Endothelial Progenitor Cells: Mainly Derived From the Monocyte/Macrophage–Containing CD34− Mononuclear Cell Population and Only in Part From the Hematopoietic Stem Cell–Containing CD34+ Mononuclear Cell Population
To the Editor:
In their very interesting paper, Rehman et al1 demonstrate that the vast majority of peripheral blood–derived acLDL+ Ulex-lectin+ cells in culture—often referred to as endothelial progenitor cells (EPC)—express monocyte/macrophage markers, indicating that they originate mainly from the monocyte/macrophage lineage. They suggest that only a small population of true stem cells/progenitor cells and endothelial cells exists that may originate directly from the hemangioblast or from hematopoietic stem cells. Our recent observations support their findings that the majority of EPC are derived from the monocyte/macrophage–containing CD34− mononuclear cell population. Nevertheless, we could also identify and quantify a minor proportion of EPC that has its origin in the hematopoietic stem cell–containing CD34+ cell population.
EPC were obtained from human umbilical cord blood (hUCB) as previously described.2 hUCB has previously been shown to contain a 10-fold excess of CD34+ cells as compared with peripheral blood. CD34+ cells were isolated, and male CD34+ cells and female CD34− cells, or vice versa, were cocultured in physiological ratios (0.5% CD34+, 99.5% CD34−). After 12 days of culture, the origin of EPC (spindle-shaped Ulex-lectin+ cells) was determined by using Y-chromosome fluorescence in situ hybridization. Our experiments showed that, even in hUCB, a rich source of hematopoietic progenitors, the majority (91±4%) of EPC originates from the CD34− mononuclear cell population. Only a minor but significant subset of cultured EPC (9±4%) originates from the hematopoietic stem cell–containing CD34+ cell fraction. Further studies should elucidate whether this subpopulation of cells contains “true progenitor cells” with a higher capacity for proliferation and differentiation into endothelial cells.
In their letter, Rookmaaker et al1 elegantly demonstrate that the vast majority of attached endothelial-like cells derived from culturing umbilical cord mononuclear cells (MNCs) originate from CD34-negative cells. These results complement our previous findings that endothelial-like cells obtained by short-term culture of peripheral blood MNCs are primarily derived from the monocyte/macrophage lineage and secrete angiogenic growth factors.1 Their findings also highlight the need to distinguish between 3 different types of adherent endothelial-like cells that can be isolated by culturing circulating MNCs in the presence of endothelial growth factors, as follows.
Monocyte/macrophage–derived cells can attain an endothelial-like phenotype in culture and do not have a significant proliferative capacity. Such cells express monocyte/macrophage markers such as CD14 and are mostly negative for stem/progenitor cell surface markers such as CD34, CD117, and AC133 (also known as CD133).
True endothelial progenitor cells (EPCs) exhibit an endothelial phenotype in culture and demonstrate significant proliferation. They do not express CD14 and are likely to express stem/progenitor cell markers as well as endothelial markers while they are in circulation. However, they probably lose the stem and progenitor markers in culture.
Circulating mature endothelial cells have either differentiated from less mature EPCs or may represent cells that have “sloughed off” from the vasculature. In culture, these can be difficult to distinguish from EPCs, because the latter may gradually cease to express stem/progenitor cell markers in culture.
Because proliferative capacity is a hallmark of EPCs that distinguishes them from monocyte/macrophage–derived endothelial-like cells, long-term culture (3 to 4 weeks) of mononuclear cells selects for true EPCs. This is the approach that has been used by Lin et al.2 Future studies that keep this distinction in mind may be able to identify the respective contribution of each of these cell types to neovascularization and vascular repair.