Circulating endothelial progenitor cells (EPCs) have been the focus of many clinical trials due to their roles in revascularisation following ischemic events such as acute myocardial infarction as well as their contribution to vascular repair during organ transplantation. Research on EPCs has been controversial due to the lack of distinct markers expressed at the cell surface and varying methods for isolation and culture have resulted in the identification of a multitude of cell types, with differing phenotype and function, all falling under the label of “EPCs”. The most widely documented EPCs isolated for cell therapy are adherent in nature and lacking the progenitor markers such as CD133 and therefore unlikely to represent a true circulating EPC, the cells mobilised in response to a vascular injury.

We recently published the isolation and extensive characterisation of a population of non-adherent endothelial forming cells (naEFCs) (Appleby et al., 2012) (Figure 1). These cells expressed the progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from ‘early’ EPCs, the ‘late outgrowth EPC’ [more recently known as endothelial colony forming cells (ECFCs)] as well as mature endothelial cells (ECs). Figure 2A exemplifies the surface expression profile of the naEFCs. Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin (Figure 2A), (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature ECs increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human ECs and have contributed to various advances in scientific knowledge (Appleby et al., 2012; Barrett et al., 2011; Moldenhauer et al., 2015; Parham et al., 2015). Here, we describe the isolation and enrichment of a non-adherent CD133⁺ endothelial forming population of cells from human cord blood.