CD34⁺AC133⁺VEGFR-2⁺ Cells Are Primitive Hematopoietic Progenitors, Not Functional Endothelial Progenitor Cells.

Endothelial progenitor cells (EPCs) are currently used for angiogenic therapies or as biomarkers to assess cardiovascular disease risk and progression. However, there is no uniform definition of an EPC, which complicates interpretation of prior EPC studies. EPCs are primarily defined by expression of cell surface antigens. The most widely cited definition of an EPC is a cell which co-expresses CD34, AC133 and VEGFR-2. Importantly, these antigens are also expressed on the most primitive population of hematopoietic progenitor cells (HPCs), including high proliferative potential- (HPP-) and low proliferative potential-colony forming cells (LPP-CFCs). Remarkably, CD34⁺AC133⁺VEGFR-2⁺ cells have never been isolated and plated in endothelial cell (EC) or hematopoietic cell clonogenic assays to determine what cell progeny can be derived from a CD34⁺AC133⁺VEGFR-2⁺ cell. Utilizing human umbilical cord blood (CB), an enriched source of both EPCs and HPCs, we isolated and purified CD34⁺AC133⁺VEGFR-2⁺ cells by FACS and assayed for the presence of clonogenic endothelial CFCs (ECFCs) plus HPP- and LPP-CFCs. Surprisingly, CD34⁺AC133⁺VEGFR-2⁺ cells do not form ECFCs under any culture conditions previously described for outgrowth of EPCs. However, consistent with a HPC phenotype, CD34⁺AC133⁺VEGFR-2⁺ cells formed both HPP- and LPP-CFCs in multiple independent assays. In addition, all CD34⁺AC133⁺VEGFR-2⁺ cells were shown to co-express the specific hematopoietic cell surface antigen, CD45, which is not present on ECs. Based on this information, we plated CD34⁺CD45⁺ or CD34⁺CD45⁻ cells to determine if EPCs could be separated from HPCs on the basis of CD45 expression. In multiple independent assays, CD34⁺CD45⁺ cells consistently formed both HPP- and LPP-CFCs but not EC colonies. In contrast, CD34⁺CD45⁻ cells form EC colonies but not hematopoietic cell colonies. Taken together, these data demonstrate that circulating CD34⁺AC133⁺VEGFR-2⁺ cells are HPCs and the biologic mechanism for their correlation with cardiovascular disease needs to be re-examined. Furthermore, studies focused on determining the angiogenic potential of CD34⁺CD45⁻ cells are needed given that this cell population harbors ECFCs.