Development 102, 471-478 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

471

Vasculogenesis and angiogenesis in embryonic-stem-cell-derived

embryoid bodies

WERNER RISAU1, HANNU SARIOLA2*, HANS-GUNTER ZERWES1, JOACHIM SASSE3,

PETER EKBLOM2, ROLF KEMLER2 t and THOMAS DOETSCHMAN2*1Max-Planck-Institut fiir Entwicklungsbiologie and 2Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, Spemannstrasse35-39, 7400 Tubingen, FRG3Shriners Hospital, Immunology section, 12502 North Pine Drive, Tampa, FI 33612-4799, USA

* Present address: University of Helsinki, Department of Pathology, Haartmaninkatu 3, 00290 Helsinki, Finlandt Present address: Max-Planck-Institut fur Immunbiologie, Stiibeweg 51, 7800 Freiburg, FRG$ Present address: University of Wisconsin, 445 Henry Mall, Madison, WI 53706, USA

Summary

Embryonic stem cells (ESC) have been establishedpreviously from the inner cell mass cells of mouseblastocysts. In suspension culture, they spontaneouslydifferentiate to blood-island-containing cysticembryoid bodies (CEB). The development of bloodvessels from in situ differentiating endothelial cells ofblood islands, a process which we call vasculogenesis,was induced by injecting ESC into the peritonealcavity of syngeneic mice. In the peritoneum, fusion ofblood islands and formation of an in v/vo-like primarycapillary plexus occurred. Transplantation of ESCand ESC-derived complex and cystic embryoid bodies(ESC-CEB) onto the quail chorioallantoic membrane(CAM) induced an angiogenic response, which wasdirected by nonyolk sac endoderm structures. Neitheryolk sac endoderm from ESC-CEB nor normal mouseyolk sac tissue induced angiogenesis on the quailCAM. Extracts from ESC-CEB stimulated the pro-liferation of capillary endothelial cells in vitro. Mito-genic activity increased during in vitro culture and

differentiation of ESC. Almost all growth factoractivity was associated with the cells. The ESC-CEB-derived endothelial cell growth factor bound toheparin-sepharose. The identification of acidic fibro-blast growth factor (FGF) in heparin-sepharose-puri-fied material was accomplished by immunoblot exper-iments involving antibodies against acidic and basicFGF. We conclude that vasculogenesis, the develop-ment of blood vessels from in situ differentiatingendothelial cells, and angiogenesis, the sprouting ofcapillaries from preexisting vessels, are very earlyevents during embryogenesis which can be studiedusing ESC differentiating in vitro. Our results suggestthat vasculogenesis and angiogenesis are differentlyregulated.

Key words: growth factor, endothelium, blood island,haemangioblast, vasculogenesis, angiogenesis, embryonicstem cell, cystic embryoid body.

Introduction

The vascular system originates from blood islandswhich differentiate from the splanchnopleuric meso-derm very early during embryogenesis. Blood islandshave also been called haemangioblasts to indicate theclose relationship between blood cell precursor cells,which are found in the centre of blood islands, andendothelial cell precursors, which differentiate at theperiphery. Growth and fusion of the numerous blood

islands in the yolk sac give rise to a capillary networkwhich, after the onset of blood circulation, differen-tiates into a regular arteriovenous vascular system(Evans, 1909; Sabin, 1920; Romanoff, 1960; Haar &Ackerman, 1971; Wagner, 1980). Development in situof blood vessels also seems to take place inside theembryo. This has long been controversial, but thework of Reagan (1915), and more recent experimentsinvolving transplantation chimaeras (Dieterlen-

472 W. Risau and others

Lievre, 1975; Pardanaud et al. 1987) indicate thatblood vessels differentiate inside the embryo. Thesevessels are connected to the yolk sac by the vitelline(omphalomesenteric) arteries and veins.

There are differences between the development ofextraembryonic and intraembryonic blood vessels.First, intraembryonic in situ endothelial cell differen-tiation seems not to be as tightly coupled withhaemopoiesis as in the yolk sac haemangioblasts.Although haemopoietic foci have been found alongthe dorsal aorta and Dieterlen-Lievre & Martin(1981) have demonstrated a diffuse intraembryonichaemopoiesis in yolk sac chimaeras, intraembryonichaemangioblasts are apparently not as abundant as inthe yolk sac. Second, the localized regression ofdefined parts of the arterial and venous vascularsystem is unique to the development of intraembryo-nic blood vessels.

Very little is known about the molecular mechan-isms that regulate the growth and differentiation ofendothelial cells. In order to approach this questionwe find it useful to distinguish between the develop-ment of blood vessels from in situ differentiatingendothelial cells, which we call vasculogenesis, andthe development and sprouting of capillaries frompreexisting ones, which is defined as angiogenesis.We have studied the latter process in the brain andkidney, because transplantation experiments usingchick-quail chimaeras (LeDouarin, 1973) had pre-viously shown that the vascular system in these organsis derived from capillaries that invade the organrudiments (Stewart & Wiley, 1981; Ekblom et al.1982). Our data suggest that these tissues produceangiogenesis factors at a defined time of development(Risau, 1986; Risau & Ekblom, 1986). Subsequently,endothelial cells differentiate in response to the tissueenvironment to the specialized endothelium (Stewart& Wiley, 1981; Risau et al. 1986).

These studies have opened the possibility of inves-tigating embryonic angiogenesis during organogen-esis at cellular and molecular levels. A similar ap-proach to studying the very early phase ofmammalian vascular development has been ham-pered so far by the paucity of tissue available, the lackof molecular probes and the lack of in vitro modelsystems. We have therefore taken advantage of ourrecently established embryonic stem cells (ESC) sys-tem as an in vitro model system to study early vasculardevelopment. ESC have been cultured directly fromthe inner cell mass cells of mouse blastocysts(Doetschman et al. 1985). When kept on a feederlayer of embryonic fibroblasts, they proliferate but donot differentiate. After withdrawal from the feeder,they spontaneously differentiate in suspension cul-ture through a series of embryoid bodies of increasingcomplexity, culminating in blood island-containing

visceral yolk-sac-like cystic embyroid bodies (CEB;Doetschman et al. 1985). We here report that these invitro differentiated ESC possess the ability toundergo vasculogenesis and induce angiogenesis.

Materials and methods

ESCMouse blastocyst-derived ESC lines were established andmaintained in culture as described (Doetschman et al.1985). The ES-D3 cell line which developed the mostadvanced embryo-like structures was used throughout.Once withdrawn from the embryonic fibroblast feeder layerthe cells were cultured in suspension culture in Dulbecco'smodified eagles medium (DMEM) containing 15 % fetalcalf serum for the first 4 days and 20 % thereafter. Days ofculture refer to days after switching the cells to differen-tiation conditions.

Transplantation experimentsAggregates of ESC and ESC-derived CEB (ESC-CEB)were collected from culture dishes, washed in PBS andeither injected into syngeneic mice or pipetted onto a 7-day-old quail chorioallantoic membrane (CAM) as described(Sariola et al. 1983). In some experiments, yolk-sac struc-tures were dissected from the differentiated nonyolk sacendoderm and separately transplanted onto quail CAMs.Yolk-sac tissue from C57 black mouse embryos (10 daysold) was similarly transplanted. CAMs were scored 24, 48,72 h and 7 days after transplantation.

ImmunohistologyFrozen sections of CAM transplants were prepared andstained as described previously (Sariola et al. 1983). Anti-bodies against FVIII-related antigens were a kind gift of DrL. Hoyer (University of Connecticut; Hoyer, 1980). Mono-clonal antibodies MB1 directed against quail endothelialcells (Peault et al. 1983) were a generous gift of Drs N.LeDouarin and B. Peault (CNRS, Paris).

Growth factor characterizationThymidine incorporation into 3T3 cells and the prolifer-ation of bovine capillary endothelial cells (gift from DrJ. Folkman, Harvard Medical School, Boston) weremeasured as described previously (Risau, 1986). ESC-CEBat the desired stage of in vitro suspension culture werecollected by centrifugation, washed twice in ice-cold PBScontaining 1 mM-phenylmethylsulphonylfluoride (Sigma)and homogenized in the same solution either in an all-glassDounce homogenizer or by six brief (5 s) sonications.Usually twelve 100 mm Petri dishes each containing100-200 ESC-CEB were used for one experiment. Thehomogenate was centrifuged at 10 000 g for 30min and theresulting supernatant was tested directly in the proliferationassays or subjected to heparin sepharose chromatographyas described (Shing et al. 1984; Risau, 1986). Heparin-sepharose-purified material was separated on 13-17 %SDS-PAGE. Proteins were electrophoretically transferredto nitrocellulose filters and incubated with rabbit anti-

Embryonic stem cell vasculogenesis and angiogenesis 473

bovine fibroblast growth factor (FGF) peptide antibodies,diluted 1:1000 in PBS containing 0 1 % BSA. One anti-serum was directed against a basic FGF-specific aminoter-minal peptide and the other against an internal epitopewhich is conserved between basic and acidic FGF (Klags-brun et al. 1986). The biotin-streptavidin-peroxidase de-tection system (Amersham) was used to visualize boundantibodies. FGF purified from bovine brain was used as apositive control.

Results

Vasculogenesis in ESC-CEBESC spontaneously differentiate in suspension cul-ture to blood island-containing visceral yolk sac-likeCEBs (Doetschman et al. 1985). The development ofblood vessels from angioblasts (vasculogenesis) wasinduced by injecting 107 ESC into the peritonealcavity of syngeneic mice. 2-3 weeks after the injec-tion CEBs formed in addition to solid tumours. ESC-CEB in the peritoneum were found to be invested bya vascularized membrane derived from the host,which could be cleanly separated from CEBs. Fig. 1shows that, in these CEBs, blood islands have fusedgiving rise to an in v/vo-like primary capillary plexus.This was consistently observed in ESC-CEB grownintraperitoneally, but not in suspension culture.Blood cells in the capillary plexus were nucleated andproduced embryonic haemoglobins (data not shown,see Doetschman etal. 1985), thus demonstrating theirorigin from ESC rather than from the host. Adulthaemoglobin was also found in intraperitoneal CEBssuggesting that capillaries derived from the host hadinvaded the ESC-CEB (see below).

ESC-CEB induce angiogenesisTo analyse further the vascularization of ESC-derivedembryoid structures, we transplanted ESC aggregates

and ESC-CEB at different times of in vitro cultureonto the quail chorioallantoic membrane (CAM,embryonic day 8). Fig. 2A shows that CAM capillar-ies were strongly attracted and grew toward the graft.Embryoid bodies at the cyst formation stage (about10 days of suspension culture) showed the highestangiogenic response on the quail CAM. The normalvascular pattern of a CAM is shown in Fig. 2B forcomparison.

The angiogenic response was directed by the differ-entiated nonyolk sac endoderm structures (i.e. em-bryonic ectoderm and/or mesodermal derivativesthereof) found within the embryoid bodies and not bythe yolk sac endoderm. ESC-CEB-derived yolk sac

Fig. 1. Formation of capillary plexus in ESC-CEB grownin syngeneic mice. Bar, 100 ̂ m.

Fig. 2. ESC-CEB transplants induce angiogenesis on quail chorioallantoic membranes (A). Blood vessels convergeupon the transplants. The normal vascular pattern of a CAM is shown in (B). Bar, 1 mm.

474 W. Risau and others

Fig. 3. Vascularization of ESC transplants grafted onto the quail CAM. ESC aggregates cultured for 12 days insuspension culture were transplanted and cultivated for 7 days on a quail CAM (Fig. 3A). Note the cysts that were notpresent before grafting and the abundant capillary net of the graft. Bar, 100^m. Capillaries in the graft express FVIII-related antigen (B), and the quail MB1 antigen (C). v, Vessels. Bars, lO r̂n.

tissue and yolk sac tissue from a normal mouse(embryonic day 10) did not give rise to an angiogenicresponse when transplanted onto a CAM. Only 2 outof 40 transplanted yolk sacs showed a mild angiogenicresponse, which was probably due to an inflammatorystimulus rather than the yolk sac tissue (data notshown).

If ESC aggregates were cultured on the CAM for alonger period of time (up to 7 days), they developedinto CEBs like in vitro or in the peritoneum. Fig. 3Ashows an abundant capillary net in those grafts.Immunohistological analysis using antibodies againstFVIII-related antigen revealed the presence of trueendothelial cells in the capillaries (Fig. 3B). Some ofthem also expressed specific markers of quail endo-thelial cells thus demonstrating their origin from thequail host (Fig. 3C).

ESC-CEB produce a heparin-binding endothelialcell growth factorSince endothelial cell proliferation is a major eventduring angiogenesis we tested whether ESC-CEBproduced growth factors for capillary endothelialcells. Cell extracts from differentiating ESC at differ-ent days of in vitro culture all contained growth

factors, whose activity increased about twofold dur-ing in vitro culture. Almost no activity was found inthe culture medium (Fig. 4).

To characterize the growth factors, we preparedextracts from ESC-CEB grown in culture for 14 days.Growth factor activity for capillary endothelial cellsbound to heparin sepharose and eluted at 1-1-1-5 M-sodium chloride (Fig. 5). Other mitogens that stimu-late thymidine incorporation into 3T3 cells were alsopresent in these extracts. One mitogen bound toheparin sepharose and eluted at 0-5M-sodium chlor-ide, whereas others did not bind.

Since the FGFs belong to the group of heparin-binding growth factors, we tested whether the endo-thelial cell growth factor from ESC-CEB mightbe related to these growth factors. In immunoblots,the heparin-sepharose-purified ESC-CEB-derivedgrowth factor reacted with antibodies raised against apeptide whose amino acid sequence is present inacidic and basic FGF (Fig. 6, lane 2). It did not reactwith an antibody against a basic FGF-specific peptide(Fig. 6, lane 4).

Discussion

The early development of the vascular system is acomplex process which involves the simultaneous

Embryonic stem cell vasculogenesis and angiogenesis 475

regression and formation of small and large bloodvessels in the embryo. Although a fair amount ofmorphological work has been performed to describethe early steps of vascular development, no in vitrosystem has been available so far to dissect and

4 8 12Day of in vitro culture

16

Fig. 4. Production of an endothelial cell growth factor byESC-CEB during in vitro differentiation and growth.Extracts from ESC-CEB (closed circles) at the indicatedday of in vitro culture were tested in the capillaryendothelial cell proliferation assay as a dose response.One unit of growth factor activity was defined as theamount of material needed to stimulate the half-maximalproliferation of capillary endothelial cells. ESC-CEBconditioned media from the corresponding cultures weretested undiluted (open circles).

manipulate these processes and to generate sufficientmaterial for biochemical analyses.

ESC established directly from mouse blastocystsdifferentiate in vitro to visceral yolk-sac-like CEBs.Blood islands develop in these embryoid bodies in asimple culture medium suggesting that ESC them-selves produce all factors necessary for the inductionof angioblasts. We found that only factors present inhuman cord serum augmented the development ofblood islands in vitro, whereas other growth orhaemopoietic factors were inactive (Doetschman etal. 1985; unpublished results).

The first event in vasculogenesis in the developingembryo, the formation of a capillary plexus fromgrowing and fusing angioblasts, could only be ob-served in ESC-CEBs growing in the peritoneum or onthe CAM, but not in vitro. This suggests that ad-ditional factors are needed for the development of acapillary plexus from blood islands. The nature ofthese factors is presently unknown. The ESC systempromises to be a useful in vitro model system to studyfactors involved in the induction of angioblasts and invasculogenesis.

In the embryo, the development of blood vessels ismore complex than in the extraembryonic mem-branes. Our results show that the differentiated non-yolk sac endoderm cells induce angiogenesis in vivo,whereas yolk sac tissue did not. Similarily, Ausprunket al. (1975) reported that capillaries from the extra-embryonic CAM connected up with embryonic capil-laries if transplanted onto another CAM. Conversely,embryonic organs like the embryonic kidney induced

••• 3 010 30 40 50

Fraction number60 70

Fig. 5. Heparin sepharose chromatography of ESC-CEB extracts. Crude extracts were applied to heparin sepharosecolumns (Ix2cm) and eluted using linear 01-2M-sodium chloride gradients. Aliquots of the fractions were tested in the3T3 cell thymidine incorporation assay and capillary endothelial cell proliferation assay.

476 W. Risau and others

X1O~3

6 8 -

45-

36-

29-20-

14- . • - < & •

1

Fig. 6. Immunoblot of heparin sepharose purified ESC-CEB-derived endothelial cell growth factor. Proteins inheparin sepharose fractions containing mitogenic activityfor endothelial cells (lanes 2 and 4) were separated on13-17% SDS-PAGE along with purified bovine brainbasic FGF (lanes 1 and 3). Blots were tested with antiseraagainst synthetic peptides, one reacting with both basicand acidic FGF (lanes 1 and 2), while the other is specificfor basic FGF (lanes 3 and 4). The arrowhead indicatesan immunoreactive ESC-CEB-derived protein. Molecularweight markers are indicated at the left.

angiogenesis (Ausprunk et al. 1975; Auerbach et al.1976; Ekblom et al. 1982). Although there are otherpossible explanations for these differences, we wouldfavour the view that intraembryonic blood vesseldevelopment requires much more sprouting and bud-ding angiogenesis than extraembryonic vasculogen-esis. The relative paucity of haemopoietic bloodislands in the embryo also supports this hypothesis.Our results raise the possibility that more endothelialcells than was previously thought invade the veryearly embryo proper. In addition, implantation andplacenta development might be regulated by angio-genesis factors produced by embryonic stem cells.

We propose to distinguish between vasculogenesis,which we define as the development of blood vesselsfrom in situ differentiating endothelial cells, andangiogenesis, which is defined as sprouting of capil-laries from preexisting vessels, because the availablemorphological (Evans, 1909; Sabin, 1920; McLeod et

al. 1987; Pardanaud et al. 1987) and experimental(Ausprunk et al. 1975; Stewart & Wiley, 1981; Ek-blom et al. 1982; Risau et al. 1986; this report)evidence indicate that different mechanisms are in-volved in the development of capillaries during theseprocesses.

We found that differentiated ESC in culture pro-duced a growth factor for endothelial cells, which bybiochemical and immunological criteria, resemblesacidic FGF, a known angiogenesis factor (Thomas &Gimenez-Gallego, 1986; Folkman & Klagsbrun,1987). We also found growth factor activity in thecystic fluid of ESC-CEB (data not shown). However,the amount of material was insufficient for furthercharacterization. The association of growth factoractivity with cultured cells has been observed in manycells, normal and transformed (Klagsbrun et al. 1986;Schweigerer et al. 1987), and is usually attributed tothe lack of a signal sequence in both acidic and basicFGF mRNA (Jaye et al. 1986; Abraham et al. 1986a).It is therefore unclear how the factors reach theirtarget cells. Embryonic tissues and tumours induceangiogenesis if transplanted onto the CAM or in therabbit cornea 1 mm distant from the limbal vascularsystem. Thus, either the angiogenic FGFs are re-leased by as yet unknown mechanisms or the angio-genic factors released by angiogenic tissues are notFGFs. Heparin-binding growth factors released byPC13 carcinoma cells in culture have been isolated(Heath & Isacke, 1984; van Veggel et al. 1987).Factors, defined by oncogenes, homologous to theFGFs have recently been identified (Taira et al. 1987;Dickson & Peters, 1987; Delli Bovi etal. 1987). Theseor still other factors might also been involved inembryonic angiogenesis.

It is conceivable that FGF alone or in combinationwith other factors are responsible for angiogenesisinduction and progression. Using the 3T3 cell thymi-dine incorporation assay, we found growth factoractivity which did not bind to heparin sepharose andmight be related to the EGF family of growth factors.Another mitogen bound to heparin sepharose andeluted at 0-5 M-sodium chloride, which is indicative ofa PDGF-like growth factor (Shing et al. 1984). Bothof these mitogens did not significantly stimulateendothelial cell proliferation in vitro.

The FGFs are very potent growth and chemotacticfactors for endothelial cells and have been shown byseveral investigators to be angiogenic in in vivo modelsystems (Folkman & Klagsbrun, 1987). Their pres-ence in the earliest cells of the embryo thereforesuggests that they play an important role duringdevelopment. It is in this respect interesting thatAbraham et al. (19866) did not find stable mRNA forthe FGFs in adult tissues, although the active growthfactor can be isolated from adult tissues. It is possible

Embryonic stem cell vasculogenesis and angiogenesis 477

that transcription of their genes ceases soon afterembryonic development, but the growth factors arestored in the tissues.

In summary, we conclude that the earliest events ofvasculogenesis and angiogenesis during embryonicdevelopment can be analysed at cellular and molecu-lar levels using embryonic stem cell differentiation invitro. Both modes of blood vessel development areclearly separate and seem to be differently regulated.

We thank Anja Tuomi and Ursula Albrecht for skilfultechnical assistance and Drs L. Hoyer, N. LeDouarin andB. Peault for their generous gifts of antibodies.

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(Accepted 9 November 1987)