Assessing Identity, Phenotype, and Fate of Endothelial Progenitor Cells

Abstract

HomeArteriosclerosis, Thrombosis, and Vascular BiologyVol. 28, No. 9Assessing Identity, Phenotype, and Fate of Endothelial Progenitor Cells Free AccessReview ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessReview ArticlePDF/EPUBAssessing Identity, Phenotype, and Fate of Endothelial Progenitor Cells Karen K. Hirschi, David A. Ingram and Mervin C. Yoder Karen K. HirschiKaren K. Hirschi From the Departments of Pediatrics and of Molecular and Cellular Biology (K.K.H.), Center for Cell & Gene Therapy and Children’s Nutrition Research Center, Baylor College of Medicine, Houston, Tex; and the Department of Pediatrics (D.A.I., M.C.Y.), Herman B. Wells Center for Pediatric Research, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis. Search for more papers by this author , David A. IngramDavid A. Ingram From the Departments of Pediatrics and of Molecular and Cellular Biology (K.K.H.), Center for Cell & Gene Therapy and Children’s Nutrition Research Center, Baylor College of Medicine, Houston, Tex; and the Department of Pediatrics (D.A.I., M.C.Y.), Herman B. Wells Center for Pediatric Research, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis. Search for more papers by this author and Mervin C. YoderMervin C. Yoder From the Departments of Pediatrics and of Molecular and Cellular Biology (K.K.H.), Center for Cell & Gene Therapy and Children’s Nutrition Research Center, Baylor College of Medicine, Houston, Tex; and the Department of Pediatrics (D.A.I., M.C.Y.), Herman B. Wells Center for Pediatric Research, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis. Search for more papers by this author Originally published31 Jul 2008https://doi.org/10.1161/ATVBAHA.107.155960Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:1584–1595Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: July 31, 2008: Previous Version 1 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.EPC Identity and PhenotypeThere are a variety of measures that one can use to assist in the isolation and quantification of EPCs, but these can be simplified into two approaches: in vitro adhesion and growth (Figure 1) and selection by cell surface phenotype using fluorescent labeled antibodies and flow cytometry (Figure 2). First, we must confess that even at the time of writing this review, to our knowledge, no one has identified specific unique cell surface molecules that permit prospective isolation of an EPC in human or any other vertebrate species. That said, remarkable progress has been made in our understanding that there are numerous cell types and lineages that participate in neovascularization during development, at homeostasis, and during disease. Despite the lack of a unifying phenotype for an EPC, an approach for addressing how all these cells and cell lineages participate in the process is emerging and has provided new strategies for enhancing or inhibiting the process of new blood vessel formation. Download figureDownload PowerPointFigure 1. Common methods of “EPC” culture. Culture of colony forming unit–Hill cells (CFU-Hill, Method A, scale bar=100 um), includes a 5-day process wherein nonadherent peripheral blood mononuclear cells (PB-MNCs) give rise to the colony. Circulating angiogenic cells (CAC, Method B, scale bar=200 um) are the adherent mononuclear cells of a 4- to 7-day culture of PB-MNCs. CAC cultures typically do not display colony formation. Endothelial colony forming cells (ECFCs, Method B, scale bar=400 um) are derived from adherent PB-MNCs cultured for 6 to 21 days in endothelial conditions, and colonies display a cobblestone morphology. Only the ECFC progeny form blood vessels de novo in vivo. Images were collected using a Zeiss Axiovert 2 inverted microscope with 10×/0.25Ph1 CP-ACROMAT (CFU-EC), 32×/0.40Ph1 LD-ACROSTIGMAT (CAC), or 5× CP-ACHROMAT/0.12Ph0 (ECFC) objectives. Images were acquired using a SPOT RT color camera (Diagnostic Instruments) with the manufacturer’s software. Images cropped and scale bars added in Adobe Photoshop version 8.0. Modified from Prater DN et al. Working hypothesis to redefine endothelial progenitor cells. Leukemia. 2007;21:1142.Download figureDownload PowerPointFigure 2. Characteristics of cells comprising the adherent population in the commonly used assays of “EPC” identification. Adherent cells that display the function are indicated by (+), that do not display the function by (−), and if the literature provides conflicting evidence (±). Those properties that distinguish cells in the ECFC assay from CFU-Hill and CAC assays are indicated in bold font. Only the ECFC and progeny display the full properties one would attribute to an EPC. VEGFR2 indicates vascular endothelial growth factor receptor 2; ALDH, aldehyde dehydrogenase; UEA-1, Ulex europeaus agglutinin-1; acLDL, acetylated low density lipoprotein; eNOS, endothelial nitric oxide synthase. The data for this figure are compiled from the articles referenced in this review.In Vitro Adhesion and MorphologyAsahara et al10 reasoned that cell surface molecules shared by angioblasts and hematopoietic stem cells (HSCs) might serve as a means to identify putative angioblasts circulating in adult peripheral blood. This rationale cannot be underestimated, as it was somehow largely underappreciated in many subsequent articles by others attempting to define human EPCs, but as new information has emerged, it has become apparent that most of the antigens currently selected as a marker for human EPCs fail to discriminate hematopoietic cells from EPCs. In the Asahara at al10 article, human CD34 expressing cells (15.7% enriched for CD34+ expression) in adult peripheral blood were interrogated as putative EPCs via in vitro and in vivo assays. While determining some of the unique aspects of putative EPCs in vitro, Asahara et al10 reported that the cells adhered to fibronectin-coated dishes with greater frequency than to type 1 collagen coated dishes and displayed a spindle-shaped morphology. Of interest, the putative CD34+ EPCs when cocultured with CD34+ depleted mononuclear cells formed clusters of round cells centrally and sprouts of spindle-shaped cells at the periphery. The adherent putative EPCs expressed a variety of cell surface proteins typically expressed by human umbilical vein endothelial cells and expression of these markers increased over time in vitro. Further studies provided evidence of these putative EPCs (CD34+ or vascular endothelial growth factor receptor 2 [Flk-1+] cells enriched to 20%) localizing to areas of neovascularization when injected in vivo into nude mice with induced hindlimb ischemia. Thus, in one article Asahara et al10 brought forth concepts of circulating EPCs, in vitro observations of EPC behavior, in vivo migration of putative EPCs to sites of vascular injury, and the paradigm of postnatal vasculogenesis.The description of a putative EPC forming clusters in vitro within 5 days from the Asahara et al10 article, was expanded on by Ito et al11 who isolated human peripheral blood mononuclear cells and plated the cells on fibronectin-coated dishes. After a 24-hour period of adhesion, nonadherent cells were removed and replated onto fibronectin-coated dishes and the number of clusters that emerged at 7 days of plating was enumerated as EPC-derived colonies. The rationale for preplating the mononuclear cells for 24 hours was to deplete the population of monocytes, macrophages, and any circulating mature endothelial cells that could contaminate the putative EPC assay system.Hill et al12 further modified this cluster-forming assay, by preplating human peripheral blood mononuclear cells for 48 hours on fibronectin coated dishes and then replating the nonadherent cells to quantify the emergence of the EPC colony–forming units several days later. This assay is commercially available, and the putative EPCs (that produce the progeny that form the colony) have been referred to as colony forming unit-Hill (CFU-Hill; Figure 1). The CFU-Hill assay has been used to demonstrate a significant inverse correlation between the circulating CFU-Hill concentration and Framingham cardiovascular risk score in human subjects.12It is important to reflect on the differences between the methods of these three early articles in the field. Whereas Asahara et al10 plated minimally enriched CD34+ putative EPCs on fibronectin-coated dishes and simply observed the adherent cells forming “blood island-like” clusters of differentiating endothelial cells, Ito et al11 modified the assay conditions to screen for nonadherent mononuclear blood cells (after 24 hours of preplating) that formed the same kind of cluster formation and counted these as EPC-derived clusters. Hill et al12 modified the assay further by preplating the blood mononuclear cells for 48 hours and then replating the nonadherent cells and observed over several days for the appearance of the EPC colony forming unit cell that Hill et al12 interpreted as a quantitative readout of the EPCs. Thus, the only variable that tied these three studies together was the cluster morphology displayed by the blood mononuclear cells in vitro (Figure 1). It is not at all clear that the original minimally enriched CD34+ putative EPCs plated by Asahara et al10 and the CFU-Hill cells are related, and yet the inference is that these very different assays measure the presence of an EPC with the same functional properties.Recently, we13 and others14,15 have demonstrated that plating human peripheral blood or umbilical cord blood mononuclear cells on fibronectin-coated plates (with or without preplating steps) in the commercial CFU-Hill media or other media optimized for growth of endothelial cells in vitro results in the appearance of colonies composed of an aggregate of round cells with spindle-shaped cells emerging from the base and radiating away from the core of the colony. These CFU-Hill appearing colonies have been demonstrated to be composed of round hematopoietic cells that include myeloid progenitor cells, monocytes, and T lymphocytes.13–15The spindle shaped cells radiating away from the core are macrophages that display many features of endothelial cells with regard to cell surface protein expression, endothelial nitric oxide production, and expression of angiogenic molecules. These spindle shaped cells expressing endothelial markers (representing previously defined EPCs) do not spontaneously form blood vessels when implanted in vivo in collagen gels (do not display postnatal vasculogenic activity) and readily ingest bacteria in keeping with their monocyte-macrophage lineage roots.13 Thus, one of the central assays in the EPC field, which arose over several experimental modifications from the original observations of Asahara et al,10 does not assay for a cell that can give rise to a lineage of endothelial progeny, but instead is actually composed of hematopoietic cells enriched for monocyte-macrophage or T cell lineage commitment. In other words, the CFU-Hill assay measures cell-to-cell interactions among hematopoietic cells, and during subsequent differentiation these hematopoietic cells are stimulated by the culture conditions to mimic many features of endothelial cells; the CFU-Hill assay fails to identify cells that directly display any postnatal vasculogenic activity and thus, by definition, CFU-Hill are not composed of EPC progeny and are not a measure of EPCs. This important clarification does not constitute an argument that CFU-Hill are not involved in neoangiogenesis or serve as a biomarker for cardiovascular outcomes, rather the point is made to simply and clearly reflect our current understanding that these colonies are hematopoietic in origin and function.A second method to isolate and enumerate EPCs based on adhesion of peripheral blood mononuclear cells to fibronectin coated culture dishes was described by Vasa et al in 2001.16,17 Low density mononuclear cells from peripheral human blood were plated on fibronectin and gelatin coated dishes in the presence of media supplemented with endothelial growth factors and fetal calf serum. After 4 days in culture, the nonadherent cells were removed, and the adherent cells were assessed for the ability to ingest acetylated low-density lipoprotein (LDL) or fluorescently labeled Ulex europaeus agglutinin 1 plant lectin (Figure 1). The adherent cells that ingested or bound both antigens were considered EPCs and were counted. These adherent cells were released from culture and confirmed to express endothelial marker proteins von Willebrand factor (vWF), vascular endothelial cadherin, and vascular endothelial growth factor receptor 2 (FLk-1 for mice and KDR for human) by flow cytometry. An inverse relationship between the circulating concentration of these EPCs and increased risk factors for development of coronary artery disease in human subjects was reported.17The above data suggest that any low-density blood mononuclear cells that adhere to the fibronectin- and gelatin-coated dishes and within 4 days express KDR, vWF, vascular endothelial cadherin, and bind acetylated LDL and Ulex lectin are EPCs. However, others have shown that similarly isolated and cultured adherent cells expressing the above antigens may also coexpress to varying degrees, CD45, CD11b, CD11c, CD14, CD68, eNOS, and E-selectin, and they avidly ingest India ink similar to macrophages.13–15,18,19 The use of a panel of cell surface antigens alone may be inadequate to discriminate EPCs from macrophages because macrophages are well known to express “endothelial-specific” proteins, particularly when cultured in medium containing certain endothelial growth factors and fetal calf serum.20–24Furthermore, direct adhesion of peripheral blood mononuclear cells onto fibronectin- and gelatin-containing substrates is a published well recognized method to isolate enriched populations of peripheral blood monocytes (>90% enriched).25 Thus, a current summation of many studies to date suggests that direct adhesion of peripheral blood mononuclear cells to fibronectin (with or without gelatin)-coated dishes and culture of the adherent layer may highly enrich for monocytes and is certainly not specific for EPCs. Whether or not the monocytes, isolated in this manner, proceed to express “endothelial-specific” markers and participate in neoangiogenesis depends on the culture conditions and growth factors used. This fact in particular has led to some considerable confusion, suggesting to some investigators that monocytes become EPCs.26,27However, a more plausible working hypothesis is that some monocyte–macrophage subsets are potent circulating regulators of the angiogenic response (and have been called circulating angiogenic cells [CAC]; Figure 1) and play important roles in vascular regulation at homeostasis, and initiation of neoangiogenesis during wound healing, tissue ischemia, tissue remodeling, and tumorigenesis without becoming an integral part of the endothelial intima.28–38It is also possible that some macrophages, like trophoblast cells in the placenta,39 may transiently adhere to areas in the vasculature in which there is no endothelial covering (because of endothelial cell death or injury) and be in direct contact with the circulating blood, express a whole host of endothelial proteins, and function in some way for some period of time like an endothelial cell, without actually ever irreversibly committing to an endothelial cell fate (with loss of all macrophage and/or trophoblast properties). This is a theoretically significant problem if a macrophage moonlighting as an endothelial cell in vivo is confronted with a bacterial challenge or a cytokine storm during an acute innate response to an invading microorganism and resumes an inflammatory macrophage fate. This is also an interesting experimental question that has important implications for disease pathogenesis such as endothelial dysfunction in diabetes, aging, hypertension, and in the development of atherosclerosis. Finally, this new insight that the preponderance of studies evaluating putative EPCs may have been examining the role of hematopoietic-derived cells reconciles the considerable amount of undeniable experimental data that putative EPCs are marrow derived (which is, of course, the predominant site of hematopoiesis in the adults).All of the above methods used mononuclear cell adhesion to a particular matrix substrate under relatively short term culture conditions to identify and enumerate populations of putative EPCs. As such, the clusters or colonies of cells that emerged were at one time referred to as early outgrowth colonies (Figure 1). Another distinctly different population of cells has been determined to emerge later in culture and at one time were referred to as late outgrowth colonies (Figure 1). This has also been a confusing point in the literature. Whereas the early outgrowth cells as described above have largely been determined to be hematopoietic cells, the later outgrowth cells are clearly endothelial cells. At present, the early and late outgrowth terms have lost their usefulness as a means to discriminate between types of colonies and have largely been retired.Although the majority of endothelial cells that can be identified in circulating human blood are thought to be mature and even senescent sloughed endothelial cells, it has been reported that some rare endothelial cells demonstrate proliferative potential. Lin et al40 isolated peripheral blood mononuclear cells from a group of patients that had undergone sex-mismatched bone marrow transplantation and reported that the endothelial cells that appeared in vitro within a week of plating the blood cells on type 1 rat tail collagen coated dishes displayed limited proliferative potential and were of host origin. In contrast, colonies of highly proliferative endothelial cells emerging 14 to 21 days later were of donor origin, suggesting a marrow origin. These blood outgrowth endothelial cells demonstrated greater than a 1000-fold expansion over a 2-month period in vitro. We13,41,42 and numerous other labs43–52 have confirmed this seminal article and moved forward to successfully isolate high proliferative endothelial colony forming cells from both umbilical cord blood and adult peripheral blood. Umbilical cord blood contains a higher concentration of these proliferative cells and the proliferative potential of the endothelial colony forming cells (ECFCs) is significantly greater (at a clonal level) than that observed for ECFCs isolated from adult peripheral blood.41Although these cells display a host of typical antigens normally expressed on vascular endothelial cells, they do not express CD45, CD14, or CD115 and do not ingest bacteria. Furthermore, when suspended in gels of differing matrix compositions and implanted in vivo in the subcutaneous space of immunodeficient mice, these highly proliferative ECFCs (Figures 1 and 2) spontaneously form human blood vessels that inosculate with nearby murine vessels and become a part of the systemic circulation of the host mouse.13,49,53 Thus, ECFCs display properties of an EPC; cells that can clonally give rise to progeny that spontaneously form human blood vessels in vivo (postnatal vasculogenesis) and become a part of a circulatory system (Figures 1 and 2).Recently, Sieveking et al54 have reported the development of a novel endothelial specific in vitro tubulogenesis assay that highlights striking differences in the properties of the putative EPCs as described by Asahara et al10 and the outgrowth endothelial colonies (emerging 14 to 21 days after initial plating of blood mononuclear cells as described above) with high proliferative potential described by Lin et al.40 Human fetal lung fibroblasts were plated to confluence, and when human microvascular, coronary artery, or umbilical vein endothelial cells were plated in coculture, tubule formation occurred by 72 hours and was maximal at 14 days in vitro (control human smooth muscle cells, hepatocytes, monocytes, and epithelial cells failed to form tubes). The ability of these endothelial cell lines to form tubes was direct contact–dependent, stimulated by VEGF, and inhibited by anti-VEGF antibody or suramin addition to the cocultures. While the putative EPCs failed to form tubes in this assay, the circulating outgrowth endothelial cells displayed tubulogenesis properties with the same kinetics as the primary human endothelial cells. Of interest the putative EPCs displayed the ability to augment primary endothelial cell tubulogenesis in the cocultures in a dose dependent noncontact manner, whereas the outgrowth endothelial cells did not display this activity. However, only the outgrowth endothelial cells possessed the ability to integrate into networks of forming tubules (established using primary endothelial cells) compared to the putative EPCs that were unable to integrate into these endothelial networks. These results are strikingly similar to those described above, by our group and others,13,15,49 for the activity displayed by ECFC in the in vivo vessel forming assay and the lack of this activity displayed by CFU-Hill (Figures 1 and 2).In sum, the properties that appear to be shared among all the in vitro adherent cell types described above are the expression of endothelial markers (KDR, CD34, vWF, eNOS, vascular endothelial cadherin, and others) under rather specific culture conditions. However, those cells that also coexpress CD45, CD14, or CD115 among other markers ingest bacteria, display limited proliferative ability, and do not form human vessels de novo in vivo are derivatives of the marrow-derived hematopoietic system and are not EPCs, while those cells that do not express CD45, CD14, or CD115 do not ingest bacteria, display high proliferative potential at a clonal level, and form in vitro tubules in coculture with human fetal lung fibroblasts or directly form human vessels de novo in vivo are reflecting those properties that best define a functional EPC (Figure 2).13 The origin of these latter high proliferative endothelial cells remains obscure, as both marrow-derived and widespread vessel-derived origins have been proposed.40,42 Thus, more extensive analyses of the cells displaying the morphology of endothelial cells in vitro has permitted a growing list of properties that can be used to discriminate hematopoietic-derived cells, such as monocyte-macrophages that have a remarkable potential to display an endothelial morphology and gene expression pattern, from ECFC which display all of the phenotypic and functional properties of true EPCs.Cell Surface Markers to Identify EPCsAs noted above, there is currently, to our knowledge, no specific identifying marker for human or murine EPCs. To trace the origin of how the many different human EPCs phenotypes emerged in the literature, we must start with the seminal article by Asahara et al10 who postulated that use of available antibodies to known proteins expressed on embryonic angioblasts and HSC might permit a method to isolate EPCs (presumably an adult angioblast). CD34 was an obvious starting molecule of interest, because it is widely recognized as the principal marker used to isolate HSC for human clinical stem cell transplantation.55,56 CD34 is a sialomucin expressed on a variety of mesoderm progeny including blood, endothelial, and fibroblast cells and by numerous epithelial lineages and some cancer stem cell populations. KDR (human) or Flk-1 (mouse), a receptor for vascular endothelial growth factor, is also widely expressed on mesoderm derived lineages including blood, endothelium, and cardiac tissues.57 Although the original studies reported by Asahara et al10 used minimally enriched populations (15% to 20%) of CD34+ and Flk-1+ cells to initiate cultures of adherent cells, the cells present in those cultures were found to localize to areas of neoangiogenesis in vivo. This association led the authors to state that circulating CD34+ and Flk-1+ mononuclear blood cells may contribute to neoangiogenesis in adult species, and this cell surface antigen pair formed the first putative marker set for identifying the EPCs (Figure 2). However, no direct evidence was presented in the manuscript to confirm that highly purified cells expressing only one or the other or both antigens directly participated in neoangiogenesis or became integrated into the endothelial intima.Pursuing the question of the origin of cells within the bloodstream that colonized experimentally implanted Dacron grafts, Shi et al58 and Bhattacharya et al59 tested the question that circulating EPCs may be the source. It had been reported for several decades that many implanted grafts can become colonized with an intact endothelial cell monolayer that was host derived.60,61 To define whether the circulating cells colonizing these grafts were simply sloughed mature endothelial cells from nearby sites, circulating EPCs (presumably derived from the endothelial intima), or EPCs derived from the marrow, Shi et al58 performed bone marrow transplantation in dogs (with defined alleles determined by polymerase chain reaction (PCR)-microsatellite analysis) and observed that donor marrow–derived cells entered the circulation and colonized implanted vascular prostheses (the cells lining these grafts expressed CD34). Because human CD34+ cells isolated by monoclonal antibody and immunomagnetic beads demonstrated the ability to give rise to adherent colonies of rapidly proliferating endothelial cells after 15 to 20 days in vitro, the authors interpreted their data to suggest that a subset of CD34+ cells localized in the marrow could be mobilized to the peripheral circulation and colonize the intraluminal exposed surfaces of vascular prostheses. However, the authors correctly stated that formal proof of this hypothesis awaited further studies in which individual endothelial cells colonizing the implanted graft could be proven to be derived from a single marrow-derived CD34+ cell.Using a similar experimental canine model of graft implantation, Bhattacharya et al59 reported that preoperative seeding of Dacron grafts with marrow-derived CD34+ cells appeared to facilitate the colonization and seeding of the grafts with endothelial-like cells, though there was no correlation between the number of seeding CD34+ cells and the degree of endothelial-like coating of the luminal surface of the grafts. Again the authors pointed out that only by labeling the marrow-derived CD34+ cells with a long-lasting marker could they provide definitive proof that the enhancement of the colonization of the grafts with endothelial cells originated from the CD34+ cells. Thus, neither of the above articles directly provided evidence that CD34+ cells directly differentiate into e