A large fraction of systemically infused MSC typically become trapped within the lungs as emboli owing to their large size and their repertoire of cell-surface adhesion receptors [20
]. Alternatively, they arrest and interrupt blood flow during the first pass through the precapillary level [24
]. Such passive arrest prevents the majority of infused MSC from homing to damaged or diseased tissues. Despite these complications, numerous animal studies and some clinical trials have reported favorable outcomes following systemic infusion of MSC [12
].The lack of specific homing is perhaps why high dosing is used in clinical trials; 150–300 million MSC are typically administered with each infusion [28
]. This prompts the questions: Can entrapped MSC transmigrate through the endothelium?; How long do the entrapped MSC survive?; and Can they provide benefit to distant organs? Several recent publications have attempted to address these questions.
Lee et al.
used a cross-species experimental design and real-time PCR (rtPCR) to track the fate of systemically administered human MSC in a mouse model [4
]. rtPCR analysis for human-specific Alu sequences (see Glossary)in blood samples showed that within 5 min of MSC infusion through the tail vein, 99% of MSC were cleared from the circulation. Within 10–30 min, a resurgence of ~2–3% of the infused MSC was observed within the blood stream. Tissue samples from various organs revealed that the majority of cells were initially found in the lung, which is consistent with previous studies [20
]. Then, 15 min after infusion, 83% of the human DNA was detected in the lung, whereas only trace amounts were detected in other tissues. The authors attempted to reduce lung entrapment by decreasing the number of infused cells, blocking key adhesion integrins and pretreating the MSC with rat white-blood cells (to sensitize them to Stromal Cell-Derived Factor-1); however, the fraction of trapped MSC remained unchanged. Histological analysis revealed that the MSC formed emboli in the afferent blood vessels of the lung, a common finding for systemic infusion of other cell types including hematopoietic stem cells and endothelial progenitor cells [9
]. No MSC were found in the bone marrow, which contradicted other studies [21
] and highlighted a potential shortcoming of PCR-based techniques, which could be approximately 10-fold less sensitive than radiolabeling techniques [31
In addition to PCR-based techniques for tracking the fate of systemically administered MSC, alternative approaches leverage the advantages of light and fluorescent microscopy that are well suited for small animal models. The Lin group has characterized tumor–cell, hematopoietic stem cell and MSC trafficking in the skull of living mice using in vivo
confocal and two-photon microscopy, which provides highresolution spatial delineation of the location of a cell [21
]. Similarly, Toma et al.
utilized intravital microscopy, which permits detailed real-time and serial imaging of in vivo
phenomenon, to examine the entrapment of MSC within a microvascular bed [24
]. In this model, the cremaster muscle of the rats was exposed and fluorescently labeled MSC were injected into the iliac artery. The density of MSC in varying depths of the vasculature was monitored over time using differential interference contrast and fluorescence imaging. All MSC arrested within 5 min of injection with 92% of the injected MSC entrapped during the first pass within the cremaster muscle. However, MSC were only trapped at the precapillary level, resulting in blockage of blood flow to the capillary bed. The number of viable MSC in the cremaster muscle decreased drastically over the next 72 h; only 14% of those originally entrapped survived, as determined by preserved nuclear morphology. As intravital microcopy is best suited for monitoring cells within a preselected location, redistribution of the MSC to other tissues is challenging to evaluate.
One method that can address this is bioluminescence imaging, which lacks single-cell resolution but enables whole-organism tracking of cell distribution. For example, Wang et al.
used MSC expressing a firefly–luciferase reporter gene in combination with bioluminescence imaging in a metastatic breast cancer model [35
]. This allowed noninvasive whole-animal tracking of intravenously injected MSC and their progeny over the course of several days. In healthy controls, MSC were initially found in the pulmonary capillaries but quickly dispersed after 1 day. The reduction of signal in the lungs can be attributed both to redistribution of MSC to other tissues as well as to cell death. Bioluminescence can be extremely valuable in characterizing MSC affinity and tropism for inflammatory and tumor microenvironments as has been reviewed by Spaeth et al.
Recent cell tracking studies have provided valuable insight into the distribution of MSC following systemic infusion and have begun to help elucidate the process of cell localization within specific tissues. However, it is critical to note that whole-animal imaging techniques such as bioluminescence lack the resolution to determine if cells remain in the vasculature or have undergone transendothelial migration. Aside from passive cell entrapment, which appears to be a dominant mechanism through which infused MSC reach their final destination, characterization of the host vasculature is required to better understand active homing mechanisms. The vascular expression of adhesion molecules and endothelial presentation of cytokines can vary substantially within a vascular bed [34
]. Thus, future studies should employ multiple methods, summarized in , to assess the final destination of the infused cells through both macroscopic distribution and microscopic spatial localization analysis.
In Vivo Cell monitoring techniques