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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Abstract-Rapid induction and maintenance of blood flow through new vascular networks is essential for successfully treating ischemic tissues and maintaining function of engineered neo-organs. We have previously shown that human endothelial progenitor cells (EPCs) form functioning vessels in mice, but these are limited in number and persistence; and also that human adipose stromal cells (ASCs) are multipotent cells with pericytic properties which can stabilize vascular assembly in vitro.In this study, we tested whether ASCs would cooperate with EPCs to coassemble vessels in in vivo implants. Collagen implants containing EPCs, ASCs, or a 4:1 mixture of both were placed subcutaneously into NOD/SCID mice. After a range of time periods, constructs were explanted and evaluated with regard to vascular network assembly and cell fate; and heterotypic cell interactions were explored by targeted molecular perturbations. The density and complexity of vascular networks formed by the synergistic dual-cell system was many-fold higher than found in implants containing either ASCs or EPCs alone. Coimplantation of ASCs and EPCs with either pancreatic islets or adipocytes produced neoorgans populated by these parenchymal cells, as well as by chimeric human vessels conducting flow. This study is the first to demonstrate prompt and consistent assembly of a vascular network by human ASCs and endothelial cells and vascularization by these cells of parenchymal cells in implants. Mixture of these 2 readily available, nontransformed human cell types provides a practical approach to tissue engineering, therapeutic revascularization, and in vivo studies of human vasculogenesis. A ssembly of new vascular networks for therapeutic purposes has been critical but elusive for tissue engineering and angiogenesis. A general requirement for preserving viability within a regenerating region is that a vascular bed is assembled or expanded sufficiently to ensure adequate tissue perfusion. Also important to success of such applications is the ability of any network to anastomose or inosculate promptly with the vessels of immediately adjacent tissues. Cell-based revascularization therapies were tested in patients with various ischemic diseases. These studies used progenitor cells from bone marrow 1,2 and skeletal muscle 3,4 to treat patients with myocardial infarction, 1,2 heart failure, 3-6 and peripheral vascular disease. 7,8 Despite accumulating data and recent metaanalyses 9,10 that support the hypothesis that progenitor cells have high potential for promoting tissue revascularization and functional recovery, invasive methods of cells harvest and their low abundance may limit adoption some of progenitor cells for therapies. Recent approaches were focused on evaluating cells derived from more available tissues including adipose tissue 11,12 and umbilical cord blood. 13,14 Endothelial progenitor cells (EPCs) isolated from adult peripheral blood, 15 bone marrow, umbilical cord blood, 13 and vessel wall 16 were intensively studied over the past decade. ...

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OBJECTIVE-Emerging data demonstrate that maternal diabetes has long-term health consequences for offspring, including the development of hypertension. In adults, circulating endothelial progenitor cells (EPCs) participate in vascular repair, and EPC numbers and function inversely correlate with the risk of developing vascular disease. Therefore, our objectives were to determine whether hyperglycemia or exposure to a diabetic intrauterine environment alters EPC function. RESEARCH DESIGN AND METHODS-We used well-established clonogenic endothelial colony-forming cell (ECFC) assays and murine transplantation experiments to examine human vasculogenesis.RESULTS-Both in vitro hyperglycemia and a diabetic intrauterine environment reduced ECFC colony formation, self-renewal capacity, and capillary-like tube formation in matrigel. This cellular phenotype was linked to premature senescence and reduced proliferation. Further, cord blood ECFCs from diabetic pregnancies formed fewer chimeric vessels de novo after transplantation into immunodeficient mice compared with neonatal ECFCs harvested from uncomplicated pregnancies. CONCLUSIONS-Collectively, these data demonstrate that hyperglycemia or exposure to a diabetic intrauterine environment diminishes neonatal ECFC function both in vitro and in vivo, providing potential mechanistic insights into the long-term cardiovascular complications observed in newborns of diabetic pregnancies. Diabetes 57:724-731, 2008

Circulating endothelial progenitor cells (EPCs) in adult human peripheral blood were originally identified in 1997 by Asahara et al., which challenged the paradigm that vasculogenesis is a process restricted to embryonic development. Since their original identification, EPCs have been extensively studied as biomarkers to assess the risk of cardiovascular disease in human subjects and as a potential cell therapeutic for vascular regeneration. Endothelial colony‐forming cells (ECFCs), which are a subtype of EPCs, were recently identified from circulating adult and human umbilical cord blood. In contrast to other types of EPCs, which display various monocyte/macrophage phenotypes and functions, ECFCs are characterized by robust proliferative potential, secondary and tertiary colony formation upon replating, and de novo blood vessel formation in vivo when transplanted into immunodeficient mice. In this unit, we describe detailed methodologies for isolation and characterization of ECFCs from both human peripheral and umbilical cord blood. Curr. Protoc. Stem Cell Biol. 6:2C.1.1‐2C.1.27. © 2008 by John Wiley & Sons, Inc.

Cell therapy with endothelial progenitor cells (EPCs) is an emerging therapeutic option to promote angiogenesis or endothelial repair. Although the release of angiogenic paracrine factors is known to contribute to their therapeutic effect, little is known about their release of proinflammatory factors and expression of proinflammatory adhesion molecules. "Early" EPCs and "late" EPCs were isolated from human peripheral blood and their release of chemokines and thromboinflammatory mediators as well as their expression of the proinflammatory adhesion molecules was assessed at baseline and with stimulation. The effect of simvastatin on monocyte chemoattractant protein-1 (MCP-1) secretion by late EPCs from patients with vascular disease was also evaluated. All groups of EPCs released chemokines and thromboinflammatory mediators. Early EPCs primarily released thromboinflammatory mediators such as tissue factor (0.5 +/- 0.1 ng/million cells, P < 0.05), whereas adult late EPCs primarily released chemokines such as MCP-1 (287 +/- 98 ng/million cells, P < 0.05). Stimulation with tumor necrosis factor (TNF)-alpha augmented the expression of proinflammatory adhesion molecules and paracrine factors by all EPC subtypes. The release of MCP-1 by late EPCs was markedly reduced by simvastatin treatment of the cells. All EPC subtypes expressed proinflammatory paracrine factors and adhesion molecules involved in atherosclerosis. Future clinical studies should therefore not only assess the efficacy of EPCs but also monitor inflammatory activation following EPC transplantation in patients. Pharmacological modulation of EPCs before and after transplantation may represent a novel approach to improve their safety.

Defining whether human circulating proangiogenic cells represent a subset of the hematopoietic system and express CD45 or are hematopoietic derivatives that do not express CD45 (and are called endothelial progenitor cells) remains controversial. We have previously developed a polychromatic flow cytometry (PFC) protocol to isolate subsets of hematopoietic cells and we now identify the circulating pool of CD34 1 CD45 dim cells representing functional circulating hematopoietic stem and progenitor cells (CHSPCs) that can be separated on the basis of AC133 expression and report that the AC133