The exact definition and characterization of EPCs is still an ongoing and unresolved issue, although researchers have been trying to standardize the definitions of EPCs [5
]. When quantifying and analyzing EPCs by flow cytometry, staining for the expression of CD34 is essential. Nevertheless, there is no clear consensus on what other markers should be mandatory. Other surface molecules, mainly CD45, have been added to the panel by some investigators to characterize EPCs [9
] and may increase the specificity for EPCs. Different technical protocols have also been recommended to stain EPCs; some recommend staining for EPCs directly from full blood after erythrocyte lysis, whereas others use peripheral blood mononuclear cells after density centrifugation, but the latter was recently associated with a loss of EPCs [10
The majority of outcome studies have quantified absolute or relative numbers of circulating EPCs within the peripheral blood. In these studies, the number of EPCs has been either expressed as an absolute number, or as a percentage of positive cells per certain volume or per acquired cells. Assays that assess and quantify the outgrowth of colonies have also emerged. It is essential to differentiate among three of the most commonly used assays – the so called ‘early outgrowth assay’ [11
], the related ‘Hill assay’ [13
], and the ‘late outgrowth assay’ [14
] – because it is now evident that the first two and the latter exhibit two different cell types. In the early outgrowth assay and the Hill assay, cells staining positive for acetylated low-density lipoprotein and Ulex europaeus
], or forming specific colonies [13
] are of hematopoietic and monocytic origin and express CD31, different endothelial markers and von Willebrand factor-secreting proangiogenic factors [15
]. In the late outgrowth assay, cells are cultivated for a longer time period of 14–21 days, do not express CD45, but do exhibit a clonal proliferative potential [14
In addition to the EPC quantities and the potential to form colonies, the characterization of their functional capacities also include proliferation, migration, adhesion and in vitro
vasculogenic capacity that can be assessed in various ways using distinct readout systems. The definition of EPCs has also undergone constant changes over the last years. Initially, EPCs were usually classified as a subtype of CD34+
hematopoietic stem and progenitor cells and characterized by the coexpression of CD133, also called prominin 1 [20
]. In various studies, EPCs were also characterized by the coexpression of KDR-1, also known as VEGF-R2. Later, accumulating evidence showed that a ‘true’ EPC, which by definition represents a cell that can differentiate into an endothelial cell, is not of hematopoietic origin, whereas the other so-called EPCs were linked to the mononuclear lineage, secreting proangiogenic factors and were referred to as EPCs due to their ability to form colonies (see above). Currently, there is evidence to suggest that ‘true’ EPCs are not of hematopoietic origin, but rather of an already differentiated endothelial origin with clonal proliferative potential, and therefore the term ‘endothelial colony forming cell’ (ECFC) has emerged [14
]. Recently, Richardson and Yoder proposed to subdivide EPCs into mainly two groups, the proangiogenic hematopoietic cell and ECFCs [22
These findings of distinct and different cell types also led to modifications in the flow cytometry staining protocols. Recently, staining for CD45dim
(the hematopoietic cell marker), CD34+
cells was recommended as a modified International Society of Hematotherapy and Graft Engineering (ISHAGE) protocol, although there is still no clear consensus about a definite panel [9
]. In another recent approach, both CD45 and KDR were excluded due to difficulties with titration [23
]. Moreover, the scaling involved in gating for cells was considered to be very important to detect ECFCs. The constantly evolving flow cytometry staining and gating protocols, of course, are additional factors that complicate comparisons with older studies.
Taken together, both historically named EPCs, which were mostly of hematopoietic origin and the ‘new’ EPCs (now increasingly known as ECFCs) exhibit a potential to form, or at least increase and support the formation of, new vessels. A major problem for researchers is the lack of a unique marker for EPCs, leaving the field in limbo and complicating precise comparisons among different studies. This is, therefore, an important caveat to mention when reviewing the literature, because not all the papers refer to the same cell type. In this review, we will use the term EPCs as it was used in the studies that are referenced. – highlight important studies and also refer to the methods used for EPC quantification.
Selected outcome and characterization studies on endothelial progenitor cells in patients at risk of cardiovascular disease.
Selected outcome and characterization studies on endothelial progenitor cells in patients with rheumatic diseases.