Human Endothelial Progenitor Cellsperspectivesinmedicine.cshlp.org/content/2/7/a006692... · 2012....

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Human Endothelial Progenitor Cells Mervin C. Yoder Department of Pediatrics, Herman B Wells Center for Pediatrics Research, Indiana University School of Medicine, Indianapolis, Indiana 46142 Correspondence: [email protected] Human endothelial progenitor cells (EPCs) have been generally defined as circulating cells that express a variety of cell surface markers similar to those expressed by vascular endo- thelial cells, adhere to endothelium at sites of hypoxia/ischemia, and participate in new vessel formation. Although no specific marker for an EPC has been identified, a panel of markers has been consistently used as a surrogate marker for cells displaying the vascular regenerative properties of the putative EPC. However, it is now clear that a host of hemato- poietic and vascular endothelial subsets display the same panel of antigens and can only be discriminated by an extensive gene expression analysis or use of a variety of functional assays that are not often applied. This article reviews our current understanding of the many cell subsets that constitute the term EPC and provides a concluding perspective as to the various roles played by these circulating or resident cells in vessel repair and regeneration in human subjects. T he importance of the systemic vasculature in mediating optimal delivery, exchange, and removal of gases, nutrients, and regulatory cells and molecules to the tissues and organs of a mature subject has long been appreciated (Aird 2007). More recently, interest in the role of the vasculature in promoting organogenesis during development, stem cell homeostasis, rescue of injured tissues following an ischemic/hypoxic challenge, and the growth and spread of cancer cells within the body has grown exponentially as investigators have probed new approaches for cellular therapies in all areas of human health and disease. Concomitant with these interests in translational research, investigators have become enthralled with the discovery of novel adult stem/progenitor cell populations that may be involved in the development, repair, or regeneration of the systemic vasculature. The first reported existence of a bone marrow– derived circulating progenitor for the endothe- lial lineage called the endothelial progenitor cell (EPC) in 1997 (Asahara et al. 1997) initiated a robust area of investigation in experimental ani- mals and human subjects with nearly 9500 papers cited in the PubMed database as of Jan- uary 1, 2011 using the search term “endothelial progenitor cell.” In contrast to the vast number of papers elu- cidating roles of putative bone marrow–derived EPC in cancer (Lyden et al. 2001; Mancuso et al. 2001, 2006, 2009; Bertolini et al. 2006; Shaked et al. 2006; Nolan et al. 2007; Gao et al. 2008; Seandel et al. 2008; Gao and Mittal 2009), car- diovascular disorders (Eizawa et al. 2004; Schmidt-Lucke et al. 2005; Werner et al. 2005; Editors: Michael Klagsbrun and Patricia D’Amore Additional Perspectives on Angiogenesis available at www.perspectivesinmedicine.org Copyright # 2012 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a006692 Cite this article as Cold Spring Harb Perspect Med 2012;2:a006692 1 www.perspectivesinmedicine.org on April 23, 2021 - Published by Cold Spring Harbor Laboratory Press http://perspectivesinmedicine.cshlp.org/ Downloaded from

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Human Endothelial Progenitor Cells

Mervin C. Yoder

Department of Pediatrics, Herman B Wells Center for Pediatrics Research, Indiana UniversitySchool of Medicine, Indianapolis, Indiana 46142

Correspondence: [email protected]

Human endothelial progenitor cells (EPCs) have been generally defined as circulating cellsthat express a variety of cell surface markers similar to those expressed by vascular endo-thelial cells, adhere to endothelium at sites of hypoxia/ischemia, and participate in newvessel formation. Although no specific marker for an EPC has been identified, a panel ofmarkers has been consistently used as a surrogate marker for cells displaying the vascularregenerative properties of the putative EPC. However, it is now clear that a host of hemato-poietic and vascular endothelial subsets display the same panel of antigens and can onlybe discriminated by an extensive gene expression analysis or use of a variety of functionalassays that are not often applied. This article reviews our current understanding of themany cell subsets that constitute the term EPC and provides a concluding perspective as tothe various roles played by these circulating or resident cells in vessel repair and regenerationin human subjects.

The importance of the systemic vasculaturein mediating optimal delivery, exchange,

and removal of gases, nutrients, and regulatorycells and molecules to the tissues and organs of amature subject has long been appreciated (Aird2007). More recently, interest in the role of thevasculature in promoting organogenesis duringdevelopment, stem cell homeostasis, rescue ofinjured tissues following an ischemic/hypoxicchallenge, and the growth and spread of cancercells within the body has grown exponentially asinvestigators have probed new approaches forcellular therapies in all areas of human healthand disease. Concomitant with these interestsin translational research, investigators havebecome enthralled with the discovery of noveladult stem/progenitor cell populations thatmay be involved in the development, repair, or

regeneration of the systemic vasculature. Thefirst reported existence of a bone marrow–derived circulating progenitor for the endothe-lial lineage called the endothelial progenitor cell(EPC) in 1997 (Asahara et al. 1997) initiated arobust area of investigation in experimental ani-mals and human subjects with nearly 9500papers cited in the PubMed database as of Jan-uary 1, 2011 using the search term “endothelialprogenitor cell.”

In contrast to the vast number of papers elu-cidating roles of putative bone marrow–derivedEPC in cancer (Lyden et al. 2001; Mancuso et al.2001, 2006, 2009; Bertolini et al. 2006; Shakedet al. 2006; Nolan et al. 2007; Gao et al. 2008;Seandel et al. 2008; Gao and Mittal 2009), car-diovascular disorders (Eizawa et al. 2004;Schmidt-Lucke et al. 2005; Werner et al. 2005;

Editors: Michael Klagsbrun and Patricia D’Amore

Additional Perspectives on Angiogenesis available at www.perspectivesinmedicine.org

Copyright # 2012 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a006692

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Fadini et al. 2006a,b; Kunz et al. 2006; Hugheset al. 2007), and diabetes (Tepper et al. 2002;Eizawa et al. 2004; Loomans et al. 2004; Fadiniet al. 2006b, 2007), little focus has been placedon fully understanding how these cells maydiffer in their roles, behavior, or function com-pared to the rare circulating endothelial cellsthat may also be involved in many of thesesame disorders. This overview will attempt tosummarize our current understanding of thevarious cell subsets that circulate in the blood-stream and are all referred to using the sameEPC terminology. Given that no specific cellsurface marker or unique gene expression pat-tern has been identified to unambiguouslymark an EPC in mouse or man, we will identifythose tools currently used to identify the puta-tive EPC phenotype and will stress the differen-ces in cell function displayed by the various“EPC” subsets.

BLOOD VESSEL FORMATION, REPAIR,AND REMODELING ARE REGULATEDBY DIFFERING MECHANISMS

Blood vessel formation in the embryo has beenexamined in numerous vertebrate model sys-tems. In the mouse, angioblast precursorsderived from posterior primitive streak-derivedmesoderm cells emerge on embryonic day(E)7.5 to initiate the process of vasculogenesis(Risau and Flamme 1995; Sabin 2002). Theangioblasts migrate into the extraembryonicyolk sac to form a primitive capillary plexus.In time other angioblasts from later primitivestreak-derived mesoderm populations migrateinto and colonize the embryo proper and com-plete the first systemic vascular capillary bed byE8.25. The first blood cells to emerge in thedeveloping mouse are the primitive erythroidprogenitor (EryP) cells that independentlymigrate into the yolk sac (Fig. 1) and segregateinto a circumferential extravascular band oferythroid cells (Ferkowicz et al. 2003). Nearthe time of onset of cardiac contractions thatpromulgate the first evidence of systemic bloodcirculation, the extraembryonic blood band iscircumscribed by adjacent endothelial cellsand the first blood-filled capillary structures

called blood islands are formed. Over the next36 h, the blood islands are remodeled via intus-suceptive angiogenesis and arteriogenesis intothe various arterial, venous, and capillary bedsof the mature yolk sac. Some of these endothe-lial cells display the capacity to form definitivehematopoietic progenitor cells that displaymultipotential hematopoietic lineage poten-tial. Of interest, the systemic vascular bed isnot completely filled with circulating bloodcells until nearly E10.5. Thus, while arisingnearly simultaneously from mesoderm, theEryP, definitive progenitor cells, and angioblastsdo not appear to be derived from a commonprecursor, the hemangioblast, as often cited(reviewed in Ferkowicz and Yoder 2005; Uenoand Weissman 2010). Recent studies suggestingthat the hemangioblast gives rise to hemogenicendothelial cells (which subsequently form thedefinitive hematopoietic lineages) only partiallyexplains the origins of the EryP and fails toexplain the origin of the earliest primitive capil-lary plexus (in which no blood cells emerge).Thus, in the earliest stages of blood vessel for-mation, hematopoiesis and vasculogenesis areindependently executed developmental eventsthat arise from mesoderm precursors. The factthat these lineages arise at nearly the sametime and place in the developing embryo nolonger infers that they arise from a commonprecursor.

Once the capillary plexus is formed, furtherexpansion of blood vessel growth occurs viaangiogenesis, as well as vasculogenesis (Risauand Flamme 1995). Angiogenesis is the forma-tion of new vessels from endothelial cells rep-resenting already existing vascular structures inthe embryo. In some cases, new vessels formfrom sprouting capillary endothelium withinthe primitive capillary plexus. In other cases,the sprouting endothelial cells arise from vascu-lar structures remodeled into arterial or venousappearing structures. It is apparent that themetabolic demands of the tissue participate inthe remodeling of the primary capillary plexusto define the final vascular bed density. Asreviewed elsewhere (Davis and Senger 2008;Iruela-Arispe and Davis 2009; Eilken andAdams 2010; Pitulescu and Adams 2010),

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genetic programmatic features and hemody-namic stressors play important roles in thecapillary plexus remodeling into arteries andveins. Some evidence supports the role ofhemodynamic stress on endothelial cells as animportant component that promotes hemato-poietic progenitor cell emergence from hemo-genic endothelium (Adamo et al. 2009). Ahost of molecular mechanisms are now wellunderstood in the regulation of the endothelialsprouting that represents a key event for newvessel formation. Subsequent steps of matrixdegradation, endothelial sprouting, endothelialcell migration, cell-to-cell interactions withadjacent endothelial cells, cytoplasmic vacuola-tion and lumenization, basement membranesynthesis, and inosculation with preexisting ves-sels to access systemic blood flow are subsequentsteps in vessel formation (Iruela-Arispe andDavis 2009).

In certain circumstances, a denudationinjury to the vascular endothelium can occurthat may not perturb the underlying endothelialbasement membrane but requires rapid cellularrecruitment to cover the otherwise thrombo-genic exposed basement membrane (Schwartzet al. 1980). For example, traumatic injurymay compromise the health of the vascularintima without harming the rest of the vesselcomponents. Localized areas of inflammationmay compromise vascular endothelial healthand survival, leading to enhanced endothelialturnover (Schwartz et al. 1981). Apparently,the first events that occur to repair denudationof the endothelium (in experimental models)include deposition of platelets to the exposedbasement membrane, increased migratorybehavior of the endothelium adjacent to theinjury, and endothelial cell spreading into theinjury site (Schwartz et al. 1975; Malczak and

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Figure 1. A late neural plate stage embryo. (A) Late neural plate stage mouse embryo with maturing Flk-1þ vas-cular plexus (green) and distinct band of CD41þ primitive erythroid progenitor cells in the proximal yolk sac(red). Note the paucity of angioblasts (green) in the blood band region. (B) Higher-magnification cross sectionof the blood band. The Flk-1þ angioblasts (green cells identified by asterisks) are located between the primitiveerythroid progenitor cells and the yolk sac visceral endoderm (blue cells indicated by endo) cells. (C) Blood islandimage depicting an angioblastic cord (a) from the same stage embryo as in panels A and B. Note the similarity ofthe cross section of the tissue in panel B to the panel C. The primary difference is the evidence that the cells high-lighted in red in panel B are now known to be primitive erythroid cells and not mesoderm (angioblastic) cords asonce thought (C). (Figure adapted from Ferkowitz 2005; reprinted, with permission, from Elsevier # 2005.)

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Buck 1977; Manderson and Campbell 1986).For small injuries, endothelial migration andspreading can result in closure of the previouslydenuded area within a day. Subsequently, endo-thelial cell proliferation (first within more distalcells but later even in those cells that hadmigrated over the injury site) ensues, often lead-ing to an eventual increased cellular density overthe primary injury site that is several times thatof the original cell density. Thus, the major celltype involved in the resolution of a denudationinjury and return of the endothelial monolayerto the original topography and function of thatvessel is the endogenous adjacent endotheliumresident within the vessel (Schwartz and Benditt1976). Whether similar events occur to replacesenescent endothelial cells, without ever expos-ing the underlying basement membrane for cir-culating blood cell attachment, is unclear. Priorstudies in rodent and pig models of normal vas-cular growth or induced vascular injury suggestthat proliferating clusters of endothelial cellsare apparent in vessels without signs of vasculardenudation, providing some support for thepresence of endogenous endothelial replace-ment (Wright 1968, 1971, 1972; Florentinet al. 1969; Caplan and Schwartz 1973; Schwartzand Benditt 1976, 1977; Schwartz et al. 1980,1981; Prescott and Muller 1983; Taylor andLewis 1986).

If large areas of endothelium are removedand/or there is damage to the underlyingbasement membrane or an artificial vasculargrafting is interposed within vessels, greaterinflux of circulating cells ensues as a firstresponse to injury. In this instance, a host of cir-culating hematopoietic cells along with plateletsreadily attach to these areas of damage or to thegrafted artificial material (Rafii et al. 1995). Insome instances, these deposited blood cellsfrom the circulating blood are soon replacedby migrating and spreading endogenous endo-thelium. In other instances, colonies of replicat-ing endothelial cells grow on the exposed areabut fail to completely repopulate the monolayerand with time these areas of exposed basementmembrane develop a fibrous and nonthrombo-genic covering (Berger et al. 1972; Herring et al.1984; Clowes et al. 1985, 1986; Zilla et al. 1994,

2007). Thus, the extent and type of vascularintima repair or regeneration is perhaps depen-dent on the extent and type of injury or implantand perhaps the age of the host. It is also wellknown that there are species-specific factorsthat may differentially regulate reendotheliali-zation of denuded vessels (Berger et al. 1972;Herring et al. 1984; Clowes et al. 1985).

Finally, tumor cell growth, expansion, andmetastasis depends on the ability of the tumorcells to secrete a variety of molecules that cooptcirculating and resident proangiogenic cells inthe tumor microenvironment to promoteangiogenesis (Mancuso and Bertolini 2010).Tumor angiogenesis is perhaps the most com-plicated context within which to understandall of the cellular elements contributing to thetumor vascular growth. Not only do tumor cellsrecruit local and resident cells that includetumor-associated macrophages, tissue macro-phages, mast cells, monocytes, neutrophils,and platelets that promote neoangiogenesiswith sprouting of nearby vascular endotheliuminto the tumor, but in some cases, tumor cellsmay become vessel-mimicking or actual ves-sel-forming cells (Hirschi et al. 2008; Ricci-Vitiani et al. 2010; Wang et al. 2010). It is withinthis complex heterogenous tumor microenvir-onment that some investigators have observedrecruitment of circulating putative EPCs thatplay a role in enhancing tumor growth andmetastasis. Defining those cells that representEPCs and truly become long-term endothelialcells that comprise the tumor vessels versusthose that enhance the vessel-forming proper-ties of recruited endothelial cells but do notdirectly become endothelial cells is an impor-tant issue in developing the most powerfuland effective drugs to inhibit all of the cellsinvolved in the most effective tumor therapy(Bautch 2010).

METHODS TO DEFINE HUMAN EPCs

In the human system, putative EPCs have beenidentified using three general approaches. Oneof the first and perhaps the simplest methodinvolves collecting low density mononuclearcells (MNCs) from human peripheral blood

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or cord blood and plating the MNCs in dishescoated with fibronectin in a commercial cell cul-ture medium containing endothelial growthfactors and fetal calf serum (Ito et al. 1999;Hill et al. 2003). After 4–5 days in culture, thenonattached cells are removed and the adherentcells examined for the ability to bind acetylatedlow-density lipoprotein (AcLDL) and Ulexeuropaeus agglutinin 1 (a plant lectin). Thus,EPCs, as defined in this assay, are characterizedby their morphologic appearance, adhesion tofibronectin, cell surface protein expression,AcLDL uptake, and lectin binding. Althoughthis is a simple method for isolating an adherentcell population, it may be flawed by the lack ofspecificity of the cells obtained. For example,blood platelets are known to contaminatemost MNC preparations and the presence ofplatelets in this culture milieu can result in non-discriminate transfer of platelet plasma mem-brane proteins to any adherent cells alsoattached to the culture matrix (including cer-tain proteins thought to be endothelial specific)(Prokopi et al. 2009). Numerous blood cells(stem, progenitor, or committed mature line-ages) express the integrin receptors for fibronec-tin and attach to plates coated with thismolecule. In fact, monocytes are known to behighly enriched from peripheral blood MNCswhen plated on fibronectin-coated dishes, andadherent monocytes cultured in media contain-ing endothelial growth factors are known toexpress a variety of proteins typically thoughtto be reserved for endothelial cells (von Wille-brand factor, endothelial nitric oxide synthase,CD31, CD144, and vascular endothelial growthfactor 2 receptor [KDR]) (Hassan et al. 1986;Schmeisser et al. 2001, 2003). Recent proteomicand mRNA profiling analyses have indicatedthat human peripheral blood MNCs culturedas adherent cells on fibronectin-coated dishesin culture medium with added endothelialgrowth factors display a gene expressionpattern that highly resembles hematopoietic(particularly cultured myeloid and T lymphoidcells) but not human endothelial cells (Medinaet al. 2010). Thus, this very straightforwardmethod of adherent MNC growth in vitrodoes not promote the unique emergence of an

EPC. Thus, given the ambiguity of this isolationmethod, one cannot recommend using thisapproach for EPC isolation or enumeration.As we will discuss later, this does not meanthat the hematopoietic cells isolated via thistechnique and cultured under these conditionsdo not display proangiogenic activity; in fact,one can readily obtain proangiogenic hemato-poietic cells using this approach that participatein ischemic hindlimb blood flow restoration inmice and even demonstrated some ability toimprove cardiac outcomes in human subjectswith acute myocardial infarction (Tongerset al. 2010). We simply point out that some ofthe lack of clarity in the field may relate to theuse of the term EPC for cells isolated usingthis procedure when, in fact, in nearly all cases,proangiogenic hematopoietic cells (of variouslineages) are selected and these cells thoughpromoting vessel repair but do not becomelong-lived endothelial lined blood vessels.

A second method of human EPC identifica-tion has relied on identification of a particularpattern of cell surface antigen expression onthe cells. As noted above, there are no uniqueor specific protein markers that can be used toprospectively isolate an EPC (reviewed in Hir-schi et al. 2008). The first description of ahuman EPC by Asahara et al. (1997) wasdependent on the selection of circulating cellswith certain cell surface markers that might beexpressed by both hematopoietic and endothe-lial cells so as to search for a putative circulatingangioblast precursor. Although this rationalewas based on the long-held recognition for theclose temporal and spatial emergence of bloodand endothelial cells during embryogenesis,the most current understanding of blood celland angioblast development indicates that theselineages may not be derived from a commonprecursor. Indeed, evidence supports the originof hematopoietic stem and progenitor cellsfrom hemogenic endothelium but not endothe-lial cells from blood cells (Yoder et al. 2007;Zovein et al. 2008; Chen et al. 2009). Asaharaet al. (1997) isolated human peripheral bloodCD34-expressing cells (15.7% enriched forCD34þ-expression) and reported that celladhesion to fibronectin-coated dishes was

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significantly greater than to type 1 collagencoated dishes and that the CD34 enriched cellsdisplayed a spindle-shaped morphology in thisculture system. Of interest, the putativeCD34þ EPCs when cocultured with CD342

MNCs on the fibronectin-coated dishes formedclusters of round cells centrally and sprouts ofspindle-shaped cells at the periphery. Theauthors pointed out that these clustered struc-tures were reminiscent of the blood island-likeclusters normally found in the developingembryonic yolk sac. The adherent putativeEPCs expressed a variety of cell surface proteinstypically expressed by human umbilical veinendothelial cells, and expression of thesemarkers increased over time in vitro. Furtherstudies provided evidence that vascular endo-thelial growth factor receptor 2 (Flk-1þ)expressing putative EPCs (enriched to 20%Flk-1þ) homed to areas of neovascularizationwhen injected into nude mice with inducedhindlimb ischemia with some evidence forimproved microvascular density after infusion.Thus, in one article, Asahara et al. (1997)brought forth concepts of circulating EPCs, invitro observations of EPC behavior, in vivomigration of putative EPCs to sites of vascularinjury, and the paradigm of postnatal vasculo-genesis whereby circulating EPCs could beinfused into an animal with subsequent contri-bution to tissue microvasculature. These wereand are important observations that havestimulated many investigators to understandthe biology of these cells, attempt to character-ize their concentration in health and disease,and isolate and infuse these cells into patientswith acute or chronic ischemic diseases. How-ever, potential limitations of this paper includedthe lack of sufficient cellular enrichment to con-stitute a purified cell population, lack of clonalanalytical studies, failure to provide any func-tional exclusionary evidence as to whether anyof the CD34þ or Flk-1þ EPCs possessed hema-topoietic potential (given the fact that many ofthe cultured CD34þ cells displayed CD45), andlack of high cellular resolution evidence that theinfused cells directly formed the new blood ves-sels in the tissues of the mice with induced vas-cular injury. Nonetheless, the use of CD34 and

Flk-1 (KDR in human subjects) as markers forthe putative EPCs were instituted with this pub-lication and have continued to be used as surro-gate markers for the presence of a circulating cellwith vascular reparative properties or in the iso-lation of this putative precursor.

The choice of CD34 as a potential marker ofthe circulating angioblast was not surprising asit is known to be expressed on endothelial cellsand is a marker used to isolate human hemato-poietic stem and progenitor cells for clinicalstem cell transplantation. But, CD34 is a widelyexpressed molecule on some mesenchymal, epi-thelial, and even cancer stem cell populations(Hirschi et al. 2008). Thus, use of CD34 as anindividual EPC marker is inadequate and wouldcertainly require the search for additionalpotentially unique markers to discriminate allthese different cellular lineages from the puta-tive EPCs. KDR (human) or Flk-1 (mouse), areceptor for vascular endothelial growth factor,is also widely expressed on blood, endothelial,and cardiac cells and thus fails to be a helpfuldiscriminator among those cells expressingCD34. Rationalizing that EPCs may sharesome cell surface antigen expression patternswith hematopoietic stem or progenitor cells,Peichev et al. (2000) chose to separate periph-eral blood cells by expression patterns forCD34, KDR, and CD133. CD133 (AC133,prominin-1) is a 5-transmembrane domaincell surface glycoprotein that localizes to mem-brane protrusions on numerous epithelial,hematopoietic, and various cancer stem cells.CD34 and CD133 were known to be highlyexpressed on hematopoietic stem cells and aredown-regulated during hematopoietic cell dif-ferentiation. Given this expression pattern inhematopoiesis, Peichev et al. (2000) rational-ized that any endothelial cells coexpressing thesemolecules may represent a more immature pro-genitor population than cells expressing eitherantigen alone. Furthermore, because KDR wasknown to be expressed by embryonic angio-blasts, Peichev et al. (2000) hypothesized thatthis antigen may be coexpressed on subsets ofCD133þ cells with angioblastic (EPC) activity.The authors reported that although 2% ofmobilized human peripheral blood CD34þ cells

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coexpressed KDR and CD133, mature CD34þ-

KDRþ human umbilical vein endothelial cells(chosen as representative of a mature vascularendothelial population) failed to expressCD133, and thus the circulating CD34þKDRþ-

CD133þ cells were thought to represent aprogenitor cell phenotype. To confirm the pres-ence of CD133 and KDR expressing cellsin vivo, Peichev et al. examined the luminal sur-faces of implanted left ventricular assist devicesin human subjects with heart failure and identi-fied some surface adhering cells expressingCD133 and KDR. Based on this evidence,the investigators concluded that a human EPCcould be defined as a circulating CD34þ cellthat coexpresses CD133 and KDR. Subse-quently, this pattern of cell surface antigenexpression has been one of the most popularto use when attempting to identify circulatingEPC in human subjects. Nonetheless, no directevidence was presented in the original manu-script that isolated and purified human circulat-ing CD34þKDRþ CD133þ cells directly par-ticipate in generating the endothelial liningof the implanted left ventricular device orwhether the cells attached to the device werehematopoietic or endothelial in origin. Becausethese antigens are all known to be expressed byhematopoietic stem and early progenitor cells,one would have liked to see the comparativeenrichments of the putative EPCs comparedto the potentially contaminating hematopoieticsubsets that may have been present.

Although CD34, CD133, and/or KDRexpression have been used to identify humancirculating EPCs in thousands of papers since2000, and many of the papers have reportedstatistically significant correlations between theblood concentration of the selected putativeEPC subset and a disease state, few haveattempted to formally compare the functionalproperties of isolated human circulatingCD34þKDRþ CD133þ cells in hematopoieticand endothelial assays (reviewed in Alaiti et al.2010). In one instance, Case et al. (2007)reported that purified CD34þKDRþCD133þ

cells were highly enriched in hematopoietic pro-genitor activity but did not give rise to anyendothelial colonies in vitro. Perhaps not

surprising given all the rationale in the abovestudies, more than 99% of the hematopoieticprogenitor CD34þKDRþ CD133þ cells coex-pressed CD45, the common leukocyte antigen(this antigen is not expressed in endothelial cellseven at the mRNA level). Timmermans et al.(2007) have also reported that colonies of endo-thelial cells that display high proliferative poten-tial are derived only from a human cord bloodor bone marrow CD34þCD452 population ofcells and not from CD34þCD45þ cells (whichwere enriched for hematopoietic colony form-ing cells). Thus, several labs have independentlydetermined that the putative EPCs expressingCD34, CD133, and KDR that express the CD45antigen are hematopoietic cells with colonyforming activity and fail to give rise to endothe-lial cells during in vitro culture or directly formblood vessels in vitro or in vivo (Fig. 2).

The question of whether a cell expressingCD45 from adult human peripheral bloodcould represent an EPC has been controversialfor some time and some of this controversycan be attributable to the underlying assump-tions of the flow cytometric approaches used.Significant changes have occurred in both thehardware and software used in flow cytometryrate event analysis over the past decade (Her-zenberg et al. 2006; Parks et al. 2006; Dudaet al. 2007a). Newer digital machines withadvanced resolution and data storage capabil-ities permit the identification of up to 20 dis-tinct cellular parameters (Perfetto et al. 2006).Inherent in the ability to discriminate such aplethora of biologic data are improvements inthe analytical software that are required toreduce spectral overlap by applying software-generated postacquisition compensation (basedon compensation bead controls) and the use ofbi-exponential scaling to properly visualizeevents below and above the zero axis (De Rosaand Roederer 2001; Parks et al. 2006). Theseadvances, termed polychromatic flow cytome-try (PFC), have permitted discovery of novelrare cell subsets, further characterized the func-tionality of established cell populations, andeven discovered gross errors in phenotyping cellswhen compared to conventional flow cytometrypractices (reviewed in Abdul-Salam et al. 2005;

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Herzenberg et al. 2006; Parks et al. 2006; Tunget al. 2007). Although numerous publicationshave proposed methods for resolution of circu-lating EPCs from circulating endothelial cells orother blood cell elements (Asahara et al. 1997;Gehling et al. 2000; Mancuso et al. 2001, 2009;Bertolini et al. 2003, 2006; Willett et al. 2004,2005; Abdul-Salam et al. 2005; Khan et al.2005; Goon et al. 2006; Duda et al. 2007b; VanCraenenbroeck et al. 2008; Widemann et al.2008), several recent publications have appliedPFC to the identification of putative EPCs andhave resolved the subsets into proangiogenichematopoietic cells (Fig. 3) and circulatingendothelial colony–forming cells (Estes et al.2010a,b; Schmidt-Lucke et al. 2010). Despitethe use of advanced PFC techniques, to demon-strate that nearly all CD133þCD34þKDRþ cellsare CD45þ and thus by definition hemato-poietic, the field will only become clarified onidentification of novel cell surface moleculesthat unambiguously identify the EPCs (seebelow for proposed definition of an EPC).

The final method to identify a human circu-lating EPC is based on colony forming ability ofthe plated MNC in vitro. The original descrip-tion of cluster-forming cells appearing within5 days of plating CD34þ cells as a putativeEPC characteristic (Asahara et al. 1997) wasexpanded on by Ito and colleagues who also iso-lated and plated blood cells on fibronectin-

coated dishes (Ito et al. 1999). One day laterthe nonadherent cells were removed andreplated onto fibronectin-coated dishes, andthe number of clusters that emerged at 7 daysof replating was used to indicate the numberof putative EPCs. The rationale for preplatingthe MNC for 24 h was to remove any mono-cytes, macrophages, or circulating mature endo-thelial cells in the MNC fraction that couldcontaminate the putative EPC assay system.Although laudable in intent, the failure toshow that all hematopoietic or endothelial ele-ments were depleted by the preplating stepdiminished the impact of the improved meth-odology. Hill et al. (2003) further modifiedthe EPC cluster assay, by preplating blood cellsfor 48 h, then replating the nonadherent cellsto quantify the emergence of the EPC-derivedcolonies. This assay has been commercializedand the putative EPC (that produce the progenythat form the colony) have been referred to ascolony forming unit-Hill (CFU-Hill; Fig. 1).The CFU-Hill assay has been used to demon-strate a significant inverse correlation betweenthe circulating CFU-Hill concentration andFramingham cardiovascular risk score inhuman subjects (Hill et al. 2003). Subsequenttranscriptome, proteomic, and functional anal-yses have determined that CFU-Hill are moreclosely related to human hematopoietic cellsthan to primary endothelial cells.

CB +++ mPB +++ ECFC

Figure 2. Formation of capillary-like structures in Matrigel coated plates. Photomicrographs (20� magnifica-tion) of freshly sorted cord blood (CB)– or mobilized peripheral blood (mPB)–derived CD34þAC133þ

VEGFR2þ cells and early passage endothelial colony–forming cells (ECFCs) plated over Matrigel. The triplepositive CB- and mPB-derived cells failed to form capillary-like structures, whereas the ECFC formed numerouslumenized structures. (Figure adapted from Case 2007; reprinted, with permission, from Elsevier # 2007.)

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Another assay system identifies outgrowthendothelial cells (OECs) possessing clonalendothelial colony–forming cell (ECFC) abilitywithin 1–3 wk of culture, when blood cells areplated on matrix coated dishes with addedgrowth factors (Gulati et al. 2003, 2004; Bom-pais et al. 2004; Hur et al. 2004; Ingram et al.

2004, 2005; Yoon et al. 2005; Guven et al.2006; Shepherd et al. 2006; Melero-Martinet al. 2007; Nagano et al. 2007; Timmermanset al. 2007; Au et al. 2008). A hierarchy of clonalproliferative potential is displayed by the ECFCwith some colonies growing to more than10,000 progeny from a single cell plated 14

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Figure 3. Frequency analysis of CD31þCD34brightCD45dimAC133þ cells using two distinct methods of analysis.In the first strategy (A–D), manually compensated data collected on a digital flow cytometer were visualized onplots with logarithmic scaling. Mononuclear cells (MNCs) (red gate in A) were identified on a forward and sidescatter (FSC/SSC) plot and subgated onto a bivariant antigen plot to identify CD34brightAC133þ cells (dark bluegate in B). CD34brightAC133þ cells were further gated to identify the CD45dim subpopulation (light blue gate inC). CD31 expression on the resulting CD34brightAC133þ CD45dim cells was confirmed on a CD31 histogram(D). In this first strategy (A–D), gate boundaries were set using Boolean gating and negative isotype controls.In the second strategy (E–I), uncompensated data was collected on a digital flow cytometer, compensated afteracquisition by using software, and visualized in plots with biexponential scaling (linear and logarithmic). MNCs(red gate in A) were identified on a FSC/SSC plot and then CD142 cells (orange gate in E) were identified. AllCD142 events were then assessed for viability (ViViD) and glycophorin A (GlyA) (F). The CD142GlyA2

ViViD2 (pink gate in F) were subgated onto a bivariant antigen plot to identify CD142GlyA2ViViD2

CD34brightAC133þ cells (dark blue gate in G). CD142GlyA2ViViD2CD34brightAC133þ cells were furthersubgated to identify the CD45dim subpopulation (light blue gate in H ). CD31 expression was confirmed on aCD31 histogram. In the second approach (E–I), fluorescence minus one gating controls were used to setgate boundaries. (Figure adapted from Estes et al. 2010; reprinted, with permission, from John Wiley & Sons# 2010.)

Human Endothelial Progenitor Cells

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days earlier (Ingram et al. 2004, 2005). The cordblood ECFC display high telomerase activityand vigorous in vivo human vessel formationwhen suspended in a matrix and implantedinto immunodeficient mice (Ingram et al.2004). The ability of these ECFCs to displayspontaneous vasculogenic properties, to inte-grate long-term into the systemic vasculatureof the host animal, and to remodel into arteriesand veins in vivo distinguishes this EPC fromall other types of cells that have been giventhis term (Bompais et al. 2004; Gulati et al.2004; Hur et al. 2004; Ingram et al. 2004,2005; Yoon et al. 2005; Guven et al. 2006; Shep-herd et al. 2006; Melero-Martin et al. 2007;Nagano et al. 2007; Timmermans et al. 2007;Au et al. 2008).

Although the general consensus purportsthat endothelial cell turnover in systemic bloodvessels is low in adult subjects, ample evidencehas also been presented to suggest that theendothelium in some blood vessels is easilydetectable (Wright 1968, 1971, 1972; Caplanand Schwartz 1973; Schwartz and Benditt1976; Kunz et al. 1978; Taylor and Lewis1986). In fact, in young experimental animals,endothelial replication rates have been reportedas high as 60% in certain focal areas within theaorta. Experimental injury to the aortic endo-thelium (direct denudation) or disorders suchas hypertension, hyperlipidemia, and endotox-emia all lead to an increase in endothelial repli-cation in rodent models (Wright 1968, 1971,1972; Caplan and Schwartz 1973; Schwartzand Benditt 1976; Kunz et al. 1978; Taylor andLewis 1986). Most evidence suggests that theproliferating cells are retained in the endothelialintimal layer; however, some circulating endo-thelial cells thought to be sloughed from thevascular endothelium may also possess prolifer-ative potential. Because human adult aorta andcord blood artery and vein endothelial cells havebeen determined to possess the clonal hierarch-ical proliferative potential similar to the circu-lating ECFCs derived from cord blood andadult peripheral blood (Ingram et al. 2005), itis plausible that the circulating ECFCs may bederived from vascular endothelium. Only fur-ther study will permit a detailed clarification

of the relationship between resident and circu-lating ECFCs.

ROLE OF EPCs IN VARIOUS HUMANCLINICAL DISORDERS

A summary of the role of EPCs in human dis-ease is complicated by the fact that so many dif-ferent EPC definitions have been used. In manyinstances, circulating EPC concentrations havebeen enumerated and correlated to a diseasestate in an effort to serve as a biomarker for dis-ease detection or staging (Alaiti et al. 2010). Insome cases, the functional role of the EPCshas been elucidated when the cells were infusedas a reparative therapy (Loomans et al. 2004).Defects in EPC function have also been identi-fied in some patients with diabetes and poten-tial therapies to restore certain aspects of EPCfunction have been proposed (Fadini et al.2007). Despite the ambiguity in fully character-izing an EPC, numerous clinical trials have beenconducted in patients with heart disease, diabe-tes, peripheral arterial disease, pulmonarydisease, and cancer in which putative EPCshave been examined as a biomarker or used asa cell therapy to treat human subjects (seewww.clinicaltrials.gov).

DEFINING A HUMAN EPC

A growing consensus is emerging that thereare many circulating blood cells that participatein the process of new blood vessel formationand vascular repair. Controversy persists as towhether cells that display numerous features ofthe hematopoietic lineage but participate innew blood vessel formation should be calledan EPC or not. If the term EPC is reserved fora progenitor cell for the endothelial lineage,we would propose that there are fundamentalproperties that this cell should display: a circu-lating cell that gives rise to progeny displayingclonal proliferative potential and differentiationrestricted to the endothelial lineage, ability toform lumenized capillary-like tubes in vitro(cells must display cytoplasmic vacuolationcapacity), and ability to form stable humanblood vessels (cells must secrete a basement

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membrane) when implanted into tissues (withor without a scaffold) that become an integratedpart of the host circulatory system and displaypotential to undergo remodeling to form theintima of arterial, venous, and capillary struc-tures. At present, the rare circulating ECFCsdisplay these features while most other bonemarrow–derived cells currently called EPCsfail to do so (Hirschi et al. 2008). Finding aunique cell surface marker that would permitprospective isolation and enrichment of cellsdisplaying the above activities would certainlyclarify the EPC identity and must remain a focusfor the field.

ACKNOWLEDGMENTS

We would like to thank Tiffany Lewallen for herexpert administrative assistance in preparationof the article. This work was supported in partby funds from the Riley Children’s Foundation.The author has no competing financial intereststo disclose.

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September 28, 20112012; doi: 10.1101/cshperspect.a006692 originally published onlineCold Spring Harb Perspect Med 

 Mervin C. Yoder Human Endothelial Progenitor Cells

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