In the present work, mBM-MSCs localization and persistency in the mouse lung was examined. mBM-MSCs were labelled via stable transduction with a replicating incompetent lentiviral vector delivering an expression cassette for luciferase and GFP. Since the luciferase ORF was in bicistronic form with GFP by an IRES sequence [22, 23], the level of GFP expression in the transduced cells, should reflect the expression level of the ORF upstream to the IRES which, in this specific case, was luciferase. However, in most of the cases, the luciferase expression level is even higher respect to that of the downstream GFP ORF [22, 23]. Using this strategy, mBM-MSCs expressing the highest amount of luciferase could be selected by simply sorting cells with the highest GFP expression. Thus, increasing the sensitivity of the system since BLI was employed for detection.
BLI is a powerful technique based on the detection of visible light produced during luciferase-mediated oxidation of a molecular substrate in the presence of the enzyme resulting from its expression in vivo as a molecular reporter. Bioluminescence arising from luciferase can be imaged as deep as several millimeters within tissues, allowing at least organ‐level resolution. Being simple to execute and minimally invasive, BLI enables monitoring and serial quantification of biological processes without sacrificing the experimental animal. This powerful technique can therefore reduce the number of animals required for experimentation because multiple measurements can be made in the same animal over time, thus also minimizing the effects of biological variations. Whole animal bioluminescent imaging is progressively becoming more widely applied by investigators with diverse backgrounds because of its low cost, high throughput, and relative ease of operation in visualizing a wide variety of in vivo cellular events. In addition, the ability to continually monitor a single individual reduces the amount of inter-animal variation and can reduce errors, leading to higher resolution and less data loss. In addition, the constant progress in the hardware and software, required for this technique, facilitates its application by researchers with little background in molecular imaging on living animals [24, 25]. Taking advantages of an integrated bioluminescent over-expressed reporter gene into the genome of transduced mBM-MSCs and of BLI, we have been able to localize mBM-MSCs in the lung in real time up to 28 days, independently from the route of administration and from the amount of cells administered. There are two main methods to introduce cells into the lung: systemic delivery, IV and local delivery, IT. Although IV represents the simplest delivery way, it rises a thromboembolic potential risk directly correlated to the number and the concentration of cells injected. In particular, concentration above 107 cells/ml was shown, at least in mice, to generate a significant increase of pulmonary embolism and consequent mortality [26]. In our case, a single dose of 105/200 µL cells was chosen, because, after several attempts, this amount of cells showed to be the best compromise between risk of embolism and signal detection. Alternatively to the IV route, IT route was employed too. The lung is the only corporeal district where compounds can be directly delivered through the airways. Although previous works reported positive [27–29] and negative [27, 30] results, cell survival levels in these studies were performed using fluorescent techniques, which could justify these strong discrepancies. In our study, to determine if the airway might be a more effective route of mMSCs delivery, 105/50 µL cells were administered. Although the number of IT delivered cells was larger respect the IV ones, the cell persistency and signal kinetic we obtained in the lung were similar, even if a fraction of IT cells is generally coughed out by mucociliary clearance.
Even if with very low efficiency, it was shown that mMSCs derived from cytokeratin-18 (K18) promoter driven GFP (K18GFP) transgenic mice, when delivered to the lung of wild-type recipient, could engraft and transdifferentiate in epithelial committed cells showing K18GFP transgene in vivo up regulation but not in vitro [28, 31]. This data suggests that the lung environment is responsible for lineage commitment and perhaps for long term survival of mMSCs [28, 31]. Based on this assumption, mBM-MSCs were pre-adapted in the lung, re-isolated and IT re-delivered to the lung. Even thought, cell survival or at least their detectability did not variate, still persisting for 28 days.
A generally relevant aspect following MSCs allogenic or xenogenic transplantation is the recipient immune response against the transplanted cells. T-cells and antibodies production has been shown not only against the cells but also against FBS derived antigens present in the cell media used to culture the cells [32, 33]. We did substitute FBS in cell culture medium with mouse autologous serum 24 hours previous cell delivering into the lung. However, no substantial differences in terms of cell survival, were observed.
C57BL/6 mice genetic background are largely used for adoptive transfer experiments and a large number of sublines with genetic polymorphisms are used around the world [34] showing defined immune-phenotypic differences [35]. Although it is difficult to define the impact that a genetic drift of a mouse specific subline could have on mMSCs survival when delivered to the lung, we limited our investigation to two sublines coming from two different mouse breeders, without observing significate differences.