Our data indicate that the majority of systemically delivered MSCs arrest at the precapillary level, resulting in cessation of flow in the microvessel and flow reduction in the feeding arteriole. Studies in large animals8,9
have shown that intracoronary delivery of MSCs leads to microinfarctions, whereas with IV infusion, most cells are trapped in the lungs.10,11
We provide direct evidence that this is attributable to precapillary MSC entrapment. This is likely a mechanical phenomenon, because MSC entrapment invariably occurred in microvessels smaller in caliber compared to the cells. We did not observe arteriolar MSC adhesion, such as that described with cancer cells in similar intravital microscopy studies.17
Unlike prior studies demonstrating increased lung clearance of MSCs with administration of SNP,10
this was not the case in the cremaster microcirculation, probably related to the reduced caliber of the pathways selectively dilated by SNP in this preparation.18
Applying a method used to measure leukocyte deformability to evaluate this property in MSCs,16
we confirmed that MSCs were as deformable as circulating MNCs (). Nonetheless, only a limited number of MSCs were able to traverse through 10-µm pores (), further supporting the concept that precapillary MSC entrapment in vivo is a mechanical phenomenon. Importantly, the rat cells used in this study were expanded to a similar degree as the human MSCs currently used in clinical trials. Intraarterial injection of human MSCs (Cambrex/Osiris, 20-µm mean diameter in our laboratory) led to similar microvascular plugging in the rat cremaster (data not shown). We posit that the large cell size is a direct consequence of the culture techniques, because freshly isolated human MSCs are smaller in size (≈10-µm diameter19
). Different methods of MSC expansion may retain the original cell size in the marrow20
and could result in improved intravascular rheology.
The tissue integration of entrapped MSCs observed in this study was a slower process compared to that reported for leukocyte transmigration,21
extending over 24 to 72 hours (). The entrapped MSCs spread on the luminal surface by 24 hours (, and ), in agreement with electron microscopy data reporting initial MSC integration within the endothelial layer.11,22
The ischemia caused by microvascular plugging is likely responsible for the cell death observed at this time point, as indicated by the predominant cell/nuclear fragmentation at 24 hours (). By 72 hours, the surviving MSCs integrate into a perivascular/intramural location, reminiscent of pericytes ( and ). This is in agreement with the recent reports of MSC-like cells in perivascular locations in various tissues,23,24
which may function as a reservoir of tissue-specific regenerative cells. We did not investigate the fate of the entrapped MSCs beyond 3 days; however, we have previously shown that a very limited number of systemically delivered human MSCs survive long term and adopt a myocyte phenotype in the mouse myocardium.25
Although we did not specifically explore the fate MSCs in the injured tissue, cell size relative to microvessel size will also likely play a determining role in the initial biodistribution and cell fate.
Figure 6 Hypothesis for the intravascular fate of culture-expanded MSCs. After intraarterial MSC injection, microvascular plugging with obstruction of flow occurs at the precapillary level. The resulting ischemia leads to loss of most of the injected cells (86%). (more ...)
In summary, when delivered systemically most culture-expanded MSCs became mechanically entrapped at the precapillary level. This leads to significant flow reduction, with predominant MSC death and a small number of cells integrating in a perivascular niche. Given that microvascular architecture is universally conserved across most organ beds, our findings in the cremaster model likely translate to other microvascular beds, as well. To enhance therapeutic success, while avoiding microembolization, future efforts should explore conditions that will preserve the putative ability of native MSCs to circulate and actively engraft.