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In the past decade, researchers have gained important insights on the role of bone marrow (BM)-derived cells in adult neovascularization. A subset of BM-derived cells, called endothelial progenitor cells (EPCs), has been of particular interest, as these cells were suggested to home to sites of neovascularization and neoendothelialization and differentiate into endothelial cells (ECs) in situ, a process referred to as postnatal vasculogenesis. Therefore, EPCs were proposed as a potential regenerative tool for treating human vascular disease and a possible target to restrict vessel growth in tumour pathology. However, conflicting results have been reported in the field, and the identification, characterization, and exact role of EPCs in vascular biology is still a subject of much discussion. The focus of this review is on the controversial issues in the field of EPCs which are related to the lack of a unique EPC marker, identification challenges related to the paucity of EPCs in the circulation, and the important phenotypical and functional overlap between EPCs, haematopoietic cells and mature ECs. We also discuss our recent findings on the origin of endothelial outgrowth cells (EOCs), showing that this in vitro defined EC population does not originate from circulating CD133+ cells or CD45+ haematopoietic cells.

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Objective We sought to identify and characterize two distinct populations of bona fide circulating endothelial cells, including the endothelial colony forming cell (ECFC), by polychromatic flow cytometry (PFC), colony assays, immunomagnetic selection, and electron microscopy. Methods and Results Mononuclear cells from human umbilical cord blood and peripheral blood were analyzed utilizing our recently published PFC protocol. A population of cells containing both ECFCs and mature circulating endothelial cells (CEC) were determined by varying expressions of CD34, CD31 and CD146, but not AC133 and CD45. After immunomagnetic separation, these cells failed to form hematopoietic colonies, yet clonogenic endothelial colonies with proliferative potential were obtained, thus verifying their identity as ECFCs. The frequency of ECFCs were increased in cord blood and were extremely rare in the peripheral blood of healthy adults. In addition, we also detected another mature endothelial cell population in the circulation that was apoptotic. Finally, when comparing this new protocol to a prior method we determined that the present protocol identifies circulating endothelial cells while the earlier protocol identified extracellular vesicles. Conclusions Two populations of circulating endothelial cells including the functionally characterized ECFC are now identifiable in human cord blood and peripheral blood by PFC.