By head-to-head direct comparison, we demonstrated a functional superiority of heart-derived cells as compared to three types of adult stem cells of extracardiac origin: bone marrow mononuclear cells, and mesenchymal stem cells from bone marrow or adipose tissue. Furthermore, the superiority of CDCs for myocardial repair was consistent with their well-balanced secretion of paracrine factors as well as their higher cardiomyogenic differentiation capacity and engraftment, although further in vivo intervention experiments (e.g., with blocking antibodies) will be required to identify the precise mechanisms of functional superiority.
We selected four cell types for comparison that have been used clinically and are considered among the most promising at present for myocardial repair/regeneration. Previous studies have shown that the implantation of BM-MNCs, BM-MSCs, and AD-MSCs into the damaged heart can improve cardiac function, likely through paracrine mechanisms, with rare events of myogenic differentiation.19-22
Despite the paucity of direct regeneration of new myocardium from these stem cells of extracardiac origin, the improvement of cardiac function reported in previous randomized clinical trials is encouraging.10-18
One distinctive feature of resident cardiac stem cells is their ability to undergo consistent cardiomyogenic and angiogenic differentiation,1-9,23-25
a finding confirmed here. Although almost 10% of CDCs expressed troponin T in vitro
, human CDCs expressing α-sarcomeric actin were infrequently observed in the infarcted hearts of SCID mice 3 weeks after treatment. Furthermore, we did not examine whether these α-sarcomeric actin positive human CDCs were physiologically integrated with the resident cardiomyocytes of mice. Therefore, regeneration of new functional myocardium directly from the implanted human CDCs is a consistent finding, but one of questionable significance; the present evidence supports the notion that mechanism of functional benefit after CDC implantation, as with other cell types, is predominantly dependent on paracrine effects and recruitment of endogenous regeneration.25
We thus compared the secretion of growth factors that generally are recognized to play critical roles in angiogenesis, anti-apoptosis, or cardioprotection. PDGF was undetectable in media conditioned by any of the four cell types, but, beyond even our own expectations, CDCs robustly produced a variety of growth factors, including angiopoietin, bFGF, HGF, IGF-1, SDF-1, and VEGF. Although BM-MSCs, AD-MSCs, and BM-MNCs could produce some of these factors in comparable levels to CDCs, the paracrine profile was uniquely well-balanced in CDCs. The robust production of diverse paracrine factors by CDCs provides a potential mechanism underlying their superiority for functional myocardial repair, but further work will be required to understand the precise role of each paracrine factor, as well as the roles of others known to be secreted but not quantified here.37
The use of the same medium (IMDM) for all cell types avoids possible confounding effects due to different culture conditions; nevertheless, the fact that cells were cultured uniformly may minimize favorable features unique to each cell type that might be more apparent with media optimized for each cell type.
Tube formation assays are commonly used to measure the angiogenic potential of endothelial cells.36
The superiority of tube formation observed in CDCs indicates their angiogenic potential, which is in agreement with CDCs’ robust secretion of pro-angiogenic cytokines (). The observation that BM-MNCs failed to form any tubes may due to the fact that MNCs need a longer time (24 hours) to form tubes,36
while we performed the assay at the 6 hour time point. We did not stain for endothelial markers (e.g. CD31), so it is unknown whether these tube structures actually represent endothelial cells differentiated from the various cell types; indeed, it has been reported that mesenchymal and fibroblast cells assist in blood vessel formation by aligning into tube networks.38
We also found CDCs were more resistant to oxidative stress-induced apoptosis in vitro
(Supplemental Figures 4&5
)) and MI-induced apoptosis in vivo
(). The superiority of CDCs to other cell types in enhancing host tissue preservation is striking. Therefore, it is conceivable that this action is responsible for the superior impact of CDCs in terms of preserving cardiac function and attenuating pathological remodeling, although the relative importance of the various favorable effects remains to be defined. It should be noted that we assayed apoptosis in vivo 3 weeks after MI/cell treatment. At that time point, the acute phase of cell death due to ischemia might have already resolved; therefore, the apoptosis we observed was a reflection of long-term remodeling and heart failure. One limitation of this experiment is that we did not perform double staining for TUNEL- and HNA-positive nuclei. Therefore we cannot distinguish whether apoptosis is reduced in endogenous cells, transplanted cells, or both. In addition, our results do not exclude the possibility that different levels of inflammatory infiltration among groups might contribute to observed differences in apoptotic rates. Nevertheless, the superior tissue preservation capacity of CDCs is consistent with their robust secretion of anti-apoptotic and pro-angiogenic factors.
We did not include a number of other stem cell types in our comparison, including myoblasts and peripheral blood-derived endothelial progenitors, although they have already been tested in clinical trials.17,18
The major reason is the relative lack of enthusiasm in the field for these cell types as candidates for further clinical development for cardiac regeneration (although endothelial progenitors do have positive effects on angina and critical limb ischemia17
). We also did not select ES- or iPS-derived cardiomyocytes for comparison, because they are far from clinical applicability at present, although both may excel in the direct production of new functional myocardium.39,40
We did, however, compare the therapeutic potency of cardiac progenitor cells purified by virtue of their c-kit-positivity,35,41
relative to unselected CDCs.42
We confirmed the reported ability of c-kit+
cardiac cells to boost cardiac function post-infarction,1,9
but we discovered that c-kit+
cells are not as potent as the CDC mixture either in terms of functional benefit or in paracrine factor secretion. One trivial explanation for this finding would be antibody-related interference with cell potency, but we believe that is unlikely: The c-kit antibody used43
but has been shown to interfere minimally with ligand binding, receptor phosphorylation, and internalization in c-kit-expressing cell lines.44
Also, magnetic-activated cell sorting for mast cells using this c-kit antibody neither induced histamine release nor did it impair the ability of cells to release histamine when stimulated.45
We must acknowledge, however, that subtle differences in specific sorting and culture methods for c-kit+
cells may affect the efficacy of the cells. Nevertheless, the finding that the natural CDC mixture is superior to the purified c-kit+
subpopulation adds a new dimension to the emerging notion that mesenchymal cells favor endogenous cardiac regeneration,10,46
at least partly via the attraction of c-kit+
cells. We conjecture that the stromal and mesenchymal cells synergize with the c-kit+
cells in the natural CDC mixture to enhance overall paracrine potency and thereby to boost functional benefit; alternatively, c-kit+
cells may be tangential to the mechanism of benefit of CDCs. More experiments are required to flesh out these ideas.