regulation of human embryonic stem cell differentiation by ...morphogenetic protein-2 (bmp-2) is a...

IntroductionThe development of human embryonic stem (ES) cells(Reubinoff et al., 2000; Thomson et al., 1998) has opened upexciting new opportunities for basic research and regenerativemedicine (reviewed by Pera et al., 2000). To exploit thepotential of human ES cells, it will be essential to understandthe molecular control of their growth and differentiation. Undercertain conditions in vitro, human ES cells differentiatespontaneously into a wide range of somatic and extra-embryonic cell types (Itskovitz-Eldor et al., 2000; Reubinoff etal., 2000). It is logical to assume that ES cell differentiation invitro mimics in a chaotic way the inductive events seen in theperi-implantation embryo in vivo. While our understandingof these processes in mammals is still incomplete, studiesof mouse development have identified several secretedpolypeptide factors which mediate critical events in cellularcommitment and development (reviewed by Beddington andRobertson, 1999).

Members of the transforming growth factor beta superfamilyplay a prominent role in driving cell commitment eventsin early development across the animal phyla. Bonemorphogenetic protein-2 (BMP-2) is a member of thetransforming growth factor beta superfamily implicated bygene ablation studies in several critical processes in earlymouse development (Ying and Zhao, 2001b; Zhang andBradley, 1996). Studies of mouse ES cell differentiation invitro adduced evidence for a critical role for BMP-2 in thedifferentiation of extra-embryonic endoderm, and in thecavitation of the embryo, the process whereby programmedcell death in a subpopulation of the pluripotent stem cells of

the inner cell mass leads to the formation of the egg cylinder(Coucouvanis and Martin, 1999). This study further showedthat BMP-2 transcripts were expressed in the extra-embryonicendoderm, a finding that has recently been confirmed in a studywhich defined a role for this molecule in the induction ofprimordial germ cells (Ying and Zhao, 2001b).

Before the development of human ES cells, humanembryonal carcinoma (EC) cells were used as a model to studycell differentiation in early human development. Human ECcells resemble primate ES cells in their morphology, surfacemarker and gene expression, and growth properties (Pera et al.,2000). A screen for factors affecting growth and differentiationof human pluripotent EC cells revealed that BMP-2 induceddifferentiation of the cells into a flattened, epithelial squamouscell displaying an immunophenotype and gene expressionprofile similar to extra-embryonic endoderm (Pera andHerszfeld, 1998). Treatment with retinoic acid had previouslybeen shown to induce EC cells to undergo a very similarprogram of differentiation (Roach et al., 1994), and cells withsimilar morphology and marker expression commonly appearin EC cell cultures undergoing spontaneous differentiation.Recently, Xu et al. (Xu et al., 2002b) reported that BMP-4induced the differentiation of human ES cells grown in serum-fee medium in the presence of FGF-2 into a different extra-embryonic lineage, the trophoblast.

Cells that morphologically resemble the flat epithelialcells induced in human EC cultures by BMP-2 also appearspontaneously in human ES cultures grown under standardconditions and are different in appearance to the cells describedby Xu et al. (Xu et al., 2002b); under suboptimal conditions

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Human embryonic stem cells differentiate spontaneously invitro into a range of cell types, and they frequently give riseto cells with the properties of extra-embryonic endoderm.We show here that endogenous signaling by bonemorphogenetic protein-2 controls the differentiation ofembryonic stem cells into this lineage. Treatment ofembryonic stem cell cultures with the bone morphogeneticprotein antagonist noggin blocks this form ofdifferentiation and induces the appearance of a novel celltype that can give rise to neural precursors. These findingsindicate that bone morphogenetic protein-2 controls a key

early commitment step in human embryonic stem celldifferentiation, and show that the conservation ofdevelopmental mechanisms at the cellular level can beexploited in this system – in this case, to provide a facileroute for the generation of neural precursors frompluripotent cells.

Key words: Human embryonic stem cell, Noggin, Bonemorphogenetic protein, Extra-embryonic endoderm, Neural,Differentiation

Summary

Regulation of human embryonic stem celldifferentiation by BMP-2 and its antagonist nogginMartin F. Pera 1,*, Jessica Andrade 1, Souheir Houssami 1, Benjamin Reubinoff 1,‡, Alan Trounson 1,Edouard G. Stanley 1, Dorien Ward-van Oostwaard ‡ and Christine Mummery 2,‡

1Monash Institute of Reproduction and Development, Monash University, 246 Clayton Road, Clayton, Victoria 3168, Australia2The Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands*Author for correspondence (e-mail: [email protected])‡Present address: Hadassah University Hospital, 91120 Jerusalem, Israel

Accepted 5 November 2003Journal of Cell Science 117, 1269-1280 Published by The Company of Biologists 2004doi:10.1242/jcs.00970

Research Article

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this differentiated cell grows rapidly and often overtakes theculture, leading to the elimination of stem cells and otherdifferentiated progeny. We speculated that the commitment toundertake primitive endoderm differentiation might be drivenby a positive feedback loop involving BMP-2, and thatmodulation of this pathway might facilitate ES stem cellrenewal or differentiation into embryonic lineages, assuggested by studies in the mouse (Lake et al., 2000; Niwa etal., 2000). In this study we sought to characterize this form ofdifferentiation and assess the role of BMP-2 in directing ESdifferentiation along this lineage.

Materials and MethodsCell culture and treatment with growth and differentiationfactorsHuman EC cells and ES cells were cultured as described in previouspublications (Pera et al., 1989; Reubinoff et al., 2000). Allexperiments were carried out at least twice on cell lines HES-2 andHES-3. The mouse embryo fibroblasts used throughout most of thisstudy came from two different lots, and they were plated at a densityof 7.5×104/cm2 for routine ES cell maintenance and experimentalprotocols. With one lot of embryo fibroblasts, BMP-2 effects wereseen in ES cells plated onto feeder cell layers at this density, but withthe second lot, effects were only apparent when ES cells were culturedon a feeder layer prepared at a lower density (see Results). Effects ofnoggin were apparent using either lot of feeder cells at the densitynormally employed for ES cell maintenance. Human EC celldifferentiation was induced by all-trans-retinoic acid or BMP-2treatment as described elsewhere (Pera and Herszfeld, 1998; Roach etal., 1994). Treatment of ES cells with noggin or bone morphogeneticproteins was begun one day following routine subculture andcontinued for 5-14 days. The medium used for conversion of nogginmonolayer cells to neural progenitors (neural progenitor medium) wasthe same as that used previously to grow neural progenitors fromspontaneously differentiating ES cell cultures (Reubinoff et al., 2001).

Recombinant human BMP-2, BMP-4 and recombinant mousenoggin were obtained from R&D Systems. Mammalian expressionplasmids containing DAN or Cerberus cDNA were describedelsewhere (Biben et al., 1998; Stanley et al., 1998). These proteinswere expressed in 293 T cells and purified by affinity chromatographyusing a FLAG affinity column.

The coding sequences of BMP-2, BMP-4 and BMP-7 wereamplified from a mouse embryonic day 7 cDNA library using a PCR-based approach. This process yielded two fragments for each of BMP-4 and -7, representing the pro-domain and cystine knot. For BMP-2,only the cystine knot region was amplified. The primers used togenerate these fragments were: BMP-4 Pro-domain (CATG-GCGCGCCTGATGATTCCTGGTAACCGAATG and ACGCGTC-TTGGGACTACGTTTGGCCCT), BMP-7 Pro-domain (ACGCGT-ATGCACGTGCGCTCGCTGCGCGCTG and ACGCGTCCAAA-GAACCAAGAGGCCCTG), BMP-2 cystine knot (GGGACGCG-TAAGCGCCTCAAGTCCAGCTGC and CAACGCGTTGCTGTGC-TAACGACACCCGCAG), BMP-4 cystine knot (CTGACGCGTAG-GAAGAAGAATAAGAACTGC and TCCGCCCTCCGGACTGC-CTGATCTC), BMP-7 cystine knot (ACGCGTCCAAAGAACCAA-GAGGCCCTG and GACGCGTGAAGAGCTAGTGGCAGC-CACAGG). MluI digested DNA fragments encompassing sequencesencoding the pro-domains of BMP-4 and BMP-7 were ligated into theAscI site of pEFBOS (Mizushima and Nagata, 1990) that had beenpreviously modified to include myc or glu/glu epitope tags.Recombinant plasmids resulting from this ligation contained the pro-domains N-terminal to the epitope tag. These plasmids were subse-quently digested withMluI and ligated to MluI fragments representingthe cystine knot regions of BMP4, 2 and 7 to yield a series of vectorsof the configuration BMP4-Pro-domain-myc-BMP-4, BMP-4-Pro-

domain-myc-BMP-2 and BMP-7-Pro-domain-glu-BMP-7. Westernblot analysis of supernatants from 293T cells transfected with thesevectors contained myc- and glu-tagged proteins of approximately 20kDa (data not shown), the predicted size of processed monomericBMPs. These supernatants also possessed BMP activity as adjudgedby their ability to induce alkaline phosphatase activity in C2C12 cells(Katagiri et al., 1994) (data not shown). To generate supernatantscontaining BMP-2/-7 heterodimers, 293T cells were transfected withthe vectors BMP-4-Pro-domain-myc-BMP-2 and BMP-7-Pro-domain-glu/glu-BMP-7, whereas supernatants containing BMPhomodimers were derived from cells transfected with only one of theabove expression vectors. Formation of heterodimers was confirmedby affinity copurification of myc- and glu-tagged proteins and by themuch higher biological activity of the heterodimers in the C2C12bioassay compared with homodimers. Purified follistatin (nativeprotein from follicular fluid) was obtained from David Phillips of thisInstitute.

Noggin-treated cells were harvested after 10-14 days using dispaseand were further subcultured on monolayers of mouse embryofibroblasts, as described for human ES cells, or under similarconditions in the absence of a feeder cell layer, or as neurospheres(Reubinoff et al., 2001). To determine the proportion of noggin orcontrol ES cell cultures that could be converted to neural progenitors,colonies were grown in control or noggin containing medium for 5days, dissected into pieces approximately 0.5 µm in diameter andtransferred to neural progenitor medium. After 1 week growth insuspension culture, the embryoid bodies or neurospheres werereplated as described previously (Reubinoff et al., 2001) on tolaminin-coated dishes in neural precursor medium lacking growthfactors, allowed to attach and grow for 2 days and then stained fornestin. The proportion of colonies showing uniform positive stainingfor nestin was determined by inspection and counting using indirectimmunofluorescence.

Gene expressionRNA isolation on magnetic oligo dT beads or on oligo dT Separosewas carried out as described elsewhere (Reubinoff et al., 2000; Roachet al., 1994). RT-PCR was carried out as described previously(Reubinoff et al., 2000). Product sizes, annealing temperatures andprimers for PCR reactions for all gene products examined in this studyare listed in Table 1. Thirty cycles of PCR were carried out for allreactions. All products were sequenced, and identity with the expectedhuman cDNA was confirmed in all cases. All RT-PCR reactions wererepeated on three separate occasions with consistent results.

Northern analysis of human EC cell RNA for BMP-2 transcriptswas carried out as described (Roach et al., 1994) using a randomprimed 32PcDNA probe consisting of a partial cDNA clonecorresponding to the region from 677-1333 bp of the cDNA sequencefor human BMP-2 (GI4557368).

Immunoblot analysis of BMP-2 expressionES cell cultures 7-14 days old containing stem cells and differentiatedcells were extracted in organ culture dishes in situ for 30 minutes intobuffer containing 150 mM NaCl, 50mM Tris and 1% Nonidet P-40,pH 8. The lysate, and the nuclei and cytoskeletal material detachedwith it, were removed from the dishes, and the remaining proteins onthe monolayer were harvested into 100-200 µl of SDS-PAGE samplebuffer, and 20 µl was added to each lane of a gel. Four to six organculture dishes were used in each experiment. The samples were runon 6% or 12% polyacrylamide gels under reducing and denaturingconditions, and the proteins were transferred to Immobilonmembranes, which were probed with mouse anti-BMP-2 (12% gel)or GCTM-2 (6% gel). Detection was carried out using rabbit anti-mouse immunoglobulin conjugated to horseradish peroxidase andenhanced chemiluminescence.

Journal of Cell Science 117 (7)

1271BMP-2 and noggin effects on human ES cells

Indirect immunofluorescenceThe sources and methods for indirect immunofluorescencemicroscopy with antibodies GCTM-2, anti-desmin and neuralmarkers, were as described previously (Reubinoff et al., 2000). Thesame methods were used with these antibodies: TG343, reactive withthe surface proteoglycan recognized by GCTM-2 and TRA1-60, fromthis laboratory (Cooper et al., 2002); TG42.1 reactive with a 25 kDasurface protein found on stem cells (this antigen copurifies with theGCTM-2 antigen and is identical to the tetraspannin CD9) (L. Stamp,A. Laslett and M.F.P., unpublished); Oct-4, mouse monoclonal againsta human Oct-4 sequence from Santa Cruz; rabbit antiserum againstSox-2 from Robin Lovell-Badge at the National Institute for MedicalResearch, London; GCTM-5 reactive with a surface protein found ondifferentiating cells of human ES and EC cultures and with fetalhepatocytes, from this laboratory (L. Stamp, H. A. Crosby, S. M.Hawes, A. J. Strain and M.F.P., unpublished); antibodies to Notch-1and Notch-2, from the laboratory of Professor Spiros Artavanis; anti-human BMP-2 from R&D Systems; antibody PHM-4, reactive withClass I HLA surface antigens, from the Department of Nephrology,

Monash Medical Centre (Hancock et al., 1982); mouse monoclonalH17 against placental alkaline phosphatase from Jose Luis Millan atthe Burnham Institute, La Jolla Ca.; mouse monoclonal antibodyagainst SPARC from Hematologic Technologies, Essex, Vermont.Assays for the beta subunit of human chorionic gonadotrophin wereperformed as described elsewhere (Reubinoff et al., 2000).

Quantitative analysis for GCTM-2 staining in control, BMP-2 andnoggin-treated cultures was carried out by harvesting colonies firstwith dispase, then dissociating them to single cells with trypsin. Cellswere stained in suspension using the primary antibody and anti-mouseimmunoglobulins conjugated to FITC, after which they were countedunder the fluorescence microscope to determine the proportion ofpositive cells. Five hundred cells from each group were counted.Alternately, when larger numbers of colonies were harvested, analysiswas carried out by flow cytometry (below).

For staining with antibodies to phosphorylated Smad1, ES cellsuspensions were plated onto glass coverslips coated with 0.1%gelatin, grown overnight then transferred to ES medium supplementedwith 0.5% FCS for 4 hours, after which 50 ng/ml BMP-2 was added

Table 1. Primers used for RT-PCRGene Primer sequence Product size (bp)Oct-4 OCT15: CGT TCT CTT TGG AAA GGT GTT

OCT26: ACA CTC GGA CCA CGT CTT TC 350

FoxD3 GENF480: GCA GAA GAA GCT GAC CCT GAGENR785: CTG TAA GCG CCG AAG CTC T

305

Cripto CRIP TOF484 : CAG AAC CTG CTG CCT GAA TGCRIP TOR668: GTA GAA ATG CCT GAG GAA ACG

185

AFP AFPF736: CCA TGT ACA TGA GCA CTG TTGAFPR1173: CTC CAA TAA CTC CTG GTA TCC

338

Sox17 SOXF17: CGC ACG GAA TTT GAA CAG TASOXR198: GGA TCA GGG ACC TGT CAC AC

181

HNF3-α HNF3- α F1939: GAG TTT ACA GGC TTG TGG CAHNF3- α R2328: GAG GGC AAT TCC TGA GGA TT

390

HNF4 HNF4F: GCT TGG TTC TCG TTG AGT GGHNF4R: CAG GAG CTT ATA GGG CTC AGA C

762

GATA4 GATA4F: CTC TAC CAC AAG ATG AAC GGGATA4R: AGT GTG CTC GTG CTG AAG

750

GATA6 GATA6F: GCC TCA CTC CAC TCG TGT CTGATA6R: TCA GAT CAG CCA CAC AAT ATG A

541

SPARC SPARCF: CTG CAG GGA GTG GAT TTA GAT CAC AAGSPARCR: CTG CAG ACC ATG AGG GCC TGG ATC

700

Transferrin TRFF1197: CTG ACC TCA CCT GGG ACA ATTRFR1765: CCA TCA AGG CAC AGC AAC TC

367

Vitronectin VNF34: TTG CAG GCA CTC AGC TAG AAVNR336: TGT TCA TGG ACA GTG GCA TT

300

BMPR-IA BMP2RI AF: GGA CAT TGC TTT GCC ATC ATABMP2RI AR: CAG ACC CAC TAC CAG AAC TTT

424

BMPR-2 BMP2RII F: TCC TCT CAT CAG CCA TTT GTC CTT TCBMP2RII R: AGT TAC TAC ACA TTC TTC ATA G

457

Activin receptor IIB Activin RII BF: CCG GGA TCC TAC GGC CAT GTG GAC ATC CAActivin RII BR: CCG CTC GAG ATG CAG GTA TGA GAG GCC TCG TGA

407

Gremlin Grem259F: AAT ACC TGA AGC GAG ACT GGTGrem500R: TTC TTG GTA GGT GGC TGT AG

241

Chordin ChdF3: AAC ACA TGC TTC TTC GAG GChdR3: CTG TGG TTC CCA GAG GTA GTG

900-1 kb (60°C)

Noggin NOGF1029: CTC GGG GGC CAC TAC GACNOGR1059: GCA CGA GCA CTT GCA CTC G

488 (60°C)

Actin ACTI NFOR: CGC ACC ACT GGC ATT GTC ATACTI NBAC: TTC TCC TTG ATG TCA CGC AC

200

Nestin Nes t 856F: CAG CTG GCG CAC CTC AAG ATGNes t 1064R: AGG GAA GTT GGG CTC AGG ACT GG

208

Pax6 PX6F1368: AAC AGA CAC AGC CCT CAC AAA CAPX6R1642: CGG GAA CTT GAA CTG GAA CTG AC

274

Brachyury Br achyF: GTG ACC AAG AAC GGC AGG AGGBr achyR: TGT TCC GAT GAG CAT AGG GGC

706 (60°C)

The PCR conditions were as follows: 94°C/4 minutes, 94°C/1 minute, 55°C/1 minute (except where specified otherwise), 72°C/1minute, 72°C/7 minutes.

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for 1.5 hours. Cells were fixed in 2% paraformaldehyde, rinsed threetimes in PBS, permeabilized in 0.1% Triton, rinsed again, thenincubated in anti-phosphorylated-Smad1 (1:50) (Persson et al.,1998)or GCTM-2 (1:10) in PBS with 4% normal goat serum for 1hour at room temp. Secondary antibodies (goat anti-rabbit-cy3-IgG1:250 and goat anti-mouse IgM-FITC 1:100, respectively) were addedfor 1 hour after washing three times in PBS/0.05% Tween. Coverslipswere mounted in Mowiol and viewed in a confocal laser scanningmicroscope.

Flow cytometryControl cells or cells treated with 25 ng/ml BMP-2 were harvestedafter 5 days of treatment following subculture. The cells werecarefully trypsinized to yield single cell suspensions, which wereincubated with monoclonal antibody GCTM-2 or an isotype-matchedcontrol antibody on ice for 30 minutes, followed by several washes inphosphate buffered saline, then a 30 minute incubation with anti-mouse immunoglobulins conjugated to fluorescein isothiocyanate.The cells were rinsed with phosphate buffered saline and fixed in 0.4%paraformaldehyde before analysis. Cells were also incubated withisotype matched control primary antibody followed by secondaryantibody.

ResultsBMP-2 treatment of human ES cellsBMP-2 treatment of human ES cell cultures grown in mediumcontaining fetal calf serum in the presence of a feeder cell layerinduced differentiation into flat, squamous epithelial cells (Fig.1A, control; B, treated cells); these cells sometimes formedfluid-filled cysts within the culture dish. They were very similarin appearance to those commonly observed during spontaneousdifferentiation of human ES cells (Fig. 1C). The appearance ofa sheet of spontaneously differentiating cells that was scrapedfrom the culture surface and processed using routinehistological methods is shown in Fig. 1D (BMP-treated cellshad a similar appearance). The flattened cells with abundanteosinophilic intracellular material resembled cells found in theprimary yolk sac of the primate embryo and in certainmorphological variants of yolk sac carcinomas; in the presenceof BMP-2 the entire culture eventually took on this appearance.By contrast, under optimal growth conditions, spontaneouslydifferentiating ES cell cultures grown in monolayer gave risenot only to these cells but also to a wide range of additionalcell types.


Fig. 1.Spontaneous or BMP-2-induced extra-embryonicdifferentiation of human EScells. (A,B) Phase contrastmorphology of control (A) orBMP-2 (B, 25 ng/ml) cells 7days after treatment. A showstypical stem cell morphology.(C) Spontaneouslydifferentiating ES cell colony.(D) Hematoxylin and eosinstained section of cells similarto those shown in C afterremoval from culture dish.(E,F) Phase contrast (E) andindirect immunofluorescence(F) image of BMP-2-treatedcells stained with antibody tocytokeratins 8, 18 and 19.(G,H) Phase contrast (G) orindirect immunofluorescence(H) micrographs of BMP-2-treated cells stained withantibody to laminin.(I) Double-label staining ofBMP-2-treated cells stainedwith GCTM-2 recognizing astem cell surface proteoglycan(red) and SPARC (green).(J,K) Phase contrast (J) orindirect immunofluorescence(K) micrographs of a cysticstructure and cells at its basestained with antiserum toalphafetoprotein. Wall of cyst(at left) and cells at base (atright) are stained. Bars,A-C, 50 µM; D, 10 µM; E-K,20 µM.


Indirect immunofluorescence examination revealed that theBMP-2-treated cells or spontaneously differentiating cellsresembling them expressed low molecular weight cytokeratinsand abundant extracellular matrix proteins (Fig. 1E,F,cytokeratin; G,H, laminin; I, SPARC staining at an earlystage of differentiation). Particularly strong staining foralphafetoprotein was observed in cystic cells and cells at thebase of cysts (Fig. 1J,K). These flat epithelial cells were notstained with the ES stem cell antibody GCTM-2, nor were theyreactive with antibodies to class I major histocompatibilitycomplex (MHC) molecules. The effect of BMP-2 on theexpression of the stem cell marker GCTM-2 could be shownquantitatively by flow cytometric analysis, with a lowerproportion of any cells showing staining after 5 days of BMP-2 treatment compared with control cultures (negative cellsincreased 1.6-fold relative to controls, Fig. 2).

Xu et al. (Xu et al., 2002b) recently showed that treatmentof the human ES cell line H-1 with BMP-4 induceddifferentiation into trophoblast cells. Under our conditions oftreatment, very few cells expressing placental alkalinephosphatase or human chorionic gonadotrophin were observedin control or treated cultures, and the morphology of the BMP-2- or BMP-4-treated cells was very different to that observedby Xu et al. (data not shown). Nevertheless, we occasionallyobserved foci of cells resembling the trophoblast precursorsdescribed by these workers; these cells appeared in less than5% of colonies treated with BMP-2, BMP-4 or BMP-2/-7under our culture conditions.

Initially our experiments used BMP-2 only and wereperformed with ES cells grown on a specific lot of mouseembryo feeder cells. When another lot of feeder cells was used,the effects of BMP-2 and other bone morphogenetic proteinswere minimal or nonexistent. Antagonism of the action of theexogenous bone morphogenetic proteins appeared to accountfor the lack of a BMP effect on ES cells cultured using thesecond lot of feeder cells, as further examination showedclearly that the outcome of treatment was entirely dependenton feeder cell density. When the second batch of feeder cellswas used at the density employed for routine ES maintenance,

the effect of BMP-2 was variable, but a threefold reduction infeeder cell density revealed a strongly inhibitory effect of theprotein on stem cell maintenance (Fig. 3A; the experiment wasrepeated three times on HES-2 and HES-3 with similar resultsand each experiment analyzed duplicate wells containing atleast four colonies per treatment). Using this density of thesecond lot of mouse embryo feeder cells, we observed thatother bone morphogenetic proteins, including BMP-4, BMP-7and BMP-2/BMP-7 heterodimers, could induce the same effectas BMP-2. To determine the possible mechanism wherebyfeeder cell layers might inhibit the action of bonemorphogenetic proteins, we carried out RT-PCR analysis forexpression of known antagonists of BMP in this system.Examination of embryo fibroblast feeder cells for transcriptsencoding known inhibitors of bone morphogenetic proteinrevealed that the antagonist gremlin was transcribed in thesecells (Fig. 3B).

Analysis of gene expression in the BMP-2-treated culturesconsisting mainly of the flat epithelial cells by RT-PCR (Fig.4A) showed reproducible loss of stem cell markers, andupregulation of a range of markers characteristic of endoderm,including transcription factors (HNF3-α, HNF-4, GATA-4 andGATA-6) and genes encoding secreted products andextracellular matrix molecules expressed in parietal endodermin the mouse and visceral endoderm in mouse and human.RNA from stem cells and BMP-2-treated cells yielded similaramounts of RT-PCR product for beta actin. Some transcriptscharacteristic of endoderm differentiation were observed atlower levels in control ES cell cultures, indicative of low levelsof spontaneous differentiation into this lineage.

Expression of BMP-2, its receptors and signaltransduction machinery in human ES and EC cellculturesThe response of human ES cells to BMP-2, and the presenceof cells resembling those in BMP-2-treated culturesin untreated ES cell cultures undergoing spontaneousdifferentiation, suggested that endogenous BMP-2 production

Fig. 2.Flow cytometric analysis of GCTM-2 stem cellsurface proteoglycan staining in control cells and cellstreated for 5 days with 25 ng/ml BMP-2. Left panelsshow side scatter versus forward scatter; middle panelsshow histograms of cell counts versus fluorescenceintensity of cells stained with isotype matched control;right panels show histograms of cell counts versusfluorescence intensity of cells stained with antibodyGCTM-2 against stem cell surface proteoglycan.

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might be modulating the spontaneous differentiation observed.We carried out RT-PCR analysis for BMP-2, and its receptorsBMPR1A, BMPR2 and the activin receptor II beta, and showedexpression of transcripts for all these genes in stem cells andin cultures consisting predominantly of flat squamousepithelial cells (Fig. 4B).

We speculated that BMP-2 production might be activated atearly stages of ES cell differentiation, driving a positivefeedback loop towards extra-embryonic differentiation. Toassess this possibility we studied BMP-2 expression indifferentiating cultures of human EC cells. Pluripotent humanEC cell line GCT 27X-1 resembles ES cells in morphology,marker expression and growth requirements. However, ECcells are easier to grow in large quantities as pure stem cellpopulation. Cell line GCT27X-1 was cultured in the absenceof a feeder cell layer and differentiation was induced bytreatment with all-trans retinoic acid or BMP-2 as describedpreviously (Pera and Herszfeld, 1998; Roach et al., 1994).Spontaneously differentiating cultures (controls) and retinoicacid treated cultures both show increased levels of transcriptsfor BMP-2, but a very striking increase in these transcripts isseen 12-48 hours after BMP-2 treatment (Fig. 4C). Thus,treatment of human pluripotent cells with BMP-2 leads to theaccumulation of transcripts for this factor, consistent with apositive feedback model.

We also determined whether or not BMP-2 protein waspresent in ES cell cultures. Attempts to identify the protein inculture supernatant or cell lysates prepared using nonionicdetergents failed. Following lysis of spontaneouslydifferentiating ES cell monolayers with nonionic detergent andremoval of the lysate, we extracted the remaining protein onthe monolayer with reducing SDS-PAGE sample buffer.Immunoblotting of this material revealed the presence of BMP-2 protein of the expected size, and a strong band corresponding

to a doublet (Fig. 4D); despite the use of reducing samplebuffer, it was difficult to convert all this material to monomericform. BMP-2 protein was also detected by immunostaining indifferentiating colonies of human ES cells (Fig. 4E-G, cellsgrown in the absence of a feeder cell layer). Recent cellbiological (Suzawa et al., 1999) and genetic (Arteaga-Soliset al., 2001) studies have highlighted the activity of bonemorphogenetic proteins bound to pericellular matrix.

Examination of feeder cell layer by RT-PCR or byimmunostaining failed to detect BMP-2 or BMP-4 transcripts,or BMP-2 immunoreactivity (data not shown).

To determine whether BMP-2 addition to human ES cellcultures resulted in activation of the Smad signal transductionpathways, and to evaluate the extent to which this signalingoperated spontaneously in ES cells, we analyzed cells for thepresence of phosphorylated Smad-1 protein in the nucleus.Before BMP2 treatment, phosphorylated Smad-1 staining wasfound at low levels in stem cell nuclei (Fig. 5A-D), whereasafter BMP2 addition, much brighter staining was detectablein the nuclei of undifferentiated cells (Fig. 5C-I). Thus, stemcells appeared to activate the Smad-1 pathway underbasal conditions, and BMP-2 treatment enhanced nuclearlocalization of this signal transduction molecule.

Results of noggin treatment of human ES cellsThe results above suggested that stem cell maintenance mightbe a dependent on a balance between expression of the BMPantagonist gremlin by the fibroblast feeder cell layer and theendogenous production of BMP-2 by the differentiating stemcells. We postulated that by strongly interfering withendogenous BMP-2 action, it would be possible to prevent thedifferentiation of the cells into the extra-embryonic phenotype,leading either to enhancement of stem cell renewal, or to


Fig. 3.Feeder cell antagonism of BMP action. (A) The effect of feeder cell density on the response of human ES cells to BMP-2. ES cells wereplated onto feeder cells prepared at a density of either 6.6×104/cm2 (high density) or 1.3×104/cm2 (low density) and were grown for 5 dayswith or without treatment with 50 ng/ml BMP-2. The wells were then fixed and stained with monoclonal antibody GCTM-2 followed bydetection with anti-mouse immunoglobulin conjugated to alkaline phosphatase; red staining against blue counterstain indicates activity. (B) RT-PCR analysis for transcripts of three BMP antagonists in ES cells, differentiating cultures of ES cells and mouse embryo fibroblasts. Gremlintranscripts are strongly expressed in mouse embryo fibroblasts.


Fig. 4.Gene expression in spontaneously differentiating and BMP-2-treated ES cells, spontaneously differentiating and BMP-2- or retinoicacid-treated EC cells, and noggin-treated ES cells. (A) RT-PCR for transcripts for stem cell markers (Oct-4, Cripto and FoxD3) or markers ofextra-embryonic endoderm differentiation (alphafetoprotein (AFP), HNF3-α, HNF-4, GATA-4, GATA-6, transferring (TRF), vitronectin (Vn)and SPARC) and beta actin in control ES cells (C2, HES-2, and C3, HES-3) or cells treated with BMP-2 (B2, HES-2 treated with BMP; B3,HES-3 treated with BMP) at 25 ng/ml for 5 days. Positive control for ES cell markers, human EC cell line GCT 27X-1 and for extra-embryonicendoderm markers yolk sac carcinoma cell line GCT 72. (B) RTPCR for BMP-2 and BMPR1-A, BMPR-2, β-actin and activin receptor β inundifferentiated control and spontaneously differentiating ES cell cultures. Controls are on the right, and differentiated cells are on the left, foreach PCR pair shown. (C) RNA blotting analysis for BMP-2 and glyceraldehyde-3 phosphate dehydrogenase transcripts in human EC cells at 0,12, 24, 48, 96 hours and 7 days after plating in the absence of a feeder cell layer (controls) with or without treatment with BMP-2 or retinoicacid. (D) Immunoblot analysis for BMP-2 in spontaneously differentiating ES cells. Tracks from left to right show 10 ng recombinant BMP-2,25 ng recombinant BMP-2, black bars indicating the position of 19, 24 and 36 kDa marker standards, cell lysate. (E-G) Phase contrast (E)image of differentiating ES cell colony showing staining by indirect immunofluourescence of BMP-2 (F) and GCTM-2 (G). Bar in E-G, 50 µM.

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an enhancement of differentiation intoembryonic lineages. We tested severalnatural and synthetic antagonists of BMP foractivity on human ES cell cultures.Follistatin, DAN, and Cer-1 were withoutany obvious morphological effect, as was asoluble form of the BMPRIA ectodomain.However, treatment with a recombinant formof mouse noggin had a profound effect onhuman ES cell morphology in the dose range100-500 ng/ml. After approximately 5 daysin culture, noggin-treated cultures consistedof colonies of small round cells, which weredifferent in appearance from ES cells, and,in contrast to control cultures, colonies innoggin-treated dishes contained no flatsquamous epithelial cells or cystic structuressimilar to those seen after BMP- 2 treatment(Fig. 6A-C). The size of the noggin-treatedcolonies was smaller than those of controls,although the same number of cells waspresent in each (not shown). In noggin-treated cultures, the percentage of cellspositive for the stem cell marker GCTM-2was much lower than controls (Fig. 6D).

The immunophenotype of the noggin-treated cells was distinguished by theirlack of expression of several markerscharacteristic of ES cells or differentiatedcells found spontaneously at early timepoints (approximately 5-7 days) followingES cell subculture under standardconditions (Table 2). Some markers of earlyneural differentiation were found in noggin-treated colonies.

RT-PCR analysis confirmed that thenoggin-treated cells expressed Oct-4transcripts at low levels, if at all (Fig. 6E,comparison of noggin-treated culture withES cells). The noggin-treated cells didexpress transcripts for markerscharacteristic of early neuroectoderm suchas Pax-6 or nestin. The noggin cells did notexpress brachyury, characteristic of earlymesoderm, and finally, they did not expressgene products characteristic of extra-embryonic endoderm, which are foundin spontaneously differentiating controlcultures or in BMP-2-treated cultures.


Fig. 6.Effects of noggin on human ES cells.(A) Area of differentiation in ES cell colony; (B)noggin-treated cells. (C) Cells from a colonysimilar to B after replating onto fresh feeder celllayer. Bar, 50 µm. (D) Proportion of GCTM-2-positive cells in human ES cultures after 5 daysof growth under control conditions or in thepresence of 200 ng/ml noggin. (E) RT-PCRanalysis of gene expression in human EScultures after 5 days of growth under controlconditions or in the presence of 200 ng/mlnoggin.

Fig. 5.Smad1 phosphorylation induced by BMPaddition to hES cells. Undifferentiated hES cellswere deprived of fetal calf serum for 4 hours thenused as controls (A-D) or treated with BMP2 (50ng/ml) for 1.5 hours (E-H). After fixing andpermeabilization, cells were stained with GCTM2(green, A and E) and anti-PSmad1 (red, F).Comparison of B and F and the overlays ofcontrol and treated cells in C and G indicate thatBMP treatment leads to nuclear translocation ofSmad1. (I) Nuclear fluorescent intensityquantified as pixel density in ten serial z-sectionsin a confocal laser scanning microscope.


The noggin-treated cells were furthercharacterized in biological studies. The addition of25 ng/ml recombinant human BMP-2 to ES cellcultures along with 250 ng/ml noggin led to theappearance of squamous cells and cystscharacteristic of spontaneously differentiating EScell cultures or BMP-2-treated cultures, indicatingthat BMP-2 could antagonize the noggin effect (notshown). The noggin-treated cells could besubcultivated under standard conditions for ES cellculture in the presence of a feeder cell layer, andretained their distinctive morphology under theseconditions for at least several passages (Fig. 6C). Tocompare the neurogenic potential of control andnoggin-treated ES cells, colonies of either cell typewere dissected under microscopic control andtransferred to medium designed to support thegrowth of neural stem cells (Fig. 7A-D). Controlcells formed embryoid bodies that displayed variablemorphology, were often cystic and frequently

reattached to the culture surface (Fig. 7A). By contrast, noggin-treated cells formed spheres which remained floating insuspension and could be serially cultivated (Fig. 7B). The fateof either control ES cells or noggin-treated ES cells followingculture in serum-free medium was examined by allowing themto reattach to a poly-L-lysine-coated surface, then stainingthem with nestin (Fig. 7C,D). The proportion of structuresforming neurospheres after 5 days of noggin treatment variedbetween 40-70%, but, in any given experiment it was at leastfivefold higher than that of untreated ES cells (Fig. 7E). Nestin-negative colonies from noggin-treated cells retained theappearance of noggin cells grown on feeder layers, whereasnestin-negative colonies from control cultures had a variedappearance typical of mixed embryoid bodies allowed toreattach to the culture surface. After 2 weeks of treatment, theproportion of noggin-treated colonies that could be convertedto nestin-positive spheres rose to 90% or higher (B.R.,unpublished; G. Peh, S. Hawes and M.F.P., unpublished).

When the spheres derived from noggin-treated cells wereallowed to attach to the dish, cells forming elongated processesmigrated out onto the monolayer (Fig. 8A). These cellsdisplayed an immunophenotype consistent with theiridentification as mature neurons, including expression of 200kDa neurofilament protein and MAP2-a,b (Fig. 8B-E).However, if noggin-treated cells were cultivated as monolayersin the absence of a feeder cell layer and in the presence ofserum, they gave rise to cultures consisting of cells withfibroblastoid morphology. At least 50% of the cells in thesecultures stained with antibodies against glial fibrillary acidicprotein and vimentin (Fig. 8F,G).

Table 2. Expression of antigens in ES cells and noggin-treated cells

Antibody Specificity Control Noggin

Transcription factorsOct-4 Human Oct-4 +++ –Sox-2 Sox-2 +++ +++

Stem cell surface markersGCTM-2 Stem cell

protoeoglycan+++ –

TG343 Stem cellprotoeoglycan

+++ –

TG42.1 CD9 +++ –

Differentiated cell surface markersGCTM-5 Embryonic liver

surface marker– –

PHM-4 HLA class I – –UJ13A Polysialylated N-CAM – –

Intermediate filament markersCam 5.2 Cytokeratin 8,18 + –AMF 17 Vimentin + –Anti-desmin Desmin – –NF-68 Low molecular weight

neurofilament– +

NF-160 160 kDa neurofilamentprotein

– –

+++, expression in 80% of cells or more; +, expression in 10-20% ofcells; –, expression in less than 5% of cells

Fig. 7.Differentiation of noggin cells into neural precursors. (A) Phase contrast micrographof control cells after transfer to neural progenitor medium; (B) phase contrast appearance ofnoggin-treated cells after transfer to neural progenitor medium; (C) phase contrastappearance of noggin-treated cells following transfer to neural progenitor medium andattachment to culture surface; (D) same field as C stained with antibody to nestin. (E) Graphshowing proportion of control and noggin-treated cells forming nestin-positive colonies after5 days of growth on a feeder cell layer in the absence or presence of 200 ng/ml nogginfollowed by 1 week of growth in neural progenitor medium. Bars, A-D, 50 µM.

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DiscussionSeveral groups have now reported the spontaneousdifferentiation of human ES cells (Itskovitz-Eldor et al., 2000;Levenberg et al., 2002; Reubinoff et al., 2000; Schuldiner etal., 2000; Xu et al., 2002a), and most of these have usedembryoid body formation to begin the initial process ofdifferentiation. ES cells allowed to differentiate on plasticsurfaces or embryoid bodies give rise to a mixture of cells, asignificant number of which express markers of extra-embryonic endoderm. The regulation of this process ofspontaneous differentiation is poorly understood. Schuldiner etal. (Schuldiner et al., 2000) showed that growth factortreatment could influence some of the outcome of spontaneousdifferentiation of ES cells but, like other studies to date, thisone did not directly elucidate the factors involved in

spontaneous differentiation. BMP-2 has a role invisceral endoderm differentiation in the mouseES cell system, and it is expressed in theextra-embryonic endoderm in this species(Coucouvanis and Martin, 1999; Ying and Zhao,2001a). We previously observed that BMP-2could induce differentiation of human EC cellsinto a cell with patterns of gene expressionresembling extra-embryonic endoderm (Pera andHerszfeld, 1998).

Here we showed that BMP-2 has a similareffect in human ES cell cultures, and that thiscell type is also seen during spontaneousdifferentiation of ES cells. The identification ofthese differentiated epithelial cells is basedchiefly on their patterns of gene expressionand on immunostaining. Thus, the cells areepithelial cells expressing low molecularweight cytokeratins, laminin, alpha-fetoprotein,transferrin, SPARC and vitronectin but not classI HLA molecules. Morphologically, the BMP-treated cells resemble a cell found in the humanperi-implantation embryo, which forms a mesh-like network within the blastocoel cavity. Thiscell was known classically in the humanembryology literature as a mesoblast, but oncomparative anatomical grounds Luckett(Luckett, 1978) argued that these cells weremore likely to represent extra-embryonicendoderm. Several further morphological studiesof the primate embryo have documented thedevelopment of cells resembling parietalendoderm and visceral endoderm from flattenedcells formed below the epiblast (Enders et al.,1990; Enders and Schlafke, 1981; Enders et al.,1986). Although detailed studies of marker

expression in these cells in the primate embryo have not beencarried out, the properties of the BMP-induced cell areconsistent with their identification as extra-embryonicendoderm. Formation of this cell in vitro may provide a sourceof multiple signals that influence human ES cell differentiation,and controlling these signals may be critical to directingdifferentiation of the ES cells themselves.

Xu et al. (Xu et al., 2002b) have shown that a different typeof extra-embryonic cell, the trophoblast, can be induced byBMP-4 treatment of ES cells cultured in serum-free mediumin the presence of FGF-2 (Fig. 9). Although we have notroutinely observed cells of this phenotype in our experiments,we have occasionally observed small foci of cells resemblingthose shown in the study of Xu et al. (Xu et al., 2002b). It ispossible that differences between the cell lines studied by our


Fig. 8.Neural derivatives of noggin-treated cells. (A) Outgrowth of cells from a sphere similarto that shown in 7B; (B,C) staining of outgrowth similar to that shown in A with antibody to 200kDa neurofilament protein (B, phase contrast; C, indirect immunfluorescence); (D,E) staining ofoutgrowth similar to that shown in A with antibody to Map 2 a,b (D, phase contrast; E, indirectimmunofluorescence); (F,G) cells similar to those shown in Fig. 6C following transfer tomonolayer culture in the presence of serum without feeder cell support (F, phase contrast;G, indirect immunofluorescence for glial fibrillary acidic protein). Bar shown in B for A-E,50µM.


two groups could account for differences in response to thesefactors. It is also possible that serum components influence theoutcome of differentiation, but it is interesting that activationof the same signal transduction pathway can result in theformation of either extra-embryonic endoderm or trophoblast.It has been shown that mouse ES cells undergoingdifferentiation can follow either of these pathways dependingon whether Oct-4 levels rise or fall (Niwa et al., 2000).

The data reported here strongly suggest that there is apositive feedback loop involving BMP-2, which acts todrive extra-embryonic endoderm differentiation of humanpluripotent stem cells. Our data show that BMP-2 can induceits own expression in human EC cells. Both human EC and EScells, and the cells that differentiate from them, expresstranscripts for receptors that enable them to respond to thefactor. Consistent with the RNA analyses, BMP-2 proteinis present in the pericellular matrix of spontaneouslydifferentiating ES cells. Data on nuclear localization ofphosphorylated Smad-1 show that under standard cultureconditions some nuclear staining is observed in ES cells; theintensity of the nuclear staining is greatly increased followingtreatment with BMP-2. All these data are consistent with anendogenous signaling system based on BMP-2 that drivesextra-embryonic endoderm differentiation.

There are several natural and synthetic antagonists to theBMPs. We showed that transcripts for the antagonist gremlinare produced by mouse embryo fibroblasts, and previousstudies have shown that rat embryo fibroblasts produce gremlinprotein, much of which is cell associated and not soluble(Topol et al., 2000). Gremlin may be one of a number of factorsproduced by feeder cells that are required for stem cell renewal.We speculate that high local concentrations of cell-associated

gremlin or other feeder cell products may have a spatiallylimited effect in blocking stem cell differentiation. We showelsewhere (S. M. Hawes and M.F.P., unpublished) that thezones of human ES cell colonies at some distance from thefeeder cell layer undergo differentiation first.

We hypothesize that stem cell fate may be determined in partby a balance between feeder cell inhibition of differentiation,mediated by gremlin, and by the production of BMP-2, whichdrives differentiation. We tested the effect of the addition ofseveral BMP antagonists in the human ES cell system andobtained a specific effect with noggin. This effect was evidentafter a short period of treatment of ES cell colonies, andappeared to affect most cells in the culture. Noggin is knownto play a role in the induction of the nervous system in severalvertebrate model systems by antagonizing BMPs (Bachiller etal., 2000; Smith and Harland, 1992) (reviewed by Streit andStern, 1999). The noggin-treated cells can give rise to bothneuronal and glial lineages, but they themselves do not appearto be equivalent to the neural progenitors that we and othershave previously derived from human ES cells. Noggin-derivedneural cells have properties expected of mature neurons andglial cells, and the noggin neural progenitors have been shownto engraft into the nervous system of experimental animalsand undergo appropriate differentiation (B.R., T. Ben-Hur, E.Reinhartz, A. Itzik, M. Idelson and M.F.P., unpublished), asshown previously for neural progenitors derived fromspontaneously differentiating ES cell cultures (Reubinoff et al.,2001).

Because antagonism of BMP signaling blocks extra-embryonic differentiation, it is interesting that noggintreatment does not enhance stem cell renewal. One plausiblehypothesis to account for our results is that extra-embryoniccells, in addition to secreting bone morphogenetic proteins andother factors that can induce differentiation, also producefactors that help maintain ES cells. In the mouse embryo, theextra-embryonic endoderm produces a variety of factors thatact locally; some are postulated to induce ectoderm, othersmesoderm, and some are known to induce germ cell formation.Thus, this tissue may have multiple effects on embryonic stemcells. If the extra-embryonic factors are not present to maintainstem cells, then the stem cells may default towardsneuroectoderm differentiation as depicted in the scheme shownin Fig. 9. In this scheme, feeder cells produce gremlin or otherfactors that act locally to maintain stem cell renewal but allowfor some degree of extra-embryonic differentiation. Completeblockade of extra-embryonic differentiation by exogenousnoggin removes a source of factors that can support stem cellrenewal and factors that drive other differentiation pathways,resulting in default differentiation into the neural lineage.

Further work will be required to determine thedevelopmental potential of the noggin cells isolated here. Itappears clear that they have neurogenic potential. By bypassingextra-embyonic endoderm differentiation, it may be possible todirect the fate of human ES cells into specific somatic lineages,as suggested for mouse ES cells (Lake et al., 2000). Theresults here indicate that BMP-controlled extra-embryonicdifferentiation is an important regulatory node in ESdifferentiation, show that addition of polypeptide regulators ofearly mammalian development can direct the fate of human EScells, and provide a facile route to the generation of neuralprecursors from ES cells.

Fig. 9.Early differentiation events in human ES cell cultures. EScells undergo spontaneous differentiation in a BMP-dependentfashion to extra-embryonic tissues; the choice of extra-embryonicendoderm or trophoblast (Xu et al., 2002b) depends onenvironmental factors. Extra-embryonic tissues can produce factorsthat either drive stem cell renewal or various differentiationpathways. Gremlin produced in feeder cells partially offsets extra-embryonic differentiation locally; addition of noggin to mediumcompletely inhibits this differentiation and allows the formation ofneural progenitor through a default mechanism. Lack of extra-embryonic endoderm in noggin-treated cultures results in a loss ofsignals for stem cell renewal and differentiation into other celllineages.

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At Monash University, this work was supported by grants from theNational Health and Medical Research Council, and the NationalInstitutes of Health (NIGMS GM68417), and by a sponsored researchagreement with ES Cell International Pte. We gratefully acknowledgethe expert assistance of Jacqui Johnson, Irene Tellis, Karen Koh andLinh Nguyen with human ES cell culture. We thank Peter ten Dijkefor the antibody to phosphorylated Smad-1and Leon Tertoolen forhelp with the confocal microscopy.

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