Human Endothelial Colony-Forming Cells

Abstract

Endothelial colony-forming cells (ECFCs) are progenitor cells that can give rise to colonies of highly proliferative vascular endothelial cells (ECs) with clonal expansion and in vivo blood vessel-forming potential. More than two decades ago, the identification of ECFCs in human peripheral blood created tremendous opportunities as having a clinically accessible source of autologous ECs could facilitate meaningful therapies with the potential to impact multiple vascular diseases. Nevertheless, until recently, the field of endothelial progenitor cells has been plagued with ambiguities and controversies, and reaching a consensus on the definition of ECFCs has not been straightforward. Moreover, although the basic phenotypical and functional characteristics of cultured ECFCs are now well established, some fundamental questions such as the origin of ECFCs and their physiological roles in health and disease remain incompletely understood. Here, I highlight some critical studies that have shaped our current understanding of ECFCs in humans. Insights into the biological attributes of ECFCs are essential for facilitating the clinical translation of their therapeutic potential.

Figure 1.

Definition, origin, and culture of human endothelial colony-forming cells (ECFCs). In vivo, human ECFCs are present in their original niche and circulating in peripheral blood (herein referred to as naive ECFCs and circulating ECFCs, respectively), whereas in vitro, cultured ECFCs are manifested as colonies of highly proliferative endothelial cells (ECs). Operationally, the criteria used to define ECFCs are only testable on cultured ECFCs, once the cells are isolated in vitro. Thus, the definition of ECFCs is intimately associated with their appearance in vitro as cultured ECFCs. (MACs) Myeloid angiogenic cells, (HSPCs) hematopoietic stem/progenitor cells.

Figure 2.

Phenotypical characterization of human cultured endothelial colony-forming cells (ECFCs). ECFCs were isolated from umbilical cord blood (cb-ECFCs) and adult peripheral blood (ab-ECFCs). (A) Indirect immunofluorescence of cultured ECFCs grown in a confluent monolayer showing positive staining for CD31, von Willebrand factor (vWF), and VE-cadherin. Cell nuclei counterstained with DAPI (scale bar, 100 μm). Binding of UEA-1 lectin to a monolayer of ECFCs (scale bar, 50 μm). ECFCs uptake of fluorescently labeled acetylated low-density lipoproteins (Dil-Ac-LDL) (scale bar, 50 μm). (B) Cloning-forming ability of cultured ECFCs. Phase contrast micrograph of a cb-ECFC colony. Arrowheads delimitate the border of the colony (scale bar, 200 mm). The endothelial nature of the colonies confirmed by binding of UEA-1 lectin (scale bar, 500 mm). (C) In vivo blood vessel–forming potential of cultured ECFCs. ECFCs were combined with human mesenchymal stem cells (MSCs), and the mixture subcutaneously injected into nude mice using a collagen/fibrin-based hydrogel. H&E from representative explants at day 8 with numerous blood vessels containing RBCs (yellow arrowheads; scale bar, 50 μm). Insets represent the macroscopic views of the explants (scale bar, 5 mm). hCD31 immunohistochemistry showed human specific lumens (scale bars, 50 μm). Perivascular coverage assessed by double immunofluorescent staining of explants with UEA-1 (red) and α-SMA (green). Nuclei counterstained with DAPI (scale bars, 50 μm). (Panels A and C adapted from Lin et al. ; reprinted, with permission, from Springer © 2013. Panel B adapted from Moreno-Luna et al. 2014; reprinted, with permission, from Elsevier © 2014.)

Figure 3.

Methods to derive endothelial colony-forming cells (ECFCs) and mature endothelial cells (ECs). (A) ECFCs are isolated from circulating mononuclear cells (MNCs) based on their robust endothelial colony-forming ability, whereas mature ECs are typically isolated based on their expression of definitive EC markers. Conceptually, ECFCs are different from mature ECs in that cultured ECFCs are differentiated, ex vivo, from circulating progenitors, and it is thus unclear whether they have previously been part of a functional blood vessel in vivo. In contrast, mature ECs are derived from the lining of blood vessels and thus have previously functioned as ECs in vivo. Despite this conceptual difference, ECFCs and mature ECs exhibit a wide range of phenotypical and functional similarities once in culture.