In this study, we present evidence that reporter gene imaging can be used to track BMMC homing, retention, and survival in a mouse model of acute cardiac ischemia and reperfusion (I/R) injury. Our data can be summarized as follows: (1) the existence of a robust correlation between cell numbers and reporter gene imaging signals; (2) preferential homing of BMMCs within the first week of I/R injury; (3) <0.1% of the total BMMCs delivered eventually engrafted into the heart by two weeks after intravenous delivery; and (4) intravenous delivery of BMMCs did not confer significant improvement of ischemia-induced left ventricular contractility.
The success of stem cell therapy will likely require novel methods to determine the dynamic biodistribution and long-term fate of transplanted cells without reliance on postmortem histology. In recent years, several imaging techniques have been developed to better understand stem cell fate in vivo
. In general, they can be divided into two broad methodologies: direct labeling and indirect reporter gene-based imaging8, 9
. The former uses a detectable probe (e.g., radioactivity or iron particles) that can be loaded into cells prior to delivery. Aicher et al.
first demonstrated tissue distribution of endothelial progenitor cells incubated with radioactive [111
In]-oxine could be successfully monitored by scintigraphic imaging11
. As [111
In]-oxine has a half-life of 67.3 hours, only ~2% of the radioactivity remained in the infarcted heart after 96 hours. A follow-up study by Kraitchman et al.
injected porcine mesenchymal stem cells labeled with [111
In]-oxine intravenously and showed cardiac engraftment up to 7 days by single photon emission computed tomography (SPECT)13
. More recently, Hoffman et al.
injected human bone marrow cells labeled with 2-[18F]-fluoro-2-deoxy-D-glucose ([18
F]-FDG) via both intracoronary and intravenous routes12
. Since [18
F]-FDG has a half-life of 110 minutes, positron emission tomography (PET) imaging needed to be performed within
2 hours after cell delivery. The authors observed 1.3% to 2.6% of [18
F]-FDG-labeled bone marrow cells present in the myocardium after intracoronary delivery and, interestingly, only background activity was detected after intravenous delivery. Taken together, these studies suggest that radiolabeling techniques are suitable for immediate, short-term tracking of delivered cells but less apt for long-term follow-up8, 9
In contrast to the short half-life of radioactive probes, iron oxide particles can be tracked for long periods of time. Amado et al
. showed that porcine mesenchymal stem cells labeled with Feridex can be delivered by endomyocardial injection and tracked by magnetic resonance imaging (MRI) for 8 weeks14
. However, the main limitation of such direct iron-labeling techniques is that the MRI signals do not
necessarily reflect cell viability, because the iron particles might persist within dead cells, leak into intercellular space, and/or be engulfed by resident macrophages22
. These factors might explain why quantitative analysis of the iron-labeled retention showed >40% of the iron-labeled mesenchymal stem cells were still present 8 weeks after delivery in the study by Amado and colleagues14
. Indeed, it is well recognized that adult stem cells have poor post-transplant viability with an estimated 99% of mesenchymal stem cells dying within 4 days after injection into healthy mouse hearts23
Notwithstanding the technical limitations of the aforementioned direct imaging techniques, the ideal cellular imaging platform should provide information regarding the following: (i) real-time, dynamic cell biodistribution kinetics; (ii) long-term cell survival; and (iii) rates of cellular proliferation. At present, both methodologies described above lack these characteristics. An alternative approach--reporter gene imaging--is playing an increasingly prominent role in monitoring stem cell fate as demonstrated by this study and other studies reviewed elsewhere8, 9
. Because reporter genes are DNA sequences that encode for reporter proteins, one can follow the signal for as long as the transplanted cells and their progeny are viable. If, for example, the stem cells are dead or apoptotic, there will be no transcription and translation of the reporter gene, and thus no imaging signal. Similarly, if cells are actively migrating away from a particular ROI, signal strength will also decrease. Likewise, if the transplanted stem cells proliferate in vivo
, or migrate into a particular ROI, there will be an increase in the imaging signal detected from that area. Using this elegant reporter gene approach, we have been able to monitor BMMC homing over a relatively protracted time period (compared to radiolabeling technique11–13
) as well as to quantify BMMC survival more accurately (compared to iron labeling technique14, 22
). However, the low energy photons (2–3 eV) from BLI can become attenuated within deeper tissues (e.g., heart) compared to more superficial locations (e.g., skeletal muscles). In our experience, the lower detection limit of BMMCs within the heart is approximately on the order of 1,000 cells compared to 100 cells in the subcutaneous tissue over the leg (unpublished data).
In our study, bioluminescence imaging of the I/R group showed significantly higher cell signal activity in the heart compared to the sham group during the first week. This difference is likely due to activation of cytokines that promotes homing of BMMCs to the ischemic sites6
. A previous study using gene expression analysis has shown that stromal cell-derived factor-1α (SDF-1), vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 (MMP-9), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) are activated after myocardial infarction24
. The exact chemoattractant factors responsible for stem cell homing remain unclear, and the process itself may be inefficient as shown by our in vivo
imaging, histological analysis, and ex vivo
real-time PCR. In fact, by 2 weeks following transplant, less than 0.1% of the total injected cells remained engrafted in the recipient hearts. The low number of cell engraftment may also explain the lack of improvement in cardiac function observed in our study. It is possible that the observed trend might have achieved statistical significance with a larger cohort of animals. Moreover, our study remains limited in that we did not follow animals out for longer than 4 weeks to observe whether the trend in functional improvement persisted or diminished. Critical evaluation of functional improvement as a function of cell dosage and time remains an area that requires further study.
In future studies, we believe in vitro
identification (e.g., by transcriptional profiling) and in vivo
validation (e.g., by reporter gene imaging) of factors important to homing and cell retention will be an attractive approach to coax exogenously administered stem cells to home in to the heart and promote long-term functional improvement. However, one of the main drawbacks of bioluminescence imaging is its restriction to small animal pre-clinical validation studies, because the low-energy photons (2–3 eV) become attenuated and scattered within deep tissues25
. In addition, the inability to perform 3-D BLI impairs ability to accurately localize signal sources from deep tissues (e.g., heart) as discussed earlier. Specifically, one of the resultant challenges from compressing 3-D data into a 2-D picture is that of increased noise to signal ratio. In the chest, for example, the summation of lung background might obscure a relatively low cell signal emitted from the heart and measured through an ROI designated over a two dimensional space. Thus, ongoing development of positron emission tomography (PET)-based reporter gene and reporter probe technique that uses high-energy photons (511 keV) and have 3-D imaging capabilities will be necessary for clinical application in the future26
In conclusion, our study suggests that reporter gene imaging can be a valuable tool for studying stem cell fate in vivo. The same imaging platform can be adopted to investigate basic mechanisms underlying myocardial cell therapy and optimize the key variables involved, such as the most efficacious cell type(s), appropriate cell dosing, and best routes of delivery (e.g., intracoronary versus intravenous). We hope that carefully designed studies using the reporter gene imaging techniques developed here and in future investigations will lead not only to advancement of stem cell research, but also to useful novel therapies and diagnostic tools for clinicians and patients.