Isolation and characterization of endothelial progenitor cells

1457Development 125, 1457-1468 (1998)Printed in Great Britain © The Company of Biologists Limited 1998DEV6304

Isolation and characterization of endothelial progenitor cells from mouse

embryos

Antonis K. Hatzopoulos 1, Judah Folkman 2, Eliza Vasile 1,*, Grant K. Eiselen 1 and Robert D. Rosenberg 1,†

1MIT, Department of Biology, Cambridge MA 02139, USA2Children’s Hospital, Harvard Medical School, Boston MA 02115, USA*Present address: Department of Pathology, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston MA 02115†Author for correspondence (e-mail: [email protected])

Accepted 12 August 1997; published on WWW 18 March 1998

The cardiovascular system develops early in embryogenesisfrom cells of mesodermal origin. To study the molecularand cellular processes underlying this transition, we haveisolated mesodermal cells from murine embryos at E7.5with characteristic properties of endothelial progenitors byusing a combination of stromal cell layers and growthconditions. The isolated embryonic cells displayedunlimited stem-cell-like growth potential and a stablephenotype in culture. RNA analysis revealed that theembryonic cells express the endothelial-specific genes tie-2and thrombomodulin(TM) as well as the early mesodermalmarker fgf-3. The GSL I-B4 isolectin, a marker of earlyendothelial cells, specifically binds to the isolated cells. Thein vitro differentiation with retinoic acid and cAMP led toa 5- to 10-fold induction of flk-1, von Willebrand Factor(vWF), TM, GATA-4 and GATA-6. Electron microscopyrevealed that in vitro differentiation is associated withincreased amounts of rER and Golgi, and a dramatic

increase in secretory vesicles packed with vWF. Whencultured in Matrigel, the embryonic cells assume thecharacteristic endothelial cobblestone morphology andform tubes. Injection into chicken embryos showedincorporation of the embryonic cells in the endocardiumand the brain vasculature. The expression of TM, tie-2,GATA-4 and GATA-6 suggests that the isolated embryonicendothelial cell progenitors are derived from the proximallateral mesoderm where the pre-endocardial tubes form.The properties of the endothelial cell progenitors describedhere provide a novel approach to analyze mediators,signaling pathways and transcriptional control in earlyvascular development.

Key words: Endothelial cell progenitor, Vasculogenesis,Endocardium, Mouse, Cardiovascular system, tie-2, thrombomodulin(TM)

SUMMARY

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INTRODUCTION

The vascular system arises early in embryogenesis to meenutritional needs of the developing organism (Risau, 19Wilting et al., 1995; Baldwin, 1996; Wilting and Christ, 1996At E7.5 in the mouse, the extraembryonic mesodermal cellthe yolk sac aggregate into clusters that constitute the instage of blood island formation. Shortly thereafter, bloislands differentiate into an external layer of endothelial ceand an inner core of blood cells (Sabin, 1920). At about same time, embryonic cells of the proximal lateral mesodeassemble into pre-endocardial tubes that connect anteriorlgenerate cardiac endocardium around the anterior intesportal (Sabin, 1917; DeRuiter et al., 1992). Posteriorangioblasts organize into the paired dorsal aortae that lassemble in the midline to form a single tube (Jolly, 1940).parallel fashion, mesodermal cells in the allantois genercords that give rise to the umbilical blood vessels. The allanexpands rapidly inside the exocoelomic cavity and eventuafuses with the chorionic extraembryonic mesoderm. Tfusion triggers differentiation of the chorionic vasculature th

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indirectly connects with the maternal vascular system of tplacenta. These early events are described by the tevasculogenesis, which signifies de novo formation of bloovessels from angiogenic precursor cells (Risau, 1991). Laterembryogenesis, the vascular tree grows by sprouting, cdivision, migration and assembly of endothelial cells derivefrom pre-existing vessels through a process termangiogenesis (Noden, 1989; Risau and Flamme, 1995).

Classical embryological investigations have sought ascertain the relationship between the different sites of origof embryonic blood vessels. According to His (1868), botintraembryonic and extraembryonic vessels are derived frothe yolk sac vasculature, whereas Rabl (1889) suggested all intraembryonic vessels sprout from the endocardiumSubsequently, Hahn (1909), Reagan (1915) and Sabin (19postulated that both somatic and splanchnic mesoderm posangiogenic potential. More recently, transplantatioexperiments in chicken/quail chimeras (Le Douarin, 1969) astaining of embryos with endothelial-specific markers havestablished that most areas of early mesoderm contendothelial cell precursors or angioblasts (Pardanaud et

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1987, 1989; Coffin and Poole, 1988; Noden, 1988; Poole aCoffin, 1989; Coffin et al., 1991; Pardanaud and DieterleLièvre, 1993).

These morphological investigations have been complemenby recent molecular studies in which genes have been identthat are expressed in endothelial cell progenitors and embryendothelial cells. These genes encode the tyrosine kinreceptors flk-1, flt-1, flt-4, tie-1 and tie-2 (for a review anreferences on endothelial-specific tyrosine kinases, Mustonen and Alitalo, 1995), the thrombin receptthrombomodulin (TM; Weiler-Guettler et al., 1996), thtranscription factors GATA-4 as well as GATA-6 (Kelley et a1993; Laverriere et al., 1994), and the adhesive protein Willebrand Factor (vWF; Coffin et al., 1991). Flk-1 and flt-1 areceptors for vascular endothelial growth factor (VEGF; Breet al., 1992; Terman et al., 1992; Millauer et al., 1993), whertie-2 is a receptor for angiopoietin-1 (Davis et al., 1996). Ttemporal and spatial patterns of expression of the above gdiffer greatly during development. In the extraembryonmesoderm, which gives rise to blood islands, flk-1 appearsshortly after gastrulation followed sequentially by tie-2 and tie-1 over the next 24 hours (Dumont et al., 1995). While flk-1 isinitially observed in extraembryonic mesoderm prior to expression in embryonic mesoderm (Eichman et al., 19Yamaguchi et al., 1993; Flamme et al., 1995), TM is first detectedintraembryonically in the proximal lateral mesoderm where pendocardial tubes form (Weiler-Guettler et al., 1996). From Eon, TM is ubiquitously expressed in endothelial cells throughoextraembryonic and embryonic tissues. The transcription facGATA-4 and GATA-6 are detected at about the same time ain the same location as TM, but are subsequently absent fother embryonic or extraembryonic blood vessels (Arceci et 1993; Kelley et al., 1993; Heikinheimo et al., 1994; Laverrieet al., 1994; Jiang and Evans, 1996; Morrisey et al., 1996). expression of the vWFgene occurs in the same location as Tand tie-2but is delayed by about 24 hours and is eventually noin most extraembryonic and embryonic blood vessels (Dumet al., 1992).

Most of the genes discussed above have been inactivatehomologous recombination in mice. A common feature these knockouts is embryonic lethality due to abnormendothelial development. However, the correspondiphenotypes are distinctive. The flk-1mutation affects bothearly endothelial development and hemopoiesis wendothelial cell precursors unable to undergo extraembryoor intraembryonic vasculogenesis (Shalaby et al., 1995). the contrary, tie-2and angiopoietin-1null embryos exhibitsevere defects in angiogenesis that appear later in develop(Dumont et al., 1994; Sato et al., 1995; Suri et al., 199Trabeculation in the heart is impaired and vasculature in bras well as yolk sac remains homogeneous in size withundergoing reorganization to generate small and large vesThe flt-1 knockout mice display defects in the assembly endothelial cells into vascular channels (Fong et al., 199while tie-1 appears to be required for proper maintenance aintegrity of the vascular system (Puri et al., 1995; Sato et 1995). In similar fashion, the VEGF knockout disrupts bloodvessel formation (Carmeliet et al., 1996; Ferrara et al., 199Interestingly, mice heterozygous for VEGF exhibit aberrantvasculogenesis implying that ligand dose gradients are crufor development of the vascular system. It is noteworthy t

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TM inactivation also leads to embryonic lethality at E8suggesting that endothelial-specific functions such thromboresistance may be critical for proper embryondevelopment (Healy et al., 1995).

The investigations described above have uncovered existence of distinct networks of gene expression intraembryonic and extraembryonic vasculogenesis. Althouthe ligands and receptors defined by these studies play a crrole in the developing vascular system, the precise intracellevents and altered cellular properties involved in endothecell differentiation remain obscure. We have establishedpotentially complementary approach for analyzing tmolecular basis of vascular development that involvharvesting of endothelial cell progenitors which, through vitro differentiation, can be used to define interactions that tplace in vivo. In this communication, we describe the isolatiand culture of early mesodermal embryonic cells that haproperties of endothelial cell progenitors with unlimited, stecell-like growth. RNA and protein analyses demonstrate tthe progenitor cells express a subset of mesodermal and eendothelial cell markers. In vitro differentiation leads to dramatic induction of endothelial-specific genes and morphological change to the mature endothelial cphenotype. Transplantation experiments show that the isolaprogenitor cells incorporate into the vasculature duriembryonic development. These endothelial cell progenitmay allow investigation of mediators, signaling pathways aaltered cellular properties that are important in early vascudevelopment.

MATERIALS AND METHODS

Isolation of mouse embryonic endothelial progenitor cellsMouse embryos were isolated at E7.5 to E7.8 of development. midday of the vaginal plug was considered as day 0.5 post coitThe egg cylinders with adjacent yolk sacs from 15-30 embryos wwashed twice in phosphate-buffered saline (1× PBS; 100 mM NaCl,4.5 mM KCl, 3 mM Na2HPO4, 3 mM KH2PO4) and incubated for 10-20 minutes with 0.05-0.1% trypsin/0.53 mM EDTA.4Na (LifeTechnologies). Dissociated cells were plated on γ-irradiated (3000rads) mouse embryonic fibroblasts (MEFs) in ES culture medi(ESCM 20%) containing 20% heat-inactivated serum (55°C minutes; Intergen), 0.1 mM β-mercaptoethanol, 1 mM MEM non-essential amino acids (Life Technologies), 100 u/ml penicillin and 1µg/ml streptomycin (Life Technologies), 2 mM L-glutamine (LifTechnologies) and 2 mM Hepes pH 7.5. Cells were grown in tisculture incubators at 37°C and 5% CO2. Within 3-4 days, cell coloniesof various morphologies emerged. When the above cells wsimilarly isolated from mouse embryos in which the TMgene wasreplaced by lacZ (β-galactosidase-producing gene; Weiler-Guettler al., 1996), one type of colonies stained intensely blue with X-gThese lacZ-positive colonies with round cell morphology werisolated using cloning rings and propagated separately on MEFs. Atwo passages on MEFs, the round cells could be maintained on 0gelatin-coated plates in the absence of feeder layers. In all subseqstudies, endothelial progenitor cells were employed that had bextensively passaged on 0.1% gelatin-coated plates.

In vitro differentiation of embryonic endothelial cellprogenitorsIn vitro differentiation of embryonic endothelial cell progenitors wainduced by addition of 1 µM all-trans retinoic acid (RA; Sigma) and

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0.5 mM dibutyryl cyclic AMP (c-AMP; Sigma). RNA was isolatedand antibody staining was performed following 4 days of treatmwith RA and cAMP. For culture in Matrigel basement membramatrix (Beckton Dickinson), endothelial progenitor cells cultured ogelatin-coated plates were resuspended in Matrigel, plated on 6-plates and placed at 37°C until Matrigel polymerized. SubsequenESCM 20% was added and cultures observed for over 14 days.

RNA analysisRNA was isolated using the RNA isolation kit from Stratagene. Tfollowing plasmids were linearized and used as templates to geneantisense RNA probes with Ambion’s MAXIscript kit; pTMPST674(TM), pVEGF-1, pvWF, p421l1 (flk-1), pflt-1, pfgfa-a1, pfgfb-b1pint-2c.28RI (fgf-3), pfgf5-5.3, pfgfk-k2, pkx10-32 (β-actin),pKFKF/PGK-Oct-3, pSK75 (brachyury), pAFPHB (α-fetoprotein),pPst232 (evx 1), pcM1x1.2Bam/BglIIA (Nkx-2.5), pGATA-1,pGATA-2, pGATA-4, pGATA-6 and p4A22.FR8 (cripto). RNaseprotections were conducted with Ambion’s RPA II kit following thmanufacturer’s specifications except that overnight (O/hybridizations were carried out at 55-60°C. Products were resolve6% denaturing polyacrylamide gels. Gels were dried aautoradiographed.

Lectin and antibody binding For the lectin staining, cells were grown in 8-chamber microscoslides. Chambers were washed twice with 1× PBS and fixed at 4°C inan 1:1 mixture of acetone:methanol for 5 minutes. Subsequently, cwere washed extensively with 1× PBS and processed for fluoresceinconjugated GSL I B4 binding (Vector Laboratories). Lectinconcentration was 20 µg/ml in 10 mM Hepes pH 7.5, 150 mM NaClAfter binding for 1-2 hours at 24°C, cells were washed extensivand analyzed under a fluorescent microscope. For control, parcultures were stained in the presence of 0.5 M galactose, a speinhibitor of GSL I B4 binding.

For detection of the von Willebrand Factor (vWF), an anti-humvWF rabbit polyclonal antibody (DAKO) that cross-reacts witmurine antigen was used. Cells were washed twice with 1× PBScontaining 1 mM MgCl2 and 0.1 mM CaCl2, fixed with 2%formaldehyde for 30 minutes, washed with 1× PBS and incubatedwith 1× PBS containing 5% fetal calf serum (FCS) for 30 minutes4°C to block non-specific binding. The cells were permeabilized w0.2% saponin, incubated at 37°C with the anti-human vWF antibo(3 µg/ml) for 1 hour, washed several times with 1× PBS containing1% albumin and then incubated with 1.5 µg/ml Cy3-conjugateddonkey anti-rabbit IgG (Jackson Immunoresearch Laboratories Inin 1× PBS containing 5% FCS for 1 hour. The samples were wasseveral times with 1× PBS containing 1% albumin, rinsed withdistilled water and mounted in Mowiol (Calbiochem). The cells weviewed with a Zeiss Axioplan microscope or with a Biorad MRC 60confocal microscope equipped with an argon krypton laser aphotographed using Kodak film.

Electron microscopyEndothelial progenitor cells were grown as described above immunofluorescent microscopy. The cells were then rapidly rinswith 1× phosphate buffer (PB), fixed with 2% glutaraldehyde in 0M sodium cacodylate-HCl pH 7.2 for 60-90 minutes at 4°C, postfixwith 2% osmium tetraoxide for 60 minutes at 4°C followed bdehydration in graded alcohol up to 70% and en block staining w70% alcoholic uranyl acetate for 30 minutes at 4°C. The samples wthen processed for Epon embedding (Vasile et al., 1983). Thin Esections were poststained with uranyl acetate and Reynold’s lcitrate and viewed with a JEOL model 1200CX electron microscooperated at 80 kV.

For immunoelectron microscopy (Brown and Farquhar, 1989), cewere rapidly rinsed with 1× PB and fixed with 2% formaldehyde, 0.05M lysine-HCl, 0.01 M sodium periodate in 0.75× PB. The cells were

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then incubated with 1× PB containing 5% normal goat serum for 30minutes at 24°C to block non-specific binding and permeabilized w0.2% saponin in the above solution. The cells were incubated wrabbit anti-human vWF antibody diluted to 3 µg/ml in PB containing1% albumin and 0.02% saponin O/N at 4°C, washed in the sabuffer and then incubated with 1.5 µg/ml goat anti-rabbit IgGconjugated to horse radish peroxidase at 24°C for 2 hours and finwashed in the same buffer. The cells were fixed with 3glutaraldehyde, incubated with 0.015% diamino benzamidine-Hand 0.01% H2O2 and processed for standard electron microscopy described above.

ELISA assayELISA assays were carried out to measure vWF secretion usreagents from DAKO. Endothelial progenitor cells before and aftstimulation with RA/cAMP for 4 days as described above weincubated O/N in a serum-free medium (ESCM) supplemented w1× Nutridoma SP (Boehringer Mannheim). The following day, thsupernatants were collected and centrifuged for 5 minutes at 1,revs/minute in a IEC model HN-SII table microfuge to removfloating cells. The supernatants were collected and processedELISA. The 96-well plates were coated with rabbit anti-human vWantibody at 10 µg/ml for several days at 4°C in 10 mM PB pH 7.2and 145 mM NaCl. The plates were washed extensively with 10 mPB pH 7.2, 0.5 M NaCl and 0.1% Tween 20 (Buffer B). Supernatandiluted 2-5 fold with buffer B were placed in the wells and alloweto bind O/N at 4°C. The wells were then washed extensively wbuffer B and the secondary peroxidase-conjugated rabbit anti-humvWF antibody was applied at 0.65 µg/ml in buffer B O/N at 4°C.Wells were extensively washed with buffer B and the chromogensubstrate (5 µl 30% H2O2 and 4 DAKOPATTS pellets in 12 ml 0.1 Mcitric acid-phosphate buffer pH 5.0) was applied for several hours24°C. When color was sufficiently intense, the reaction was stoppwith 1.5× volume of 1 M sulfuric acid and optical density wasmeasured in a DuPont microplate reader. For control, wells weincubated with either buffer B or ESCM that was incubated O/N 37°C without cells and processed in parallel. Samples were prepaand measured in quadruplicates.

Xenograft of mouse cells in chicken embryosMurine embryonic progenitor cells transfected with the PGKβ-geoconstruct were injected in chicken embryos at day 9 or 10 development (β-geo is a fusion between lacZ (β-galactosidase gene)and neomycin resistancegenes; Friedrich and Soriano, 1991). Murineembryonic fibroblasts, isolated from a ROSA gene-trap mouse lthat ubiquitously expresses the lacZgene, were used as a control. Thtwo cell types displayed similar intensity of blue color when stainewith X-gal. 150,000-350,000 cells in 3-7 µl were injected into anextraembryonic vein. The chicken embryos were isolated 1, 3 odays later, dissected and frozen on dry ice. Subsequently, frozen tiswas mounted and cryosectioned. Sections were stored at −80°C untiluse. For X-gal staining, sections were fixed at 4°C in 1:1 acetomethanol for 5 minutes and washed twice for 10 minutes with 1× PBS.After fixation, sections were stained O/N at 37°C in a staining solutiof 1× PBS containing 0.02% NP40, 0.01% SDS, 2 mM MgCl2, 5 mMpotassium ferricyanide, 5 mM potassium ferrocyanide and 1 mg/X-gal (Boehringer Mannheim). After staining, sections were washtwice for 10 minutes with 1× PBS and photographed.

For antibody staining, cryosections of chicken embryos injectwith murine cells were removed from −80°C, air dried for 10-20minutes and fixed at 4°C in 1:1 acetone methanol for 5 minutes. Sliwere then washed twice for 10 minutes in 1× PBS and blocked with1% BSA and 0.05% saponin at 24°C for 2-3 hours. Subsequenprimary antibodies were added after dilution in blocking buffer. Thrabbit polyclonal anti-human vWF antibody from DAKO and a ramonoclonal anti-mouse TM antibody were used at 3 µg/ml and 30ng/ml respectively. Staining was O/N at 4°C. The slides were th

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washed twice for 10 minutes in 1× PBS and secondary antibodiewere applied in blocking buffer for 2-3 hours at 24°C. The secondantibodies were fluorescein (DTAF)-conjugated donkey anti-rabIgG (H+L) at 3.75 µg/ml and Cy3-conjugated donkey anti-rat IgG(H+L) at 2.5 µg/ml (both from Jackson Immunoresearch LaboratoriInc.). The slides were washed twice for 10 minutes in 1× PBS,mounted with Vectashield (Vector Laboratories) and examined uneither fluorescent or confocal microscopes.

RESULTS

Isolation of cells from early mouse embryosWe sought to isolate and grow endothelial cell progenitors frmurine embryos at a stage when the first signs of vascdevelopment are detected. Our approach was to identify cfrom trypsin-dissociated E7.5-E7.8 embryos, confirm thidentity with cell-type- and stage-specific genetic markerswell as characteristic morphological properties and th

Fig. 1. (A) Mouse embryos were isolated between E7.5 and E7.8.stained with X-gal. The decidua, trophectoderm, parietal endoderline including the ectoplacental cone and chorion, were removed.placed on γ-irradiated mouse embryonic fibroblasts as a feeder laymorphologies were observed. Colonies of distinct morphologic typcell morphology isolated from E7.5-E7.8 TMlacZ murine embryos anE7.8 wild-type mouse embryos after transfer to gelatin-coated pla

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establish conditions for their maintenance in culture. Initiattempts to grow cells directly on plates coated with substrasuch as laminin, gelatin and collagen failed. The addition the growth media of various combinations of aFGF, bFGTGFβ, PDGF, LIF and VEGF also proved insufficient tosupport the proliferation of murine cells from early embryoHowever, after incomplete trypsinization, cells weroccasionally noted attached to the plate. This observatraised the possibility that a feeder or a stromal cell layer wrequired to provide an appropriate substratum for sustained proliferation. Indeed, in subsequent experiments, dissociaE7.5-E7.8 embryos were plated on γ-irradiated mouseembryonic fibroblasts and displayed a robust cell growth standard tissue culture medium containing 20% serum.

The experimental scheme for this procedure is depictedFig. 1A employing murine embryos in which the lacZgene hadbeen targeted into the TM locus (Weiler-Guettler et al., 1996).At E7.5, X-gal staining demonstrates that expression confined to the proximal lateral mesoderm where the fi

The photograph at the upper right corner shows an E7.5 TMlacZembryom and Reichert’s membrane, as well as the tissues above the indicated broken The remaining egg cylinder with the adjacent yolk sac were trypsinized ander. Within a week, two types of colonies of round and fibroblastoide were separated and grown individually. (B) Picture of a colony of round

d stained with X-gal. (C) Endothelial cell progenitors isolated from E7.5-tes.


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Fig. 2. GSL I-B4 isolectin strongly binds to the isolated endothelialcell progenitors (upper panel). Binding is completely blocked by 0.M galactose, a specific inhibitor of GSL I-B4 binding (lower panel).

intraembryonic endothelial cell progenitors originate (insephotograph). TMlacZembryos were isolated at E7.5-E7.8, anthe entire decidua with trophoblast cells, ectoplacental coparietal endoderm and Reichert’s membrane was removedmicrodissection. The remaining egg cylinders with the adjacyolk sacs were trypsinized and plated on γ-irradiated mouseembryonic fibroblasts. Within 3 to 4 days, manmorphologically distinct colonies were observed. After aboa week, only round cells and fibroblastoid cells remained.

The round cell colonies derived from TMlacZ mice stainedintensely blue after X-gal treatment, indicating originatiofrom proximal lateral mesoderm (Fig. 1B). The round ccolonies were isolated using cloning rings and growseparately. Although the initial isolation required the use feeder layers, once cultures were established, cell growth cbe sustained on gelatin-coated plates. The morphology ofcloned round cells is shown in Fig. 1C.

Employing the isolation protocol described above, simiresults have been obtained with other murine strains includC57BL, 129/J and BALBc. The round cells show unlimitegrowth potential, which is an unusual property of primacultures. At the present time, the initially cloned round cehave been continuously propagated in culture for 3 yewithout diminished growth or phenotypic changes.

The isolated round cells express early endothelialcell markers In earlier investigations of vascular development, the GSL I4isolectin has been demonstrated to bind to endotheprogenitors and mature endothelial cells between E7.5

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The above observations strengthen the notion that these cmight represent murine embryonic endothelial cell progenitoTo further examine this possibility, we investigated the genexpression profile of these cells by RNase protection asswith embryonic stem (ES) cells as a control. For thesexperiments, 25 genes were selected for study that wmembers of the following groups: (1) endothelial-specifigenes expressed early in development, (2) genes exprespostgastrulation in lateral and primitive streak mesodermendoderm and ectoderm, and (3) genes expressed in lineages that develop in close association with endothelcells, i.e., myocardium and blood.

Fig. 3 shows representative data for a selected set of gewith RNase protection assays. The endothelial progenitoexpress high levels of endothelial cell markers such as tie-2andTM as well as the early mesodermal marker fgf-3(int-2;Wilkinson et al., 1988). These cells also exhibit small amounof VEGFmRNA as well as trace quantities of vWFmRNA, butpossess no detectable flk-1 mRNA or flt-1mRNA. Finally, theendothelial cell progenitors show no expression of mesodermmarkers associated with primitive streak and axial mesodesuch as brachyury(Wilkinson et al., 1990) and evx 1 (Bastianand Gruss, 1990), the endodermal marker α-fetoprotein(Tilghman, 1985), the ectodermal marker fgf-5 (Hébert et al.,1991), early myocardial markers such as Nkx-2.5(Lints et al.,1993), MLC-2a (Kubalak et al., 1994) and cripto (Dono et al.,1993), early blood markers like GATA-1 (Pevny et al., 1991),GATA-2(Tsai et al., 1994), embryonic or adult globins or othegenes including fgf-1, fgf-2, kfgf (Hébert et al., 1990) andoct3/4(Schöler et al., 1990).

In vitro differentiation of endothelial cell progenitorsInvestigations mainly in Xenopusembryos have identifiedgrowth factors and other agents that regulate formation adifferentiation of early mesoderm. These components inclumembers of the FGF and TGFβfamilies, which induceventralization, and retinoic acid (RA) and noggin, whicinduce dorsalization (Slack, 1994). Based upon these daendothelial cell progenitors were exposed in culture to TGFβ,bFGF or RA together with cyclic AMP (RA/cAMP) to testwhether these factors might influence growth and/or indudifferentiation. These experiments demonstrated that TGFβ1inhibits cell growth without affecting cell morphology or thepattern of gene expression, except for a moderate increasVEGFmRNA (data not shown). These results also showed thbFGF had no obvious effect other than slightly enhancing taction of TGFβ1 (not shown). In contrast, exposure toRA/cAMP for 4 days led to dramatic morphological changeThe cells became flat, elongated and assumed an endothcell-like shape. RNA was isolated from RA/cAMP-treated celas well as untreated cells, and RNase protection assays wcarried out using the same battery of genes described aboIn addition, two newly identified members of the GATA familyGATA-4and GATA-6were included in these studies becausboth genes are expressed early in pre-endocardial tubes.

We observed a dramatic elevation in the levels ofvWF and

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Fig. 3. RNase protectionanalysis of gene expression inisolated endothelial cellprogenitors. The probe used ineach experiment is indicated tothe left. Asterisks markprotected fragments. The firsttwo lanes of each panelrepresent incubation of the probewith yeast RNA in the absence(−) or presence (+) of RNases.ES, RNA from Embryonic Stemcells; MEEPs, RNA from mouseembryonic endothelialprogenitors. In all cases, 10 µgof total RNA was incubated withabout 50-100,000 cts/minute of32P radioactively-labeledantisense RNA probes. β-actinwas used as a control to monitorRNA levels in each lane.

TM mRNAs upon treatment with RA/cAMP (Fig. 4)Interestingly, the expression of flk-1was activated whereas thaof tie-2 was unaffected. The existing low levels of GATA-4aswell as GATA-6were also greatly increased by exposure RA/cAMP. Normalized for β-actin, the quantitativemeasurements demonstrate elevated message levels of abfold for GATA-4, 8-fold for vWF, TMas well as GATA-6, andmore than 10-fold for flk-1. The expression of GATA-4, GATA-6 and TM in uninduced endothelial cell progenitors suggeorigination from the proximal lateral mesoderm where pendocardial tubes arise. The lack of expression of flk-1 in theuninduced cells implies that the isolated cells may be dist

Fig. 4. RNase protection assays with RNA isolated from murineendothelial cell progenitors exposed to retinoic acid and cAMP. Thprobes used in each experiment are outlined at the top of each paUN, RNA from untreated endothelial cell progenitors; RC, RNAfrom RA and cAMP-treated cells. In all cases, 10 µg of total RNAwas incubated with about 50-100,000 cts/minute of 32Pradioactively-labeled antisense RNA probes. β-actinwas used as acontrol to monitor RNA levels in each lane.

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from extraembryonic ventrolateral mesoderm, which gives rto blood islands. The presence of fgf-3mRNA substantiates theprimitive nature of endothelial cell progenitors as comparedmature endothelial cells, which do not possess this markTable 1 contains a list of the genes analyzed with specprobes in the RNase protection assays.

Analysis of vWF gene induction The induction of vWF gene expression constitutes an earmolecular marker of endothelial cell development (Coffin al., 1991). The biosynthesis, processing and secretion of

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Table 1. A list of the genes analyzed by RNase protectionassays in the undifferentiated and differentiated murine

endothelial cell progenitors Expressed or induced genes† Non-expressed genes

flk-1* flt-1tie-2 GATA-2Thrombomodulin* GATA-1von Willebrand Factor* globinsGSL I B4 ligand(s) Nkx-2.5GATA-4* criptoGATA-6* MLC-2afgf-3 (int-2) Oct 3/4VEGF a-fetoprotein

aFGF (fgf-1)bFGF (fgf-2)fgf-5kfgfbrachyuryevx 1

†The expression levels of genes marked with an asterisk are increased upondifferentiation.


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adhesive protein represents a characteristic property of maendothelial cells (Jaffe et al., 1973; Wagner and Marder, 198To evaluate whether endothelial cell progenitors are ablecarry out the full sequence of events, differentiated aundifferentiated cells were stained with a rabbit anti-humvWF antibody that recognizes the murine form of the adhesprotein. As shown in Fig. 5A, vWF protein accumulates in tcytoplasm of differentiated cells. There is strong perinuclestaining indicative of rough endoplasmic reticulum (rER) well as the staining of large granules dispersed throughoutwhole cytoplasm.

The cytoplasmic localization of vWF protein was furthedefined using electron microscopy (EM). In the untreated cesubcellular organelles associated with the secretory pathwi.e. rER and the Golgi apparatus, are scarce suggesting very little secretion takes place. Instead, a large number of ribosomes are present (Fig. 5B, left panel). Followindifferentiation, a remarkable increase is observed in number of both rER and Golgi cisternae documenting a sto the secretory phenotype (Fig. 5B, right panel). A horsradish-peroxidase-linked secondary antibody revealed presence of vWF protein in large vesicles that appear readysecretion. Although additional intracellular sites were staineWeibel-Palade bodies were not conclusively identified (Wagnet al., 1982). The appearance of large secretory vesicindicated that the differentiated cells secrete vWF protein. Tsurmise was verified by measuring the levels of vWF in tmedia of RA/cAMP-treated and untreated cells. As shownFig. 5C, a marked increase in the amount of secreted vprotein was documented, substantiating the transition towafunctional endothelial cell phenotype. In vitro labeling wit[35S]methionine and [35S]cysteine, followed byimmunoprecipitation and SDS-PAGE demonstrated that vWprotein was correctly processed with removal of thprepeptides and propeptides (data not shown).

Isolated round cells assume endothelial morphologyin MatrigelThe characteristics of endothelial cell progenitors were aassessed by culture in Matrigel, an extracellular matbasement membrane material that can be used to promdifferentiation of endothelial cells (Grant et al., 1991). Ashown in Fig. 6, the cells gradually lose their round appearaand assume the characteristic cobblestone morphologymature endothelial cells. Further incubation in Matrigproduced tube-like structures, a property of mature endothecells. These results provide additional support for the notthat the round cells represent endothelial cell progenitors.

The isolated endothelial cell progenitors incorporateinto the embryonic vasculatureTransplantation experiments are difficult to perform in earmouse embryos because of the small size and surrounthick decidual tissue. Chicken embryos are receptive xenografts at early stages of development until a functiothymus has been formed and are easily accessible transplantation studies. For these reasons, the developmepotential of the isolated cells was examined by injection inthe extraembryonic veins of chicken embryos between daand 10 of development. The endothelial cell progenitoemployed for the injection experiments were previous

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transfected with a plasmid carrying the PGKβ−geoconstruct.After G418 selection, several neomycin-resistant colonies wisolated and RNA prepared. RNA analysis using most of tprobes described above demonstrated that each colexhibited the same expression profile as the wild-type isolacells normalized to β-actin (data not shown). Theseobservations showed that isolated endothelial progenitrepresent a homogeneous population and provided an eadetected marker to identify areas engrafted with murine ce

After 3-4 days following injection, chicken embryos werisolated, frozen and cryosectioned. The sections were fixed stained with X-gal to locate murine cells. The analysis showthat large numbers of endothelial cell progenitors had survivin the chicken embryos and that the majority of murine cewere present in the developing heart, brain and liver (nshown). The location and relationship of endothelial ceprogenitors to the host chicken endothelium was analyzusing two endothelial-specific antibodies: (1) an anti-mouTM rat monoclonal antibody that recognizes only mouse bnot chicken antigen in conjunction with a secondary Cyconjugated donkey anti-rat antibody (red color), and (2) tpreviously mentioned rabbit polyclonal anti-human vWantibody that recognizes both mouse and chicken antigenassociation with a fluorescein-conjugated donkey anti-rabsecondary antibody (green color). The cryosections were texamined for antibody staining by confocal microscopy shown in Fig. 7. The endothelial cell progenitors are preswithin trabeculae in the ventricles of the developing heawhere murine cells became part of the host endocardium (F7A-C). Indeed, chicken myocardium is occasionalcompletely surrounded by murine cells. Many murine cells aalso found around the developing retina, between the sensand pigment layers, an area that harbors host endothelial c(Fig. 7F). In certain cases, endothelial cell progenitors aintegrated into the brain microvasculature (Fig. 7D,E). Murincells in the liver did not appear to associate with endothecell sinusoids. No incorporation of progenitor cells was notin the extraembryonic membranes or in large, well-establishintraembryonic vessels. The experiment was repeated sevtimes using different cell clones with identical results. Acontrols, similar numbers of murine embryonic fibroblasexpressing lacZ were injected at the same stage odevelopment. Early time points (1 day) revealed the preseof fibroblasts throughout the embryo and the circulatiowhereas later points (4 days) showed no localization to specorgans and no incorporation into chick heart or brain. Theresults demonstrate that the isolated round cells exhibit potential to form vasculature and support the conclusion tthese cells represent an early progenitor cell.

DISCUSSION

During the past few years, considerable progress has bmade in elucidating the molecular basis of vasculdevelopment. Specific tyrosine kinase receptors have bshown to be expressed in endothelial cell precursoInactivation of these genes and their corresponding ligandshomologous recombination has revealed the crucimportance of these signaling systems in the developmeassembly and maintenance of the vascular system (Musto

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Fig. 5. Analysis of von Willebrand Factor induction. (A) Confocal microscopy of undifferentiated (UN) and RA/cAMP differentiated cells (RC)stained with a rabbit anti-human vWF polyclonal antibody. The secondary antibody is a donkey anti-rabbit IgG conjugated to Cy3. (B) Electronmicroscopy of undifferentiated (UN) and differentiated endothelial cell progenitors (RC). After differentiation, a dramatic increase of rER(arrow) and Golgi cisternae number in the cytoplasm indicates a transition to a secretory cell type (N, nucleus). Electron microscopy wasperformed following vWF antibody binding coupled to secondary anti-rabbit antibody-conjugated to horse radish peroxidase. The colorreaction (shown as dark gray staining) reveals that vWF is found in large secretory vesicles (arrowheads). Bars are 1 µm. The insert shows anarea of secretory vesicles containing vWF magnified 1.7× compared to the right panel. (C) The immunoassay of vWF in differentiated andundifferentiated endothelial cell progenitor tissue culture supernatants reveals increased secreted protein upon differentiation.

and Alitalo 1995). Moreover, components of the extracellumatrix and their cellular receptors such as fibronectin aintegrins are also indispensable to the formation of blovessels (George et al., 1993; Yang et al., 1993, 1995). Howeit remains to be established if a single or multiple endothecell precursors exist for different vascular beds. It is aunclear whether a common set or multiple sets of molecusignals are required to induce differentiation of endothelial cprogenitors in the yolk sac, allantois and endocardial tubes, if the various endothelial cell-specific signaling systems plthe same role at each site. The nature of molecular interactthat establish the structural and functional diversity endothelium as well as the linkage between endothelial development and hemopoiesis remain to be defined.

We have isolated and maintained in culture embryonendothelial cell progenitors that arise on E7.5 to E7.8 priorthe formation of the cardiovascular system. The procedudeveloped to obtain cell populations with the characteristicsendothelial progenitors are simple as well as reproducible generate homogenous cell populations with unlimited grow

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properties. The homogeneous nature of endothelial cprogenitors was established by isolation/expansion of singcolonies obtained with cloning rings, and the analyses of geexpression in multiple independent clones followintransfection with neomycin-resistant plasmids. The stem-celike unlimited growth of endothelial cell progenitors is shareby only one other primary culture system, the embryonic stecells.

The isolated progenitor cells express high levels of eaendothelial cell-specific genes, such as TM and tie-2, and stainwith the GSL I B4 lectin, a marker for endothelial cells andtheir precursors (Coffin et al., 1991; Schnurch and Risau, 19Weiler-Guettler et al., 1996). These cell populations alsexpress genes of early mesoderm such as fgf3, but not thspecific for primitive streak mesoderm, notochord, endoderectoderm, extraembryonic mesoderm or myocardium. Tendothelial cell progenitors undergo in vitro differentiatiowith a dramatic induction of flk-1, vWF, GATA-4, GATA-6andTM genes. Differentiation is accompanied by a transition to tmature endothelial cell phenotype with the correct process


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Fig. 6. The isolated endothelial cell progenitors wereplaced in Matrigel, a substratum that enhancesdifferentiation of endothelial cells. (A) 2 days inculture, (B) 3 days in culture, (C) 5 days in culture and(D) 8 days in culture. The pictures show that theendothelial cell progenitors progressively lose theirround shape, assume the characteristic cobblestonemorphology of mature endothelial cells and form tube-like structures.

Fig. 7. Injection of murine endothelial cellprogenitors into the extraembryonic veins of chickenembryos. The presence of murine cells in variousorgans was documented by confocalimmunofluorescence microscopy on antibody stainedcryosections. Chicken endothelial cells are markedwith a primary rabbit polyclonal anti-vWF antibodyin association with a secondary fluorescein-conjugated donkey anti-rabbit IgG (green color).Murine cells are stained with a rat anti-TMmonoclonal antibody in conjunction with a secondaryCy3-conjugated donkey anti-rat antibody (red color).Computer-assisted superposition graphically revealsthe incorporation of murine cells in the hostcirculatory system (yellow color). (A-C) Heart. Thesame section is stained with anti-vWF (A) and anti-TM (B) antibodies; computer-assisted superpositionof A and B is shown in C (L, lumen; M, myocardium;bar is 40 µm). (D-F) Brain. In D and E, computer-assisted superposition of anti-vWF and anti-TMstaining demonstrates incorporation of murine cells inthe brain vasculature; bars are 20 µm. A large numberof murine cells were present around the developingeye as shown in F; bar is 30 µm.

and secretion of vWF. The endothelial cell progenitors whplaced in Matrigel assume the characteristic cobblestmorphology of mature endothelial cells and assemble in tulike structures. Finally, even after prolonged culture, the abcloned cell population retains the potential to contribute to developing vascular system as shown by xenogrtransplantation in chicken embryos.

Given that the isolated progenitor cells possess the X-

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reaction product when derived from TMlacZmice and expressGATA-4 as well as GATA-6, it appears likely that this cellpopulation originates from the proximal lateral mesoderm. previous studies of embryonic development, two patternsvasculogenesis have been described (Noden, 198Intraembryonic round cells have been observed that assemlocally to form endothelium in the midline of the embry(Dumont et al., 1992). A mesenchymal fibroblast-like cell fro

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the splanchnopleure or ventral mesoderm has been identthat migrates widely and forms vascular cords (Jolly, 194We presume that endothelial cell progenitors isolated in tstudy represent the first type, i.e., cells that assemble to fpre-endocardial tubes and eventually differentiate inendocardium, dorsal aorta and other portions of the cenvascular system. In the light of the above discussion, extensive incorporation of endothelial cell progenitors in ttrabeculae of the developing chicken heart may reflect affinity for myocardium and/or the presence of the tie-2 liganangiopoietin-1, in this tissue. It is also interesting to note taccumulation of injected cells around the developing eye mibe explained by the recent observation that angiopoietin-1also highly expressed in neighboring epithelium (Davis et 1996).

A second endothelial progenitor cell type might then responsible for generating blood islands. Consistent with tnotion, we have isolated from E7.5 to E7.8 embryosfibroblastoid cell population that responds to bFGF aexpresses endothelial markers including flk-1 and flt-1, as wellas scavenger receptor that leads to acetylated Low DenLipoprotein uptake (ac-LDL; unpublished data). Thembryonic cell population resembles early endothelprecursors isolated from quail blastodiscs with regard to thmorphology, ac-LDL uptake and dependence on bFGF growth (Flamme and Risau, 1992). It is noteworthy thtransplantation experiments in chicken/quail chimeras suppthe existence of two distinct populations of endothelial cprogenitors; one that contributes to formation of the doraorta and dorsal vessels and the other that contributes to vevessels and also gives rise to hematopoietic precurs(Pardanaud et al., 1996).

The absence of flk-1 expression in endothelial cellprogenitors with induction of the gene upon differentiation vitro is somewhat surprising. It is possible that flk-1expressionin endothelial cell progenitors is maintained in vivo by aexogenous factor and thus lost upon isolation. Alternativelyis conceivable that, unlike blood islands, the expression of 1 in endocardial tubes occurs later than other endothelial cspecific genes. From published reports and our own whomount in situ data with flk-1 probes, this later possibilityappears reasonable because widespread expression ofreceptor gene in the intraembryonic mesoderm obscuresearly endocardial tube expression. Indeed, the stainingparallel sections of E7.8 embryos with antibodies against Tand flk-1 reveal that TMexpression precedes flk-1 expressionin endocardial tubes (data not shown).

While this manuscript was in preparation, the isolation putative human endothelial cell progenitors was reported fradult human blood (Asahara et al., 1997). In contrast to thfindings, the endothelial cell progenitors described in tpresent study could only be isolated from E7.5 to E7embryos. Employing identical conditions of isolation, we hafailed to obtain the same cells from older embryos or adtissues. At present, the relationship between the two progencell types is unclear. Differences exist in morphology apatterns of gene expression. The isolated adult endothelialprogenitors are spindle shaped, express both flk-1 and thave a finite life span in vitro and appear to differentiate to cepositive for ac-LDL uptake. Thus, these progenitors resemthe cells dissociated from quail blastodiscs (Flamme and Ris

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1992). The adult progenitor cells isolated from blood integrainto sites of active angiogenesis after venous injection. similar fashion, it has been previously shown that humumbilical vein endothelial cells can also incorporate arounangiogenic sites after injection (Ojeifo et al., 1995). It will bof great interest to compare the behavior of the embryonendothelial cell progenitors in response to angiogenic signin adult pathological situations (Folkman, 1995).

The endothelial cell progenitors isolated in this investigatioshould provide a new tool for elucidating regulatory events vasculogenesis. The ease of isolation, unlimited growwithout detectable phenotypic changes, feasibility of genemanipulation and in vivo developmental potential oprogenitors constitute major advantages of this approach. are currently searching for signals, besides RA/cAMP, thinduce genes like flk-1and vWFand hope to determine whethersuch factors have comparable functions in vivo. In this regasignals of this type are known to originate from the anteriintestinal portal endoderm that induce both endocardial- amyocardial-specific genes in isolated mesodermal cells (Sand Markwald 1996). The endothelial cell precursors can employed in differential screens to identify new genes that ainduced or suppressed after differentiation in vitro and similchanges can then be sought in vivo. It should also be possto isolate progenitors from mice with knockouts of endothelicell-specific receptors which cause later embryonic lethalitthe behavior of these genetically altered cells can be thassessed by a combination of cell culture and transplantatexperiments. The above studies should help to define regulation of vasculogenesis and shed light on angiogemechanisms activated in adult pathological situations affectithe vascular system, e.g., tumor-induced angiogenesis, diabretinopathy, psoriasis, myocardial infarction and endothelcell growth during tissue remodeling.

We are greatly indebted to many of our colleagues for sendingcDNA probes and antibodies for analysis: Dr S. Kennel for the Tantibody; Dr B. G. Hermann for the brachyurycDNA, Dr D. Dumontfor flk-1 and tie-2, Drs G. Breier and W. Risau for flt-1 and VEGF,Drs G. Martin and U. Deutch for fgf1, fgf2, fgf3, kfgfand fgf5, Dr S.H. Orkin for GATA-1 and GATA-2, Dr M. S. Parmacek for GATA-4and GATA-6, Drs P. Gruss and H. Bastian for evx 1, Dr P. Soriano forβ-geo, Drs K. Chien and S. Kubalak for MLC-2a, Dr R. P. Harvey forNkx-2.5, Dr M. Persico for cripto, Dr H. Schöler for oct3/4and DrTilghman for α-fetoprotein. We would like to thank Drs D. Fischmanand T. Mikawa for helpful comments and suggestions, Dr H. Raybufor advice on animal husbandry, Dr Cesario Bianchi for his help aadvice on the lectin and antibody stainings, Dr S. Chatterji and Smith for their help in using the confocal microscope, and J. Jacksand Mrs C. Battlefield for preparing chicken embryos and cells fmicroinjection. We would like to thank Drs M. Houssain, W. Aird, MKrieger and E. W. Knapik for insightful comments on the manuscripThis work was supported by HL 41434.

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