Although it was hoped that revascularization by angiogenic growth factor therapy might be an alternative for surgical interventions in no-option PVD patients, results from clinical trials have been disappointing (3
). The other alternative, cell transplantation, might offer better perspectives, because it may overcome the problem of lack of response of endogenous vascular cells to growth factors and it may combine revascularization with replacement of lost tissue. Here, we tested these paradigms by delivery of exogenous vascular progenitors (mMAPC-VP) and by evaluating cell populations that have the ability to differentiate into vascular and muscle cells, namely mBMCs (18
) and mMAPC-U (22
). We found that only mMAPC-U durably improved muscle function and perfusion due to the favorable combination of effects on revascularization and muscle regeneration, in both moderate and severe limb ischemia models. Furthermore, similar results were seen when hMAPCs were used in the severe model.
mMAPCs engrafted robustly up to 5 weeks, even though cell number decreased over time. As mMAPC expressed GFP — a well-known immunogen (30
) — we believe that this decrease might be due to immune rejection by the host. Consistent with this is the presence of CD45+
cells surrounding implanted cells. The hypoxic (5% O2
) and low-serum (2%) conditions under which mMAPCs are maintained (31
) may have conditioned them to survive in ischemic environments. Such a favorable preconditioning effect was also documented for BMCs (32
). Although mMAPCs only express low levels of CXCR4
), known to assist in perivascular positioning and retention (33
), the grafted cells were consistently found in the immediate vicinity of vessels, a strategic position from which they could affect vascular bed expansion. Possibly, chemokine receptors and other adhesion receptors may be upregulated once mMAPCs are grafted in tissues.
mMAPCs differentiated into ECs and SMCs and contributed to SkMBs, consistent with their in vitro pluripotent differentiation properties (22
). While we did not observe fusion of mMAPCs with vascular cells, muscle contribution in vivo was at least in part due to fusion with host muscle cells; however, it remains to be determined whether the latter was via direct fusion with host myoblasts or differentiation to a muscle satellite stage and subsequent fusion (Supplemental Figure 2). In vitro, mMAPCs can differentiate into myotubes independent of fusion when cocultured with C2C12 cells and also fuse with C2C12 cells (Supplemental Figure 2). As expected, vascular-committed mMAPCs did not contribute to skeletal muscle. Despite the presence of skeletal muscle precursors in BM (18
), mBMCs also failed to differentiate in situ into SkMBs. This is in agreement with the observation that cells with high fusion capability mainly reside in the stromal fraction, while only a small fraction of the hematopoietic lineage is fusigenic (34
). Other non–muscle-derived cell types have shown the potential to contribute to SkMBs in ischemic muscle, e.g., CD34+
cord blood cells (10
), the immortalized CD34–
R26 cell line generated from peripheral blood (35
), or ADSCs (36
A recent report has documented that cotransplantation of ECs with mesenchymal precursor cells in a cranial window model leads to the formation of durable, functional blood vessels (37
). Although we demonstrate that mMAPC-VP induced an improvement in perfusion and function, this was less pronounced than for mMAPC-U, and the improvement was no longer seen beyond 9 days after treatment. Moreover, we did not find durable contribution of mMAPC-VP to vessels. The differences between our results and those in the cranial window model may be due to the different tissue environments in the 2 models. Similar to mMAPC-VP and in line with other studies (7
), injection of mBMCs resulted in vascular bed expansion and improved perfusion and function of hind limb muscles; however, this too was temporary and less pronounced compared with mMAPC-U. Whether the lack of direct contribution of mBMCs to vessels and muscle tissue, or mMAPC-VP to skeletal muscle or their lower level of engraftment, underlies the significantly lower degree of improvement in perfusion or swim endurance compared with that of mMAPC-U remains to be determined. Also, what the contribution is of SkMB generation from mMAPC-U or the trophic support of endogenous myogenesis provided by mMAPC-U to the improved function is unclear. Nevertheless, muscle function of mMAPC-U–treated animals far exceeded that of the other study groups, in which no effects on muscle regeneration were seen.
It should be noted that the majority of mMAPC-U did not directly contribute to ECs, SMCs, or SkMBs. Although some earlier studies have suggested that grafted cells directly contribute to a much greater degree to vessels and/or tissue in ischemia models (40
), recent studies have demonstrated that direct contribution is minimal (9
), or even absent (11
), and that grafted cells improve vascularization via trophic effects (8
). In our study, it is highly likely that trophic factors secreted by the long-term engrafted mMAPC-U, such as VEGF and IGF-1, contributed to the improved perfusion and limb function seen in mMAPC-U–treated animals.
A surprising finding was the progressive necrosis and fibrosis and the associated decrease in muscle function of mBMC-treated animals after day 9. It is noteworthy that aside from endogenous CD45+
cells, donor-derived immunological cells (or their progenitors) were also present in foci of inflammatory dendritic and T cells. In some animals, we saw an extensive infiltration of necrotic gastrocnemius muscle with T lymphocytes and dendritic cells, some of which were GFP+
and hence donor in origin. Many preclinical studies have evaluated the benefits of mBMC transplantation in limb ischemia (5
); however, in most studies, cells were injected only in the upper part of the limb, where inflammation due to necrosis is limited, which may have precluded the detection of the contribution of grafted immune cells to the inflammatory reaction. While we used unfractionated BMCs, in most clinical studies, only the mononuclear fraction or subsets of these are used (Supplemental Table 1). Nevertheless, given the fact that these fractions still contain mature immune cells or precursors thereof, and that they are injected into the gastrocnemius muscle in the majority of PVD patient trials (Supplemental Table 1), our observation should serve as a note of caution, in that grafting of such cell populations in the ischemic, necrotic, and inflammatory limb muscle may contribute to and exacerbate the inflammatory response with potentially detrimental consequences later on.
Finally, we demonstrate that hMAPCs had a beneficial effect on limb perfusion and muscle regeneration similar to that of mMAPCs in a severe limb ischemia model. While these studies were done in immunocompromised mice, in a clinical setting, human recipients will be immunocompetent; if one is to consider allogeneic cells as graft, allogeneic hMAPCs, expressing HLA molecules upon differentiation, will likely also be rejected due to HLA incompatibility, as we have seen here for the neoantigen-containing GFP+
mMAPCs. Studies wherein mice with a “humanized” immune system are used will be needed to address the effect of HLA mismatch on the efficacy of hMAPCs in limb ischemia (45
In summary, we show here that locally injected undifferentiated mMAPCs have the potential to remedy ischemic problems of the lower extremities in both moderate and severe ischemia models. Importantly, their human counterparts had a similar beneficial effect in the severe model. In the moderate model, the effect was superior to that of vascular-committed MAPCs or mBMCs, in both early and later phases. This greater potential may be related to the ability of the cells to stably engraft and induce vascular and skeletal muscle regeneration via direct contribution to vascular and skeletal muscle cells and via trophic effects on the endogenous vascular and skeletal muscle cells.