The limited vessel-forming capacity of infused endothelial progenitor cells (EPCs) into patients with cardiovascular dysfunction may be related to a misunderstanding of the biologic potential of the cells. EPCs are generally identified by cell surface antigen expression or counting in a commercially available kit that identifies “endothelial cell colony-forming units” (CFU-ECs). However, the origin, proliferative potential, and differentiation capacity of CFU-ECs is controversial. In contrast, other EPCs with blood vessel-forming ability, termed endothelial colony-forming cells (ECFCs), have been isolated from human peripheral blood. We compared the function of CFU-ECs and ECFCs and determined that CFU-ECs are derived from the hematopoietic system using progenitor assays, and analysis of donor cells from polycythemia vera patients harboring a Janus kinase 2 V617F mutation in hematopoietic stem cell clones. Further, CFU-ECs possess myeloid progenitor cell activity, differentiate into phagocytic macrophages, and fail to form perfused vessels in vivo. In contrast, ECFCs are clonally distinct from CFU-ECs, display robust proliferative potential, and form perfused vessels in vivo. Thus, these studies establish that CFU-ECs are not EPCs and the role of these cells in angiogenesis must be re-examined prior to further clinical trials, whereas ECFCs may serve as a potential therapy for vascular regeneration.

From the paradigm shifting observations of Harvey, Malpighi, and van Leeuwenhoek, blood vessels have become recognized as distinct and dynamic tissue entities that merge with the heart to form a closed circulatory system.1 Vessel structures are comprised predominantly of a luminal layer of endothelial cells that is surrounded by some form of basement membrane, and mural cells (pericytes or vascular smooth muscle cells) that make up the vessel wall. In larger more complex vessel structures the vessel wall is composed of a complex interwoven matrix with nerve components. Understanding the cellular and molecular basis for the formation, remodeling, repair, and regeneration of the vasculature have been and continue to be popular areas for investigation. The endothelium has become a particularly scrutinized cell population with the recognition that these cells may play important roles in maintaining vascular homeostasis and in the pathogenesis of a variety of diseases.2 Although it has been known for several decades that some shed or extruded endothelial cells enter the circulation as apparent contaminants in the human blood stream,3 only more recent technologies have permitted the identification of not only senescent sloughed endothelial cells,4 but also endothelial progenitor cells (EPCs), which have been purported to represent a normal component of the formed elements of circulating blood5 and play roles in disease pathogenesis.6–9 Most citations refer to an article published in 1997 in which Asahara and colleagues isolated, characterized, and examined the in vivo function of putative EPCs from human peripheral blood as a major impetus for generating interest in the field.10 This seminal article presented some evidence to consider emergence of a new paradigm for the process of neovascularization in the form of postnatal vasculogenesis. Since publication of that article, interest in circulating endothelial cells, and particularly EPCs, has soared, and one merely has to type the keyword search terms, endothelial progenitor cell, to recover more than 8984 articles including 1347 review articles in PubMed (as of June 2008). What can we possibly add in the form of another EPC review that will be considered of significant value for the reader? We will attempt to review some of the early article in the field and reflect on how information in those articles was gradually derivatized into perhaps more conflicting rather than unifying concepts. We will also attempt to concisely address some of the important determinants and principles that are now leading to a new understanding of what functionally constitutes an EPC and outline some of the current measures used to identify, enumerate, and quantify these cells. Finally, we give our opinion of the best definition for an EPC based on some comparative analyses performed primarily in human subjects

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

Endothelial progenitor cells (EPCs) can be isolated from adult peripheral and umbilical cord blood and expanded exponentially ex vivo. In contrast, human umbilical vein endothelial cells (HUVECs) or human aortic endothelial cells (HAECs) derived from vessel walls are widely considered to be differentiated, mature endothelial cells (ECs). However, similar to adult- and cord blood–derived EPCs, HUVECs and HAECs derived from vessel walls can be passaged for at least 40 population doublings in vitro. Based on this paradox, we tested whether EPCs reside in HUVECs or HAECs utilizing a novel single cell deposition assay that discriminates EPCs based on their proliferative and clonogenic potential. We demonstrate that a complete hierarchy of EPCs can be identified in HUVECs and HAECs derived from vessel walls and discriminated by their clonogenic and proliferative potential. This study provides evidence that a diversity of EPCs exists in human vessels and provides a conceptual framework for determining both the origin and function of EPCs in maintaining vessel integrity.