventions that target stem cell survival at later stages may prove useful. Another potential explanation is that reporter genes might have experienced a certain degree of gene silencing (previously described with full-length CMV promoter) [37
]. However, this is unlikely as gene silencing has been shown to be more prominent at later stages after cell transplantation [37
]. Nevertheless, use of a constitutive mammalian promoter (such as ubiquitin or α-actin) may help to address this issue. Furthermore, we have not observed any effect of tempol on the level of fluc expression under control conditions. While no gross morphological changes or alterations in proliferation rate were observed after stable cell transfection, we cannot completely exclude that introduction of reporter genes, or the CMV promoter might be associated with alterations in cell genotype. In fact, previous reports have suggested that introduction of reporter genes may result in upregulation and downregulation of certain genes and even changes in cell growth characteristics [39
]. Because most, if not all, of these studies used a strategy of random DNA integration, it is possible that different studies may have different responses in terms of cell alterations, what may translate in differences in protein expression. In addition, in plasmid-mediated integration of DNA, variable number of copies of the exogenous DNA (i.e., reporter gene) may get integrated into the genome, what may result in changes of the cell’s phenotype. Thus, further studies are needed to clarify this issue, as molecular imaging strategies come closer to clinical applications. In addition, it is possible that the reporter genes were relatively weak, and that after the first week, the amount of light emission was below the detection limit for the cooled CCD camera.
In the current study, we delivered cells using an epicardial approach. This route of delivery is the most commonly used route in small animal models [6
] and has also been used in clinical studies. Furthermore, we have shown that the use of US guidance for cell delivery is reproducible and consistent [22
] and likely results in less perturbation of the microenvironment (compared to open chest injections) as well as less morbidity and mortality. However, it may not be the cell delivery route of choice when the target is a certain coronary distribution (and a large area of myocardium), rather than a specific area of the myocardium (e.g., intracoronary delivery in large animals or patients after myocardial infarction). In the present study, we used intact unperturbed rats, so results from this study should not be extrapolated to other models of disease (e.g., myocardial ischemia/infarction). Nevertheless, the results reported here open novel avenues to investigate the effect of metabolic intervention in ischemic myocardium. Furthermore, while the use of ischemic or infarcted models may be closer to clinical practice, even in intact subjects (like the ones used in this study), stem cells will face a noxious microenvironment after transplantation to the myocardium and have been shown to have short cell survival [6
]. Furthermore, the potential noxious environment that stem cells will face after transplanted to diseased tissue (e.g., infarcted myocardium) is likely more significant than that observed in healthy tissue (as used in this study). Thus, modulation of the microenvironment in that setting may translate into a more pronounced beneficial effect on stem cell survival.
In summary, in the current study, we demonstrated that modulation of the cell and myocardial microenvironment can have an impact on stem cell survival. We showed that antioxidants preserved cell survival after transplantation to the myocardium of intact animals. Furthermore, we showed that reporter gene imaging can be used to monitor stem cell biology of transplanted cells to the myocardium. Increased understanding of the interaction between stem cells and their local microenvironment will likely leads to more successful therapeutic strategies for CAD.