Fate of culture-expanded mesenchymal stem cells in the microvasculature: in vivo observations of cell kinetics

Abstract

Vascular delivery of mesenchymal stem cells (MSCs) following myocardial infarction is under clinical investigation. Little is known about the microvascular fate of MSCs. We used intravital microscopy of rat cremaster muscle microcirculation to track intraarterially delivered MSCs. Rat MSCs (average diameter, 23 microm) were bolused into the ipsilateral common iliac artery. Interrogation of an arteriole-venule pair revealed that 92+/-7% (n=6) of MSCs arrest and interrupt flow during first pass at the precapillary level, resulting in decreased flow in the feeding arteriole (velocity decreased from 6.3+/-1.0 to 4.6+/-1.3 mm/sec; P<0.001). MSC deformability evaluated using filtration through polycarbonate membranes revealed that the cortical tension of MSCs (0.49+/-0.07 dyne/cm, n=9) was not different from that of circulating mononuclear cells (0.50+/-0.05 dyne/cm, n=7). When intravital microscopy was performed 3 days following injection, the number of MSCs in the cremaster further decreased to 14% of the initial number, because of cell death in situ. In vivo labeling of the basement membrane revealed that at 1 day, the surviving cells were spread out on the luminal side of the microvessel, whereas at 3 days, they integrated in the microvascular wall. Despite their deformability, intraarterially delivered MSCs entrap at the precapillary level because of their large size, with a small proportion of surviving MSCs integrating in a perivascular niche.

Figure 1

Rheological effects of intraarterially delivered MSCs. a, MSCs fluorescently labeled with CMFDA were injected above the origin of the cremaster artery, leading to diffuse precapillary cell entrapment. b, Microvascular plugging significantly decreased the RBC velocity in the feeding arteriole. c, High magnification of MSCs arrested in a microvessel with interruption of flow (arrow indicating the flow divergence point; see supplemental Video 1). d, Three entrapped MSCs observed for up to 90 minutes: no morphological changes suggestive of extravascular migration. Scale bar = 100 µm.

Figure 2

Physical properties of MSCs. a and b, Ramp–filtration curves from experiments to measure deformability of MNCs and MSCs, respectively. c, Cortical tensions (stiffness) of MSCs and circulating MNCs were comparable; after disrupting the MSC cytoskeleton with cytochalasin B (cytoB), MSC cortical tension decreased. After oxidative stress (H₂O₂), cell stiffness nonsignificantly increased.

Figure 3

Time course of MSC persistence in cremaster microcirculation. The density of fluorescent cells (numbers of cells/field normalized to the number of injected cells in millions) decreased significantly over time (■); when MSCs underwent oxidative stress (H₂O₂) to induce apoptosis, virtually no cells were identified at 24 hours (◆) (n = 3, P<0.05), indicating that the remaining fluorescent cells were MSCs and not an artifact from dye transfer.

Figure 4

Initial fate of entrapped cells. Multicolor in vivo fluorescence imaging of MSCs in cremaster muscle. The CMFDA-labeled MSCs are green fluorescent (third row), whereas the microvascular basement membrane was stained red with rhodamine-labeled BSL I (second row). The MSCs were double-labeled with DAPI for nuclear staining (blue, top row). The bottom rows represent fused images. a, At 24 hours, the MSCs were spread out on the luminal side of the microvessels with preserved cytoplasmic and nuclear morphology. b, Heavily fragmented cells at 24 hours, with nuclear condensation (arrows) suggestive of apoptosis. c, At 72 hours, most persisting cells were morphologically intact, with preserved nuclear morphology. Scale bar = 50 µm.

Figure 5

MSC localization relative to the vessel wall. Multicolor in vivo fluorescence imaging of MSCs and basement membrane in the cremaster muscle, as in Figure 4. At 24 to 48 hours, the cells were spread out on the luminal side (a and b), with restoration of flow (depicted by the column of blood in the middle of column b, representing DIC imaging at 48 hours). At 72 hours, some of the cells appear perivascular, with presence of a basement membrane between the MSC and the microvessel (c). Scale bar = 50 µm.

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%). The few surviving cells initially spread out on the luminal side of the vessel and then localize in a perivascular niche.