Working hypothesis to redefine endothelial progenitor cells

Leukemia (2007) 21, 1141–1149 & 2007 Nature Publishing Group All rights reserved 0887-6924/07 $30.00 www.nature.com/leu REVIEW Working hypothesis to redefine endothelial progenitor cells DN Prater, J Case, DA Ingram and MC Yoder Since 1997, postnatal vasculogenesis has been purported to be an important mechanism for neoangiogenesis via bone marrow (BM)-derived circulating endothelial progenitor cells (EPCs). Based on this paradigm, EPCs have been extensively studied as biomarkers to assess severity of cardiovascular disease and as a cell-based therapy for several human cardiovascular disorders. In the majority of studies to date, EPCs were identified and enumerated by two primary methodologies; EPCs were obtained and quantified following in vitro cell culture, or EPCs were identified and enumerated by flow cytometry. Both methods have proven controversial. This review will attempt to outline the definition of EPCs from some of the most widely cited published reports in an effort to provide a framework for understanding subsequent studies in this rapidly evolving field. We will focus this review on studies that used cell culture techniques to define EPCs. Leukemia (2007) 21, 1141–1149. doi:10.1038/sj.leu.2404676; published online 29 March 2007 Keywords: endothelial cell; progenitor cell; angiogenesis; vasculogenesis Introduction and initial discovery of endothelial progenitor cells (EPCs) By the early 1950s, human patients with cardiovascular disease were having diseased segments of large blood vessels replaced by artificial vascular grafts, which upon later excision were covered in a layer of endothelial cells (ECs).1,2 It was assumed that the endothelium observed on these vascular grafts grew from the original vessel ends at the site of anastomosis, and any patches of endothelium discontinuous with this endothelium arose from microvasculature penetrating the interstices of the graft (perhaps through development of vaso vasorum).1,3 Stump et al.4 directly addressed the question of whether circulating cells would colonize an artificial substrate implanted intravascularly by suspending a piece of Dacron material within a pig aortic graft, thereby isolating the surface of the Dacron material from any opposing microvasculature. After 14 days in the animal, they observed patches of endothelium on the graft, and by 20 days, the graft segment was completely lined by endothelium (identified with silver nitrate staining). The authors concluded that circulating endothelium must have attached and proliferated to cover the suspended material. Gaynor et al.5 measured the number of circulating endothelial cells (CECs) in the blood of rabbits administered a dose of endotoxin, and Correspondence: Dr MC Yoder, Departments of Pediatrics, Biochemistry and Molecular Biology, Indiana University School of Medicine, Herman B Wells Center for Pediatric Research, Cancer Research Institute, 1044 W Walnut Street, R4/402E, Indianapolis, IN, USA. E-mail: Received 7 February 2007; revised 28 February 2007; accepted 1 March 2007; published online 29 March 2007 reported an increase in the CEC concentration, which suggested that CECs could serve as a marker to measure vascular injury. Indeed, CEC concentrations have been found to be altered in patients with a variety of vascular injury and disease, including coronary angioplasty,6 acute coronary syndromes and unstable angina,7 peripheral vascular disease,8 and inflammatory vasculitis,9 when conditioning for bone marrow transplantation,10 and during organ transplant rejection.11 More recently, CECs have been measured by CD146 expression, and changes in the concentration of CD146 þ cells have been used as a biomarker for many human disorders.12 The concentration of CECs in the blood of normal human subjects is low, typically about three cells per ml, but this number may significantly increase in many disease states.13–15 Prior to 1997, the predominant paradigm for new vessel formation in adult subjects was believed to be endothelial sprouting from pre-existing vessels, and regional hypoxia was believed to be abated through a process termed angiogenesis.16 In 1997, Asahara et al.17 purified a population of circulating cells which displayed properties of both ECs and progenitor cells. Termed ‘endothelial progenitor cells’ (EPCs), they were purported to give rise to differentiated ECs in a process termed postnatal vasculogenesis.17 Before this seminal contribution, vasculogenesis was thought to occur only in the developing fetus when progenitors of endothelium (angioblasts) differentiate to form nascent vessels in developing tissues.18–20 The de novo formation of vessels from uncommitted precursors sets vasculogenesis apart from both angiogenesis and arteriogenesis.19 It now appears that aside from angiogenesis and arteriogenesis, circulatory system remodeling in the adult may indeed involve the recruitment of not only endothelial progenitors but also a plurality of cells in an intricate collaboration.21–39 Sprouted endothelium may line the vessel lumen while other cells support vessel formation through secretion of cytokines and modification of the extracellular space.40–45 Although each cell type appears to be fundamentally different, all cell types involved in postnatal vasculogenesis have been lumped into the single term ‘EPC’. Here, we attempt to clarify study results by closely examining and comparing the methods used to define different cell populations and in the end, propose some unifying working hypotheses. EPC definition and characterization – colony-forming unit-EC EPCs represent a minor subpopulation of blood mononuclear cells (MNCs), and in vitro cell culture methods have been developed to select and expand this population. In general, three culture methods for isolating EPCs have been described (Figure 1). The original method by Asahara et al.,17 which has subsequently been modified,46,47 can now be performed using a commercially available kit (Figure 1a; Method A) (Endocult, StemCell Technologies, Vancouver, British Columbia, Canada). SPOTLIGHT Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN, USA Defining endothelial progenitor cells DN Prater et al 1142 SPOTLIGHT Figure 1 Common methods of EPC culture. Culture of colony-forming unit – endothelial cells (CFU-EC, Method A, scale bar ¼ 100 mm) includes a 5-day process wherein non-adherent MNCs give rise to the EPC colony. Circulating angiogenic cells (CAC, Method B, scale bar ¼ 200 mm) are the adherent mononuclear cells of a 4- to 7-day culture procedure. CAC cultures typically do not display colony formation. Endothelial colonyforming cells (ECFCs, Method B, scale bar ¼ 400 mm) are derived from adherent MNCs cultured for 7–21 days in endothelial conditions and colonies display a cobblestone morphology. Images were collected using a Zeiss Axiovert 2 inverted microscope with  10/0.25Ph1 CPACROMAT (CFU-EC),  32/0.40Ph1 LD-ACROSTIGMAT (CAC), or  (...truncated)