In this study we investigated BMSC survival and ischemia-directed homing using two in vivo reporter gene expression imaging techniques, BLI and confocal endomicroscopy. Endomicroscopy has superior sensitivity in visualizing transplants at low cellular concentrations or with fading tracer signals. Its high optical resolution enables the study of cellular morphology, delineation of the cell infiltrate as well as homing capacity of individual cells. Drawbacks of the technique are its invasive nature requiring direct tissue contact with limited deep tissue penetration. Autofluorescence did occur at times but the chance of false positive findings in our study was rather limited because of the typical cell size and morphology of BMSC. BLI, on the other hand, is a non-invasive and hence more practical technique to use in follow-up studies of transplant survival, but it lacks the high optical resolution of endomicroscopy. It also has superior specificity for detecting Luc+ cells. However, BLI is unable to detect single cells or transplants with low cell concentration or limited cell numbers. Nevertheless, a combination of the high specificity of BLI joined with the high optical resolution of confocal endomicroscopy, overcomes several constraints of either technique alone and enables to study the biology of BMSC survival and homing with more precision and accuracy.
Studying cell homing using reporter genes has several advantages over conventional cell tracking agents, such as cell labeling dyes, microparticles, radioisotopes or magnetic resonance imaging (MRI) contrast agents. An important drawback of these conventional methods is that the agent used can become decoupled from the original cell transplant and/or ingested by other cell types, such as macrophages. Macrophage ingestion is a major confounder in transplantation studies because many cells of donor origin die shortly after cell transplantation, triggering a local immune reaction with macrophage infiltration
Because reporter genes are integrated into the cell genome, cell tracking studies using reporter gene expression is much less likely to yield false positive results: the expression of the reporter gene is limited to viable cells of the original cell transplant and is preserved in successive cell divisions. When cells do not survive or are ingested by other cells, the signal diminishs over time. However, since the reporter gene is integrated in the genomic DNA, cell proliferation will increase rather than decrease signal intensity
, as such preventing dilution of the original labeling signal.
Despite these benefits, the use of reporter genes also entails a number of caveats. A first problem could arise from the induction of immune responses directed against non-host alloantigens expressed on the cell surface of the transfected cells that express non-self proteins, such as Luc or eGFP. However, Bergwerf et al.
 demonstrated that the immune reactive cytotoxic T-cell response following BMSC transplantation was not specific for the non-self eGFP or Luc genes. These authors suggested that the observed T-cell mediated transplant rejection could be due to the presence of xenogeneic constituents of the growth medium, such as fetal calf and horse serum, which render the cultured cells more immunogeneic. Another concern is gene silencing, which is a mechanistically very complex phenomenon that can vary with specific experimental conditions due to a site-dependent adaptation of the expression of the reporter gene in vivo.
We demonstrated in our hindlimb ischemia mouse model that cell transplant survival after IM injection was limited in time and that CsA immunosuppression alone was not sufficient to maintain long-term survival of the allogenic BMSC graft. Our data are in line with the BLI results of Swijnenburg et al
. In this study, immunosuppression with mycophenolate mofetil, sirolimus or tacrolimus, both in monotherapy or in combination, failed to induce long-term survival of embryonic stem cell xenografts. On the other hand, Swanger et al.
 showed that, at least in the central nervous system, high dose CsA significantly increased allogeneic BMSC graft survival compared to standard dose CsA, indicating that higher doses of CsA could be sufficient to maintain long-term survival of allogeneic BMSC at immunologically privileged sites. Furthermore, evidence from BLI experiments from Zangi et al.
 about survival and rejection of IV or intraperitoneally injected allogeneic MSC transplants, indeed showed that, at least in mice, survival of a systemically injected MSC transplant is limited in time. Moreover, it was demonstrated that even IM injection of BMSC in immune competent syngeneic mice did not result in permanent graft survival
We used a hindlimb ischemia model for the study of ischemia-directed homing firstly for reasons of good accessibility of the tissues of interest for the optical probe of the confocal endomicroscope. Therefore, in vivo endomicroscopy would be less suitable to study homing towards deeper anatomical structures or to moving organs, such as the heart. Secondly, hindlimb ischemia mouse models have already been validated extensively as experimental set-up for the study of ischemia-directed MSC homing. A better understanding of survival and homing characteristics of MSC in this experimental model is of particular relevance for the early-phase clinical trials on the use of human MSC for the treatment of critical limb ischemia that are currently ongoing
We caution against CV injection of large amounts of BMSC because this leads to massive pulmonary infarction. Gao and co-workers already reported that systemic injection of MSC initially results in retention in the lungs, after which a gradual redistribution could be seen to the liver, bone marrow and other organs. These authors showed that the pulmonary retention could be reduced using the vasodilator sodium nitroprusside
. This finding, i.e. initial intrapulmonary cell trapping with gradual tissue redistribution, was confirmed by Daldrup-Link et al.
, who used MRI imaging, and by in vivo BLI studies by Schrepfer et al.
. In the latter study, episodes of tachypnea, apnea and hemodynamic alterations, characteristic of pulmonary embolism, were reported when MSC were directly infused into the inferior vena cava. Pulmonary entrapment was also evident in a myocardial infarction study with BrdU-labeled MSC
. When the lung circulation was bypassed through direct intracavitary administration of MSC, significantly more cells were observed within the infarction zone within the first hours after transplantation. In a later report by the same authors, long-term engraftment of the infused MSC, as initially suggested on the MRI images, could not be verified with Y chromosome gene detection. Further analysis revealed that the positive MRI signals originated from cardiac resident macrophages that had ingested the MRI contrast particles. Intriguingly, although cells of donor origin were absent at 4 weeks after infusion, ventricular function was still improved in the MSC-treated animals
. The authors attributed this phenomenon to possible paracrine effects. Another tissue distribution study in the clinical setting of myocardial infarction, showed initial lung uptake of intravenous (IV) injected 18F-FDG-labeled bone marrow-derived cells, followed by a gradual redistribution to the reticulo-endothelial system (spleen, liver, bone marrow)
. Stem cell homing towards the infarction site, however, could not be demonstrated. Evidence for the pulmonary entrapment of stem cells was also reported after IV injection of neural stem cells
, leading to massive pulmonary inflammation and apoptosis. Together, these studies indicate that the lung might act as a barrier for the passage of different stem cell subtypes
, especially for those that are relatively large and express abundant adhesion molecules
. Our study adds further evidence to the finding of intrapulmonary cell trapping after CV injection of BMSC and questions the safety of this route for BMSC transplantation.
As indicated by our results, direct intracavitary injection into the left ventricle is a feasible approach to circumvent this pulmonary “first-pass” effect, but it is rather impractical to use in small animal experiments. However, direct IA administration of BMSC in the arterial circulation, e.g. by intracoronary injection after myocardial infarction, thereby avoiding the lung circulation, would in our opinion prove to be the method of choice for BMSC cell application in large animal and human clinical trials. Furthermore, considering that active ischemia-directed BMSC homing of unstimulated BMSC was a rare biological phenomenon in our studies, this also favours direct IA injection of BMSC into the vasculature of the tissue of interest. Alternatively, the homing process could potentially be improved with use of stimulated rather than naive BMSC. BMSCs can easily be ex vivo manipulated by pretreatment, preconditioning and genetic modification, which was reported to increase their therapeutic benefit
. For instance, hypoxic proconditioning has been shown to stimulate the induction of several chemokine receptors, such as CXCR4 and CXCR7. This led to improved migration and recruitment of BMSC into ischemic tissues and stimulated secretion of proangiogenic and mitogenic factors improving therapeutic potential of the transplanted BMSC
As a limitation of our work, we are aware that we could have underestimated the BMSC homing process because of the specific limitations of our detection techniques and experimental set-up. However, by combining the advantages of BLI with endomicroscopy, we feel confident about our results. However, it is possible that we underestimated the homing potential of BMSC, because of the limited graft survival time or the use of naive rather than stimulated or manipulated BMSC. Also, the use of CsA could have interfered with BMSC kinetics or altered the BMSC microenvironment, leading to an impaired homing respons. Transgenic immunodeficiency models, rather than immunosuppression with pharmacological agents, would be an interesting alternative to use in future studies on BMSC survival and homing. Finally, increasing the number of observations in each group would further strengthen our statements.
In summary, in this study a combination of BLI and confocal endomicroscopy was applied for dual reporter gene-expression imaging of BMSC survival and homing in a clinically relevant mouse model of peripheral tissue ischemia. We report that allogeneic BMSC graft survival time was limited in the ischemic muscle and that CsA immunosuppression alone was not able to sustain long-term survival of the allograft. We did not find abundant evidence for ischemia-directed BMSC homing as a common biological process and caution against the direct CV injection of large amounts of BMSC.