A novel adult human stem cell population, hBMSCs, which appears distinct from previously described populations of adult stem cells, was clonally isolated, beginning at the single-cell level, from mixed total BM cell culture. Clonally isolated hBMSCs differentiated into cells of 3 germ layers (endoderm, mesoderm, and neuroectoderm) and self-renewed in culture for more than 140 PDs without obvious loss of plasticity or onset of replicative senescence. The transplantation of hBMSCs into a model of MI ameliorates the functional and pathologic changes following MI. The mechanism of improved cardiac function not only consists of differentiation of transplanted stem cells into essential myocardial tissues such as CMCs, ECs, and SMCs but also involves paracrine effects of the transplanted stem cells, which stimulate the proliferation of host myocardial tissues, including ECs and CMCs, and prevent apoptosis of endangered cells following ischemic injury.
The hBMSCs that we have identified are a unique population of adult BM-derived multipotent stem cells in that they express minimal levels (less than 3%) of CD90, CD105, and CD117. None of the characteristic marker panels defining HSCs, MSCs, and MAPCs matches the profile of hBMSCs. hBMSCs do not express the well-known MSC marker proteins CD29, CD44, and CD73 (also called SH3 or SH4) (33
). The minimal expression of surface molecules appears to be a prerequisite for the plasticity of hBMSCs as shown in other adult multipotent stem cells (11
). In contrast to MAPCs, hBMSCs do not express genetic markers of ES cells, such as Oct4, which are believed to be essential for MAPC function. Another distinguishing feature is that rat BM-derived stem cells, which we isolated previously, did not require leukemia inhibitory peptide for culture expansion (data not shown) (35
), which has been suggested to be essential for murine MAPC cultures.
Recent studies have demonstrated an important role of cell fusion in stem cell plasticity. Both in vitro (25
) and in vivo (26
) experiments using ES cells and/or BM-derived stem cells showed that cell fusion is responsible for a certain percentage of phenotypic changes observed following stem cell transplantation. Cell fusion occurs in normal mammalian development during the formation of osteoclasts (36
) and myoblasts (37
) or during tumor progression (38
). Recent in vivo studies suggest that hepatocytes derived from cell fusion of HSCs and host hepatocytes can correct underlying metabolic disorders and that fused cells can undergo reduction division (26
). The present in vitro data indicate that both fusion and transdifferentiation could be responsible for the phenotypic changes of hBMSCs to ECs, SMCs, and CMCs, and furthermore that the prevalence depends on cell type. Although no definitive studies were performed in vivo to quantify the contribution of cell fusion to phenotypic changes observed, the results of in vitro studies implied that both fusion and differentiation can play a role. Although some recent studies raised controversy regarding the transdifferentiation potential of HSCs (12
), another recent study still claimed this potential (39
). More importantly, these studies were performed using specifically defined subpopulations of HSCs, while our studies have used an entirely different stem population derived from BM.
In the past, recovery of cardiac function following MI has been considered to be completely dependent on the integrity of the remaining noninfarcted portion of the LV. Following MI, the viable myocardial tissue bordering the infarct area can undergo hypertrophy to compensate for lost CMCs (40
). However, replenishment of lost CMCs was thought to be impossible. Moreover, although neovascularization within the infarcted tissue appears to be an integral component of the remodeling process, under normal circumstances the capillary network cannot keep pace with tissue growth and is unable to support the greater demands of the hypertrophied but viable myocardium, which subsequently undergoes apoptosis and necrosis due to inadequate oxygenation and nutrient supply, leading to further deterioration of cardiac function (41
). Thus far, no studies using human stem cells derived from BM have documented both therapeutic neovascularization and cardiomyogenesis following MI. Our experiments show that transplantation of a clonal adult human stem cell population can replenish both vascular networks and cardiac muscle, and they also reveal that the therapeutic effect consists of endogenous tissue repair (angiogenesis and endogenous cardiomyogenesis) as well as exogenous tissue regeneration (vasculogenesis and exogenous cardiomyogenesis). A key observation is that the rate of engraftment and cell survival was remarkably higher following hBMSC transplantation compared with TBMC transplantation. It seems highly probable that this prolonged interaction between host and transplanted hBMSCs, by promoting secretion of multiple paracrine factors and augmenting direct cell-to-cell contact, could lead to a significantly higher degree of endogenous cardiomyogenesis and angiogenesis as well as the differentiation of local stem cells (27
To induce angiogenesis and/or arteriogenesis, tight orchestration of monocytes/macrophages, ECs, and SMCs/pericytes is critical (22
). PDGF-B is prerequisite for the investment of stable vessels with pericytes (31
). Given the complex structure of formed mature vessels with periendothelial matrix and pericytes/SMCs, a combination of various angiogenic growth factors may be advantageous (22
). In addition to documenting direct vasculogenesis from transplanted stem cells, these studies suggest a potential additional benefit of hBMSC transplantation into ischemic myocardium, augmenting neovascularization by supplying various angiogenic cytokines (VEGF, angiopoietin-1 and -2, bFGF, HGF, PDGF-B).
Moreover, as IGF, VEGF, bFGF, and HGF are known to function as survival factors for the endangered CMCs (45
), these paracrine effects may be as important as the differentiation potential of stem cells for neovascularization.
Interestingly, our study demonstrated no functional improvement in unselected total BM–transplanted rats, which apparently differ from those of the previous studies using BM cells (47
). The discrepancy of therapeutic effects could result from the difference in animal models and the cells used. One study (47
) used cultured BM cells in a pig model of chronic MI, and the other study (48
) used mononuclear cells in porcine MI model. Our study used unselected
BM cells in a large infarction model in rats. The size of infarction created is much smaller in pigs than in rats because pigs cannot tolerate large infarctions as a result of mechanical dysfunction and arrhythmia. In the large infarction we induced in rats, use of unselected BM cells may result in a failure of therapeutic effect because of an insufficient dose of therapeutic cells or by the inclusion of cells that may actually have a detrimental effect.
Since the loss of CMCs plays the most direct role in the development of heart failure, cardiomyogenesis has been the central aim of cardiac regenerative therapy using stem cells. Our results demonstrate direct differentiation of hBMSCs into CMCs, which we define as exogenous cardiomyogenesis. Our data also reveal significant endogenous cardiomyogenesis, i.e., generation of new CMCs from the host myocardium after hBMSC transplantation. Upregulation of cardiac transcription factors further supports cardiomyogenesis after hBMSC transplantation. This phenomenon of endogenous cardiomyogenesis after stem cell transplantation has not, to our knowledge, been previously reported. The recent discovery of cardiac progenitor or stem cells (27
) supports the possibility of stimulation of endogenous cardiac progenitor or stem cells by engrafted hBMSCs. Whether this effect is a general mechanism in other stem cell transplantation remains to be determined.
One issue that must be considered in the realm of transplantation of cells into the heart is the provocation of arrhythmia. In the present study, 14 of 15 rats survived in the hBMSC group, compared with 12 of 15 in both the TBMC and the PBS groups. Although we could not monitor the animals continuously for the occurrence of arrhythmias, repetitive echocardiography with electrocardiographic monitoring was performed, which did not reveal arrhythmias in any of the treatment groups. In this regard it is noteworthy that the arrhythmic events that have been reported thus far have occurred following skeletal myoblast transplantation (49
), while no events have been reported following transplantation of BM or circulating cells. We think this may be the result of the fact that skeletal muscle and cardiac muscle cells depolarize and repolarize actively, while marrow-derived progenitors are passive. Nevertheless, caution is mandated in the early phases of all studies of intramyocardial cell transplantation.
Compared with the other human stem cells or progenitor cells, hBMSCs have certain potential advantages for regenerative therapy of cardiac diseases. Among the human stem cells shown to improve cardiac function are endothelial progenitor cells (14
), and angioblasts (15
), which have been shown to promote neovascularization in vivo. Human ES cells have demonstrated the potential to differentiate into CMCs in vitro (50
), but recent human ES cell transplantation into murine ischemic hearts has been shown to result in teratoma formation. Human MSCs have been shown to transdifferentiate into CMCs. However, the number of transdifferentiated cells was small, transdifferentiation into ECs and SMCs was not demonstrated, and functional data in vivo were not generated (20
). In contrast, hBMSCs have the required multipotency in vitro and in vivo to regenerate damaged myocardium, are culture-expandable, and disclose the functional capability for therapeutic application. Finally, considering the scope of multipotency of the hBMSCs and the robust nature of engraftment, it is possible that the use of hBMSCs may extend to a therapeutic arena including other degenerative or inherited diseases.
Coronary artery disease accounts for 50% of all cardiovascular deaths and nearly 40% of the incidence of heart failure (1
). The current findings have provided evidence that hBMSC transplantation could have relevant implications for the treatment of human disease. We have been able to demonstrate here, for the first time to our knowledge, that adult human
stem cells can augment both therapeutic neovascularization and cardiomyogenesis, thereby enhancing functional and anatomic regeneration after MI. This new form of cardiac repair may improve the immediate and long-term outcome of ischemic heart disease and may therefore merit clinical investigation in patients with ischemic heart disease.