Myocardial infarction is a leading cause of heart failure and death in developed countries. Although postinfarction survival rates have improved in recent years, reduced heart function due to excessive loss of cardiomyocytes remains a major problem. The lack of resident stem cells in the heart has led to an intensive search for alternative sources of cardiomyocyte progenitors. Embryonal stem cells have been shown to differentiate into cardiomyocytes that can form stable intracardiac grafts (19
). Skeletal myoblasts or cardiomyocytes from fetal or neonatal mice have also been shown to take up residence in cardiac tissue after injury (20
). Although demonstrating the potential of cellular engraftment as a means to augment the number of myocytes in cardiac muscle, these previous studies have not identified a progenitor population readily accessible from an adult patient’s own tissue that can be adapted to clinical therapy.
Here we have assessed the ability of purified stem cells derived from adult mouse bone marrow to participate in cardiac muscle regeneration following the induction of ischemia by coronary artery occlusion and reperfusion. These stem cells are a highly enriched stem cell population that can be reproducibly isolated from adult tissues. When transplanted into the bone marrow of lethally irradiated mice, purified SP cells marked with the lacZ gene regenerated the hematopoietic system. At 2 or 4 weeks after coronary artery ligation, lacZ-positive cells had migrated into cardiac myofibers, where they participated in the regeneration of healthy muscle. This level of myocardial engraftment, 0.02% of all cardiomyocytes, was similar to that seen with use of whole bone marrow or purified stem cells to regenerate skeletal muscle (2
). Importantly, there was no evidence of SP cell migration into the hearts of sham-operated animals. Finally, the presence of lacZ-positive hematopoietic cells in the cardiac tissue (measured with Ab’s against CD45 and macrophages) was not a problem in this study, as shown in Figure . This is consistent with previous observations that the major inflammatory response is observed immediately after infarction and has largely resolved by 2 to 4 weeks (22
We also found that lacZ-positive cells could participate in neovascularization in regenerating heart tissue. Many small vessels, comprising one to three endothelial cells (cell number determined by 4′,6-diamidine-2-phenylindole dihydrochloride [DAPI] staining), clearly expressed the marker gene. Some large- or medium-sized vessels also appeared to have incorporated marked cells, although such structures consisted primarily of host cells. The overall level of neovascular engraftment was approximately 3%. This is lower than the engraftment of more differentiated endothelial cell progenitors derived from whole blood (7
). The majority of engraftment also appeared to be adjacent to the region of infarct. We did not attempt to determine whether these vessel structures were arterioles, venules, or lymphatic vessels.
We present evidence here that bone marrow hematopoietic stem cells, purified as SP cells, have transdifferentiated and engrafted into myocardium and endothelial cells. However, future studies can extend and confirm these conclusions using additional criteria. First, we cannot exclude the possibility that small numbers of contaminating nonhematopoietic stem cells are responsible for the cardiac and endothelial cell engraftment. Such contaminants could arise from imperfect cell sorting or could even be due to stem cells copurified by our SP isolation procedure. Clonal studies will be essential to rule out the possibility that multiple stem cell populations are present within the donor cell population. Second, current studies in the field are limited by the use of markers that are present ubiquitously in donor cells, such as lacZ, green fluorescent protein (GFP), or Y chromosome fluorescence in situ hybridization (FISH) (12
). While one could argue about differences in sensitivity and specificity of these marker systems, each will identify any donor cell, not just one that has differentiated into the cell type of interest. This necessitates the use of secondary criteria, such as morphology and colocalization with specific markers, to establish cell identity. Perhaps more definitive will be using donor cells that express a nuclear-localized marker gene under the control of a cell type–specific promoter. In this case, donor cells will not express the marker until they differentiate into the cell type of interest (2
). Although such studies should generate less disputable data, they are also flawed in that expression of any one gene, at any given time, is not a reliable indicator of a distinct phenotype. Hence, determining donor cell localization into complex tissues in this manner will still require colocalization with other markers, as well.
Our detection of angioblast markers on purified SP cells may bear on the developmental fate of these stem cells. Before transplantation took place, we observed no expression of genes indicative of a mature, differentiated endothelial cell phenotype in SP cells. Instead, our analyses revealed, as expected, expression of genes reported to be activated in a common progenitor of both hematopoietic cells and endothelial cells, namely PECAM-1 (24
) and Tal-1 (25
). Surprisingly, other genes thought to be specifically reflective of an embryonic endothelial cell progenitor, such as tyrosine kinase receptors Flk-1 and Flt-1 (28
), were not expressed in SP cells; however, expression of their ligand VEGF-A was detected.
Perhaps expression of VEGF-A in the endogenous population of SP cells plays a role in “directing” these multipotent cells to the surface of mature endothelial cells expressing VEGF receptors in adult tissues when needed. Such a role for VEGF-A, via Flk-1–receptor signaling, has been described for angioblast migration during embryonic vascular development (29
), supporting a role of VEGF-receptor binding in directing the positioning, as well as differentiation, of multipotent progenitors. Alternatively, it is also possible that VEGF-A expression by multipotent stem cells may serve a unique functional role in the stem cell microenvironment, independent of its well-documented effects on endothelial cell behavior (30
), perhaps in the maintenance of a “progenitor” state.
In addition to expressing VEGF-A, the bone marrow SP cells also expressed RNA for Ang-1 and its receptor, Tie-2. Ang-1 and Tie-2 are thought to be involved in an important paracrine-signaling pathway needed for the recruitment, or maintenance, of smooth muscle cells and pericytes to stabilize newly forming endothelial tubes (31
). Embryos lacking either Ang-1 (31
) or Tie-2 (16
) die at embryonic day 11.5 and 10.5, respectively, with severe vascular malformations and hemorrhage. The role of Ang-1 and Tie-2 in the SP cells is uncertain. Since SP cells express the ligand, as well as the receptor, both autocrine and paracrine regulatory pathways can be envisioned. In a paracrine manner, perhaps Ang-1 or Tie-2 bind to their respective partner in the bone marrow stroma to modulate cell survival or responses to other soluble factors in the microenvironment, as has been proposed for mature endothelial cells (32
). Certainly, much remains to be determined regarding the role of factors such as Ang-1 and VEGF-A in the modulation of SP survival, growth control, and interactions with the surrounding microenvironment.
Although endothelial cells and hematopoietic cells are thought to be related developmentally, possibly originating from a common hemangioblast precursor (33
), evidence for such a cell in the adult is lacking. We suggest that the SP cells are bona fide hematopoietic stem cells with the capacity for endothelial or cardiomyocytic differentiation under circumstances of acute injury. Our findings underscore the developmental versatility of adult hematopoietic stem cells and suggest that their functional role is ultimately determined by their migration into particular microenvironments, such as myocardium, and their exposure to locally generated signals at those sites.
What cell type participates in myocardial regeneration? In our experiment, the purified stem cells stably engrafted into the bone marrow of recipient mice. The presence of lacZ-marked cells in damaged cardiac tissue could reflect direct incorporation of migrating SP cells or their progeny. Since the surface phenotypes of lacZ-positive cells that had integrated into cardiac tissues were those of differentiated myocytes and endothelial cells, we are unable to deduce the exact identity of the cells that first migrated into the heart.
The incorporation of bone marrow–derived SP cells into both regenerating endothelial cells and cardiomyocytes suggests that circulating stem cells may naturally contribute to repair of these tissues. The lack of sensitive markers and similar experimental design has likely impeded previous observation. Although the contribution of our stem cells to cardiac regeneration was low, improvements in efficiency that can be achieved through a better understanding of the process promise to offer new therapeutic avenues in the long term.