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 vesselforming ability, termed endothelial colonyforming 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 CFUECs 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. IntroductionNew blood vessel formation occurs via angiogenesis, vasculogenesis, or arteriogenesis. 1,2 Since 1997, postnatal vasculogenesis has been purported to be an important mechanism for angiogenesis via marrow-derived circulating endothelial progenitor cells (EPCs). 3 Based on this paradigm, EPCs have been extensively studied as biomarkers of cardiovascular disease and as a cell-based therapy for repair of damaged blood vessels. [4][5][6] However, administration of EPCs or bone marrow-derived cell populations enriched for EPCs into subjects with cardiovascular disease has had limited efficacy, with regard to new vessel formation. Many investigators speculate that the paracrine effects of cultured EPCs are responsible for the modest effects in patients because there is no evidence of long-term engraftment of EPCs into newly formed vessels. 7-9 These clinical observations are surprising given animal studies where EPC administration partially rescued cardiovascular dysfunction following ischemic hind limb or myocardial injury with some evidence for EPC contribution to new vessel growth. 5,9 In most studies, EPCs are identified and enumerated via flow cytometric identification of cells expressing CD34, CD133, or the VEGF receptor 2 (KDR). 3,10,11 Because these molecules are also expressed on hematopoietic stem/progenitor populations, 12-15 the presence of hematopoietic contamination of EPCs should be expected. EPCs are also quantitated by counting in a commercially available kit that identifies "endothelial cell colony-forming units" (CFU-ECs). Identification of CFU-ECs from peripheral blood by use of colony-forming ...

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Endothelial progenitor cells (EPCs

Biomechanical forces are emerging as critical regulators of embryogenesis, particularly in the developing cardiovascular system 1,2 . After initiation of the heartbeat in vertebrates, cells lining the ventral aspect of the dorsal aorta, the placental vessels, and the umbilical and vitelline arteries initiate expression of the transcription factor Runx1 (refs 3-5), a master regulator of haematopoiesis, and give rise to haematopoietic cells 4 . It remains unknown whether the biomechanical forces imposed on the vascular wall at this developmental stage act as a determinant of haematopoietic potential 6 . Here, using mouse embryonic stem cells differentiated in vitro, we show that fluid shear stress increases the expression of Runx1 in CD41 + c-Kit + haematopoietic progenitor cells 7 ,concomitantly augmenting their haematopoietic colony-forming potential. Moreover, we find that shear stress increases haematopoietic colony-forming potential and expression of haematopoietic markers in the paraaortic splanchnopleura/aorta-gonads-mesonephros of mouse embryos and that abrogation of nitric oxide, a mediator of shear-stress-induced signalling 8 , compromises haematopoietic potential in vitro and in vivo. Collectively, these data reveal a critical role for biomechanical forces in haematopoietic development.In the mouse, the first haemogenic areas appear in the yolk sac starting at day 7.5 of development (E7.5) 9 . After the establishment of circulation and the onset of vascular flow at day 8.5, additional haemogenic sites appear between day 9 and 10.5 as Runx1 + regions within

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.

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Endothelial progenitor cell (EPC) nomenclature remains ambiguous and there is a general lack of concordance in the stem cell field with many distinct cell sub-types continually grouped under the term “EPC”. It would be highly advantageous to agree standards to confirm an endothelial progenitor phenotype and this should include detailed immunophenotyping, potency assays, and clear separation from haematopoietic angiogenic cells which are not endothelial progenitors. In this review, we seek to discourage the indiscriminate use of ‘EPCs’, and instead propose precise terminology based on defining cellular phenotype and function. Endothelial colony forming cells (ECFCs) and myeloid angiogenic cells (MACs) are examples of two distinct and well-defined cell types that have been considered ‘EPCs’ because they both promote vascular repair, albeit by completely different mechanisms of action. It is acknowledged that scientific nomenclature should be a dynamic process driven by technological and conceptual advances; ergo the ongoing ‘EPC’ nomenclature ought not to be permanent and should become more precise in the light of strong scientific evidence. This is especially important as these cells become recognised for their role in vascular repair in health and disease; and, in some cases, progress towards use in cell therapy.

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