Absence of Suppressor of Cytokine Signalling 3 Reduces Self-Renewal and Promotes Differentiation in Murine EmbryonicStem Cells

ARIEL FORRAI,a KRISTY BOYLE,a ADAM H. HART,a LYNNE HARTLEY,a STEVEN RAKAR,b TRACY A. WILLSON,a

KEN M. SIMPSON,a ANDREW W. ROBERTS,a WARREN S. ALEXANDER,a ANNE K. VOSS,a LORRAINE ROBBa

aThe Walter and Eliza Hall Institute of Medical Research, Parkville, Australia; bZenyth Therapeutics Limited,

Richmond, Australia

Key Words. SOCS-3 • Embryonic stem cells • Endoderm

ABSTRACT

Leukemia inhibitory factor (LIF) is required to maintainpluripotency and permit self-renewal of murine embryonicstem (ES) cells. LIF binds to a receptor complex of LIFR-�and gp130 and signals via the Janus kinase–signal trans-ducer and activator of transcription (JAK–STAT) pathway,with signalling attenuated by suppressor of cytokine signal-ling (SOCS) proteins. Recent in vivo studies have high-lighted the role of SOCS-3 in the negative regulation ofsignalling via gp130. To determine the role of SOCS-3 in EScell biology, SOCS-3–null ES cell lines were generated.When cultured in LIF levels that sustain self-renewal ofwild-type cells, SOCS-3–null ES cell lines exhibited lessself-renewal and greater differentiation into primitiveendoderm. The absence of SOCS-3 enhanced JAK–STATand extracellular signal–related kinase 1/2 (ERK-1/2)–mito-

gen-activated protein kinase (MAPK) signal transductionvia gp130, with higher levels of phosphorylated STAT-1,STAT-3, SH-2 domain–containing cytoplasmic protein ty-rosine phosphatase 2 (SHP-2), and ERK-1/2 in steady stateand in response to LIF stimulation. Attenuation of ERKsignalling by the addition of MAPK/ERK kinase (MEK)inhibitors to SOCS-3–null ES cell cultures rescued the dif-ferentiation phenotype, but did not restore proliferation towild-type levels. In summary, SOCS-3 plays a crucial role inthe regulation of the LIF signalling pathway in murine EScells. Its absence perturbs the balance between activation ofthe JAK–STAT and SHP-2–ERK-1/2–MAPK pathways, re-sulting in less self-renewal and a greater potential for dif-ferentiation into the primitive endoderm lineage. STEMCELLS 2006;24:604–614

INTRODUCTIONPropagation of pluripotent murine embryonic stem (ES) cells ismaintained by the cytokine leukemia inhibitory factor (LIF)[1, 2]. LIF induces dimerization of the LIFR-�–gp130 receptorcomplex, activating multiple cytoplasmic signalling proteins[3]. The cytoplasmic Janus kinase (JAK)–signal transducer andactivator of transcription 3 (STAT-3) pathway and extracellularsignal–regulated kinase 1/2 (ERK-1/2)/mitogen-activated pro-tein kinase (MAPK) pathway are two major signalling cascadesactivated by LIF. In the presence of LIF, JAKs phosphorylatetyrosines on the intracellular portion of gp130 and LIFR-�,which then act as docking sites for STAT-1 and STAT-3 pro-teins that also become phosphorylated [4]. Dimeric phospho-STAT-3 enters the nucleus, binds to specific DNA-bindingelements, and activates transcription. STAT-3 is a critical me-diator of LIF-induced signalling pathways that regulate ES cell

self-renewal. Mutation of STAT-3–interacting tyrosines ongp130, targeted deletion of Stat3, or overexpression of a dom-inant negative STAT-3 all abrogate the ability of LIF to main-tain self-renewal of ES cells, and constitutive expression ofactivated STAT-3 prevents differentiation of ES cells after thewithdrawal of LIF [5–7].

In ES cells, activation of the Ras–ERK-1/2–MAPK signal-ling pathway via gp130 is dependent on phosphorylation of theSH2 domain–containing cytoplasmic protein tyrosine phospha-tase 2 (SHP-2). Phosphorylation of a single tyrosine residue(Y757) in murine gp130 is necessary and sufficient for recruit-ment of SHP-2, leading to its tyrosine phosphorylation in aJAK-1–dependent manner [4, 8]. SHP-2 acts as an adaptorprotein, associating with growth factor receptor–bound protein(GRB)-2 and thereby activating the Ras–ERK-1/2–MAPK path-way [9]. Unlike STAT-3 signalling, the SHP-2 signalling path-

Correspondence: Lorraine Robb, M.D., Ph.D., The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville,Victoria 3050, Australia. Telephone: 61-39-345-2527; Fax: 61-39-347-0852; e-mail: robb@wehi.edu.au Received July 16, 2005;accepted for publication August 12, 2005; first published online in STEM CELLS EXPRESS August 25, 2005. ©AlphaMed Press1066-5099/2006/$20.00/0 doi: 10.1634/stemcells.2005-0323

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way does not promote ES cell self-renewal. ERK-1/2 activationhas a differentiative effect on ES cells, and inhibition of theERK-activating enzyme MAPK/ERK kinase (MEK) has beenshown to enhance self-renewal of ES cells [10]. Mutation of theSHP-2–binding tyrosine on gp130 prolongs STAT-3 activation,diminishes ERK activation, and increases ES cell self-renewal[10]. ES cells homozygous for a SHP-2 mutation demonstrateLIF hypersensitivity and greater LIF-stimulated STAT-3 phos-phorylation, greater self-renewal capacity, and less differentia-tion [11].

An increasing number of studies has shown that the sup-pressor of cytokine signalling 3 (SOCS-3) protein is a keyregulator of signalling mediated via gp130. SOCS proteinscontain a central SH2 domain and a carboxy-terminal SOCSbox. Each of these domains is thought to have a separatefunction in regulating cytokine signalling. The SH2 domaininteracts with phosphorylated tyrosine residues in tyrosine ki-nases and cytokine receptors and can negatively regulate theiractivity [12]. The current model suggests that SOCS-3 is re-cruited to the signalling complex by interaction with Y757 ofgp130, leading to inhibition of JAK activity [13]. Y757 is alsothe binding site for SHP-2, although the binding affinity ofSOCS-3 is higher than that of SHP-2 [12, 14]. The SOCS boxforms part of the Elongin B/C-Cul5-SOCS-box (ECS) proteincomplex [15–17]. The ECS complex is a member of a family ofubiquitin ligases that share a Cullin-Rbx module. SOCS box–containing proteins recruit substrates to the ECS complex, tar-geting them for polyubiquitination and proteasomal degrada-tion, and thereby controlling a variety of cellular processes.SOCS-3 binds specifically to Cul5–Rbx2 complexes in mam-malian cells, but to date only insulin receptor substrate (IRS)-1and IRS-2 have been shown to be substrates for degradation[17, 18].

Gene-targeting experiments have reinforced the notion thatSOCS-3 is a major physiological regulator of signalling viagp130. SOCS-3–null embryos die at midgestation as a result ofplacental failure, and this can be rescued by a reduction ofsignalling via LIFR-� [19–21]. When the Socs3 gene wasablated in a cell type–specific manner, this resulted in prolongedSTAT-3 activation and consequent changes in the functionaloutcome of gp130 signalling in response to interleukin 6 [22–24]. In ES cells stimulated with LIF, Socs3 is a major transcrip-tional target of STAT-3 [25]; however, little is known about thefunction of SOCS-3 in ES cells. Overexpression of SOCS-1 orSOCS-3 has been reported to block self-renewal and promotedifferentiation [25, 26]. Recently, SOCS-3 was identified as oneof a number of genes upregulated in SHP-2–null ES cellsstimulated with LIF, and overexpression of SOCS-3 in wild-type (WT) ES cells was shown to promote ES cell differentia-tion to hemangioblasts and primitive erythroid progenitors [27].In order to understand the role of SOCS-3 in ES cells, wederived SOCS-3–null ES cell lines. In standard culture condi-tions, SOCS-3–null ES cell lines show less proliferation andgreater differentiation to endoderm. In steady state, STAT-3phosphorylation is greater in SOCS-3–null ES cells and, incontrast to WT cells, SHP-2 is phosphorylated. In response toLIF stimulation, STAT-3, SHP-2, and ERK-1/2 activation areprolonged in SOCS-3–null ES cells. Overall, we demonstratethat SOCS-3 plays a crucial role in regulating LIF signalling in

ES cells and that its absence results in alterations in self-renewaland differentiation.

MATERIALS AND METHODS

Generation of SOCS-3–Null Mice and ES Cell LinesThe SOCS-3 gene-targeting vector has been described previ-ously [20]. A second SOCS-3 targeting construct was created byreplacing the neomycin resistance cassette with a hygromycinresistance cassette. Generation and verification of SOCS-3 het-erozygous ES cell lines were as described elsewhere, except thatthe W9.5 ES cell line was used [20]. Two targeted clones wereinjected into C57BL/6 blastocysts and chimeric offspring werebred to C57BL/6 mice to derive mice heterozygous for theSOCS-3 mutation. To obtain SOCS-3–null ES cell lines,SOCS-3 heterozygous mice were intercrossed, and blastocystswere collected and cultured as described previously [28]. Thegenotype of the ES cell lines was determined by Southernblotting of BamHI-digested DNA with 5� and 3� genomic DNAprobes.

Cell CultureES cells were maintained on a layer of irradiated primary mouseembryonic fibroblasts in ES medium—Dulbecco’s modifiedEagle’s medium (DMEM) supplemented with 4.5 g/l glucose,3.4 g/l NaHCO3, 15% (vol/vol) batch-tested fetal calf serum(FCS) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 0.1 mM 2-mercaptoethanol (Sigma), and 0.1 mM nones-sential amino acids (Invitrogen, Carlsbad, CA, http://www.invitrogen.com)—as described elsewhere [29]. Feeder-indepen-dent lines were maintained on gelatin-coated tissue culturedishes. To quantitate proliferation, viable single cells wereplated at 100 cells per well in gelatin-coated 24-well tissueculture plates in triplicate, and the number of cells per well wascounted daily. To assess self-renewal, the number of coloniespresent at day 3–6 after plating was ascertained by stainingplates with methylene blue and scoring all colonies, in a blindedfashion, as undifferentiated, partially differentiated, or fullydifferentiated. Undifferentiated colonies were tight, roundedcolonies, composed purely of stem cells, partially differentiatedcolonies contained stem cells with, flattened, differentiated cellsat the periphery of the colony, and differentiated colonies werecomposed entirely of differentiated cells. Embryoid bodies wereformed by seeding a single-cell suspension of ES cells at 2000–5000 cells/ml in a differentiation medium as described else-where [30]. To test the effect of MEK inhibitors, ES cells wereplated at 0.5 � 106 cells per well of a six-well tissue cultureplate in standard ES cell culture medium and were supple-mented with either 10 �M UO126 (Promega, Madison, WI,http://www.promega.com), 50 �M PD98059 (Promega), or 1�l/ml dimethylsulfoxide (DMSO). The medium was changedevery 2 days.

Reverse Transcription-Polymerase Chain ReactionTotal RNA was extracted from feeder-independent ES cell linesusing the RNeasy Mini RNA kit (Qiagen, Valencia, CA, http://www1.qiagen.com). DNaseI-treated samples were reverse tran-scribed (RT) using Superscript III (Invitrogen), the resultantcDNA preparations were standardized, and polymerase chainreaction (PCR) was performed as described elsewhere [31].

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References for primers are as follows: Oct4, Nanog, Hprt: [32],Mixl1: [33], Sox1, Otx2: [34], Afp Gata1, T: [35], and Zfp42.Isl1, Dab2, Lamb1–1, Ttr, COUPTF1 COUPTF2, Gata4,Gata6, Tcf2, and HNF4a: [36]. Primers for Socs3 were: 5�-AGATTTCGCTTCGGGACTAGC-3�5�-CTGGGTCTTGACG-CTCAAGCT-3�.

Indirect ImmunofluorescenceSOCS-3–null and WT ES cells were plated onto gelatinizedpotassium hydroxide (KOH)-treated glass coverslips at a densityof 1 � 105 cells per coverslip. Cells were cultured for 48 hours,washed with phosphate-buffered saline (PBS), and fixed in 4%paraformaldehyde (PFA) for 10 minutes at room temperature.Immunofluorescent staining was performed as previously de-scribed [37] using antibodies specific for Oct4 and GATA-4(Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com). After application of the secondary antibody (AlexaFluor 594 goat anti-mouse IgG, Molecular Probes Inc., Eugene,OR, http://probes.invitrogen.com), the cells were washed, coun-terstained with 4�,6-diamidino-2-phenylindole (DAPI) mountedin DAKO (Glostrup, Denmark, http://www.dako.com) mount-ing media and viewed with a Zeiss Axioplan 2 (Carl Zeiss, Jena,Germany, http://www.zeiss.com) microscope. Images were cap-tured with a Zeiss Axiocam (Carl Zeiss) and processed withAxiovision software (Carl Zeiss).

Microarray AnalysisTotal RNA was extracted from two independent WT and SOCS-3–null ES cell lines grown on primary mouse embryonic fibro-blasts in standard culture conditions using Qiagen RNeasy MiniRNA purification columns. The RNA from each cell line wasquantitated using an Agilent 2100 Bioanalyser (Agilent Tech-nologies, Palo Alto, CA, http://www.agilent.com). cDNA wassynthesized from 5 �g of RNA according to Affymetrix meth-odology, and biotin-labeled cRNA was synthesized using theAffymetrix Enzo BioArray High Yield RNA Transcript Label-ing Kit (Affymetrix, Santa Clara, CA, http://www.affymetrix.com). Affymetrix mouse genome (MOE 430 2.0, Affymetrix)GeneChips were hybridized with 10 �g of biotin-labeled cRNAaccording to the manufacturer’s protocol. Expression levelswere calculated using Robust Multichip Analysis (RMA) [38].A moderated t-statistic was computed for each gene in thetwo-group comparison (SOCS-3 null vs. WT). This is of theform t* � M/SE*, where M is the log2 fold change and SE* isa smoothed estimate of the standard error, calculated accordingto the empirical Bayes procedure as described elsewhere [39].We selected, as differentially expressed, those genes with anabsolute value of t* � 5. This threshold was derived by exam-ining a plot of the quantiles of t* against the quantiles of thet-distribution on the appropriate degrees of freedom, and look-ing for departures from linearity. All calculations were done inR, using the “affy” and “limma” packages. The microarray datacontained in this manuscript have been submitted to the GEOdatabase, available at http://ncbi.nlm.nih.gov/geo.

Chimera Generation, Induction of Teratomas, andTumor HistologyChimeric embryos were generated by injection of ES cells intoblastocysts obtained from intercrosses of mice carrying theROSA-26 lacZ gene trap [40]. Chimeras were collected at

embryonic day 9 and analyzed for ES cell contribution, aspreviously described [41]. To generate teratomas, 4-week-oldnude mice were injected s.c. in opposing flanks with 1 � 106

WT or SOCS-3–null ES cells in 100 �l of PBS, and tumors wereharvested 4–6 weeks later. Tissue was fixed in Bouin’s fixative,dehydrated, embedded in paraffin, sectioned at 0.4 �m, andstained with hematoxylin and eosin. Eight WT and eight SOCS-3–null tumors from four ES cell lines were examined. Animalstudies were approved by the Melbourne Health Research Di-rectorate Animal Ethics Committee.

Immunoprecipitation and Western BlottingFor cytokine stimulation experiments, feeder-independent EScell lines were grown to near confluence in standard ES cellmedium and then washed three times in PBS and cultured in EScell medium with 0.5% FCS and without LIF for 4 hours priorto restimulation with LIF (1000 units/ml). For immunoprecipi-tation, cells were lysed in KALB lysis buffer (1% TritonX-100(vol/vol), 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mMEDTA) supplemented with 1 mM Na3VO4, 1 mM NaF, 1 mMphenylmethylsulfonyl fluoride (PMSF), and complete proteaseinhibitor mixture (Roche Applied Science, Indianapolis, https://www.roche-applied-science.com). Total cell lysates were pre-cleared with 50% slurry protein A sepharose (PAS), incubatedwith 2 �g SH-PTP2 antibody (c-18, Santa Cruz), and immuno-precipitated with PAS. For Western blotting, near confluentcells were lysed in RIPA lysis buffer (1% Triton X-100(vol/vol),0.1% SDS(w/vol), 1% sodium deoxycholate, 150 mM NaCl, 20mM Tris-HCl pH 7.5, 0.01% sodium azide) supplemented asabove. Lysates or immunoprecipitates were subjected to SDS-PAGE separation and immunoblotting using antibodies specificfor SOCS-3 (Immuno-Biological Laboratories Co., Ltd.,Gunma, Japan, http://www.ibl-japan.co.jp), STAT-3 (SantaCruz), STAT-1 (BD Signal Transduction, Franklin Lakes, NJ,http://www.bdbiosciences.com), and ERK-1/2 (Cell SignallingTechnology, Beverly, MA, http://www.cellsignal.com) and an-tibodies specific for the phosphorylated forms of STAT-3,STAT-1, ERK-1, ERK-2, and SHP-2 (Cell Signalling). Thedensitometry analysis was performed on scanned HyperfilmECL autoradiographs using a Molecular Dynamics Densitome-ter and ImageQuant software (GE Healthcare Life Sciences,Piscataway, NJ, http://www.amersham.com).

Online Supplemental MaterialSupplemental online Figure 1 shows the generation and valida-tion of SOCS-3–null ES cell lines. Supplemental online Figure2 shows alterations in global gene expression in SOCS-3–nullES cells, using an MA plot. Supplemental online Tables 1 and2 list genes with fivefold or greater altered expression in SOCS-3–null ES cell lines.

RESULTS

Generation of SOCS-3–Null ES Cell LinesTo explore the function of SOCS-3 in ES cells, we initiallyattempted to generate SOCS-3–null ES cell lines by seriallyinactivating each allele using targeting constructs with selec-tion cassettes for neomycin resistance or hygromycin resis-tance [20, 42]. When electroporated into W9.5 ES cells [43],the frequency of ES cell clones with a single, correctly

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targeted Socs3 allele (Socs3�/�) was approximately one innine for each construct. To generate doubly targeted clones,2748 G418- and hygromycin-resistant ES cell clones, gener-ated from three independent, karyotypically normal Socs3�/�

cell lines, were screened by Southern blotting. ES cell linesbearing targeted deletions of both Socs3 alleles were notobtained (data not shown). This suggested that ES cells wereunable to survive homozygous targeting of SOCS-3. Themost rigorous step during gene targeting is the requirementfor a single, targeted cell to survive electroporation andantibiotic selection to proliferate into an undifferentiated ES

cell colony. To circumvent this, we derived SOCS-3–null EScells de novo from blastocysts. Socs3�/� mice, derived fromSocs3�/� W9.5 ES cells, were intercrossed, blastocysts were col-lected and cultured and, after hatching, the inner cell mass waspicked and expanded to generate ES cell lines [28]. From 96blastocysts, 55 ES cell lines were obtained, of which 12 werehomozygous for targeted deletion of Socs3. RT-PCR and immu-noblotting demonstrated that SOCS-3 RNA and protein were notdetectable in the SOCS-3–null ES cell lines (supplemental onlineFig. 1). In all experiments reported here, WT ES cell lines out-grown from blastocysts in the same experiment as the null lines

Figure 1. SOCS-3–null ES cells exhibit altered morphology, proliferation, and clonogenicity. (A–H): Morphology of two independentwild-type (WT) and SOCS-3–null ES cell lines, grown in standard ES cell culture conditions (15% fetal calf serum and 1000 units/ml LIF),on (A–D) and after weaning off (E–H) PMEFs. Note the partially differentiated appearance of the colonies arising from the null lines.Differentiated cells present in SOCS-3–null ES cell lines cultured on or off PMEFs are indicated by arrows. ES indicates undifferentiated EScell colonies. (I): Growth of WT and SOCS-3–null ES cell lines. At each time point, the mean and standard deviation for triplicate wells isshown. Similar results were obtained with four WT and four SOCS-3–null lines. (J): Number of colonies present at day 3 after plating 100cells in standard ES cell culture conditions. Colonies were fixed, stained, and scored as undifferentiated, mixed, or differentiated. Means andstandard deviations for triplicate wells are shown. (K): WT and SOCS-3–null colonies were enumerated as for (J) after 3 days of culture instandard ES cell medium with concentrations of LIF as shown. Abbreviations: SOCS-3, suppressor of cytokine signalling 3; ES, embryonicstem cell; PMEF, primary mouse embryonic fibroblasts; W and WT, wild type; N, SOCS-3 null; LIF, leukemia inhibitory factor. Results areshown for a single representative experiment. Scale bars � 25 �m.

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were used as controls, and all results shown are representative of3–6 WT and SOCS-3–null ES cell lines.

SOCS-3–Null ES Cell Lines Exhibit ReducedProliferation and Self-RenewalSOCS-3–null ES cell lines could be continuously passaged inculture and were recoverable after cryostorage. Initially, all lineswere maintained on a feeder layer of primary mouse embryonicfibroblasts. To facilitate biochemical analysis, feeder-independentlines were established. During routine culture, the feeder-dependentand feeder-independent SOCS-3–null ES cell lines were noted togrow more slowly than the WT lines, but could be serially passagedfor at least 3 months. In SOCS-3–null ES cell cultures, around 50%of the colonies were observed to be partially differentiated, withcells at the periphery of the individual colonies adopting a flattened,refractile morphology. (Fig. 1A–1H). To quantitate the prolifera-tive defect in the null lines, viable single cells were plated at lowdensity (100 cells per well in 24-well tissue culture plates) and thenumber of cells per well was counted daily. In comparison withWT ES cells, the SOCS-3–null cells demonstrated markedly lowerproliferation (Fig. 1I). To assess self-renewal, the number of col-onies present at day 3 after plating was ascertained and the colonieswere scored as undifferentiated, partially differentiated, or fullydifferentiated. As shown in Figure 1J, clonogenicity of the nulllines was markedly lower and, in addition, SOCS-3–null ES cellsgave rise to a higher proportion of colonies containing differenti-ated cells. To assess LIF responsiveness in the null lines, cells wereplated in different concentrations of LIF, and colony morphologywas scored daily. At 10 units of LIF, both WT and SOCS-3–nullES cell cultures contained �5% undifferentiated colonies. In1000–5000 units of LIF, �5% of WT ES cell colonies weredifferentiated but 50% of SOCS-3–null colonies were partiallydifferentiated (Fig. 1K). Culture of the SOCS-3–null ES cells in

high concentrations of LIF (up to 50,000 units) did not alter thepercentage of partially differentiated colonies (not shown).

SOCS-3–Null ES Cells Differentiate into Endodermin the Presence of LIFWhen murine ES cells are propagated in standard conditionswith FCS and LIF, minimal differentiation is observed. Incultures of SOCS-3–null ES cell lines, however, we observed agreater proportion of partially differentiated ES cell colonies,together with dispersed, refractile cells with stellate morphol-ogy, reminiscent of primitive endoderm cells [44]. To establishthe identity of the differentiated cell types we used RT-PCR toexamine the expression of genes associated with the undiffer-entiated state or with differentiation into different cell lineages.In keeping with the observation that SOCS-3–null ES cellscould be cultured continuously and formed colonies containingcells with typical ES cell morphology, the pluripotential cellmarkers Nanog, Oct4, and Zfp42 were expressed. Markers ofmesoderm (Brachyury, Mixl1), neuroectoderm (Isl1, Sox1,Otx2), and trophectoderm (Hand1) were not detected in theSOCS-3–null ES cell lines. In contrast, there was a strikingupregulation of endoderm-specific gene expression. Endoder-mal transcription factors Tcf2, Gata4, Gata6, Hnf4a, andCOUPTF1 were upregulated. Markers of parietal endoderm,Lamb1–1 and Dab2, were expressed, but visceral endodermmarkers, Afp and Ttr, were not, suggesting that the differentiatedcells arising in the SOCS-3–null ES cell cultures were of theprimitive endoderm type (Fig. 2A). Immunohistochemical anal-ysis detected Oct4 protein in WT and SOCS-3–null ES cells(Fig. 2B–2D). GATA-4 protein was not detectable in WT EScells, but in SOCS-3–null cultures was detectable in cells at theedge of partially differentiated colonies (Fig. 2E–2G). GATA-4,but not Oct4, was detectable in the dispersed cells present in the

Figure 2. In the absence of SOCS-3, ES cells undergo differentiation to primitive endoderm. (A): Reverse transcription-polymerase chain reactionanalysis demonstrates that SOCS-3–null ES cells express typical stem cell markers and show increased expression of primitive endoderm markers.(B–G): Indirect immunofluorescence was utilized to detect Oct4 (red) and GATA4 (green) in wild-type and SOCS-3–null ES cell cultures. In mergedimages (H–J) and (B, E, H) Oct-4 but not GATA-4, is detectable in WT ES cell colonies. (C, F, I): In a partially differentiated SOCS-3–null EScell colony, Oct-4 is readily detectable and GATA4 is seen in cells at the periphery of the colony. (D, G, J): Undifferentiated Oct-4 positiveSOCS-3–null ES cell colony and dispersed, differentiated cells with typical endodermal morphology (arrows) in which GATA-4 protein is detected.Nuclei of all cells in (B–G) are stained with 4�,6-diamidino-2-phenylindole. Abbreviations: SOCS3, suppressor of cytokine signalling 3; ES,embryonic stem cell; �ve, positive control (embryoid body or embryonic day 9 cDNA); �ve, negative (no cDNA) control; WT, wild-type. Scalebars � 50 �m.

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SOCS-3–null ES cell cultures, thereby establishing their identityas endoderm. (Fig. 2H–2J).

To extend the analysis of transcriptional differences betweenSOCS-3–null and WT ES cells, gene expression in WT and SOCS-3–null ES cell lines grown in standard ES cell culture medium ona feeder layer was compared. The gene expression levels of 45,000known and predicted gene transcripts present on the AffymetrixMOE430 GeneChip were assayed, and expression of a large num-ber of genes was significantly different in the null lines (supple-mental online Fig. 2). The 161 genes upregulated and 61 genesdownregulated more than fivefold in the SOCS-3–null ES cell linesare listed in supplemental online Table 1 and Table 2. As indicatedby the RT-PCR results, early endoderm-specific genes were highlyupregulated in the SOCS-3–null ES cell lines, but genes character-istically expressed in embryonic definitive endoderm (e.g., Afp,Serpini1, Alb1, Ipf1) were not upregulated, further demonstratingthat a major consequence of the absence of SOCS-3 is the inductionof a proportion of ES cells to adopt a primitive endodermal fate(supplemental online Table 1). Of the 161 upregulated genes, 25%had previously been documented to be expressed in endoderm ofthe preimplantation embryo, or during differentiation of embryoidbodies or embryocarcinoma cell lines to form endoderm [45–48](supplemental online Table 1). In keeping with the role of

endoderm in the production of basement membrane, there was aprominent increase in the expression of genes encoding secretory/extracellular matrix (ECM) proteins, and enzymes associated withthe post-translational modification of ECM molecules [48]. Theexpression of genes associated with trophectoderm was similar inWT and mutant cell lines, and genes marking the neuroectodermaland mesodermal lineages were not upregulated. Expression ofgenes associated with pluripotency, including Nanog, Oct4, Zpf42,and Foxd3, was not significantly different in the WT and SOCS-3–null ES cell lines. None of the other seven members of the SOCSgene family showed altered gene expression in the SOCS-3–nullES cells, indicating that functional compensation by other SOCSgenes was unlikely to be contributing to the observed phenotype(supplemental online Table 2; data not shown).

SOCS-3–Null ES Cell Lines Can Differentiate intoCells Derived from All Three Germ LayersTo determine whether the absence of SOCS-3 affects ES celldifferentiation potential, expression of cell lineage–specific markergenes was assessed in WT and SOCS-3–null ES cell cultures afterLIF withdrawal. In addition, the lines were cultured at low densityin differentiation medium to allow formation of embryoid bodies(Fig. 3A). Embryoid bodies formed at 5- to 10-fold lower frequen-

Figure 3. SOCS-3–null ES cells retain the capacity to differentiate into cells derived from all three germ layers. (A): WT and SOCS-3–null embryoidbodies. (B): Reverse transcription-polymerase chain reaction analysis showing gene expression in WT and SOCS-3–null ES cells maintained in LIF(�LIF) and after LIF withdrawal (�LIF) and in day 6 embryoid bodies (D6 EB). (C–H): SOCS-3–null ES cells were injected s.c. into nude mice,and the resulting teratomas were fixed, sectioned, and stained with hematoxylin and eosin. Tissues derived from neuroectoderm (C), mesoderm (E,F), and endoderm (G) were present. (C): Primitive neuroepithelial tubes. (D): Cartilage. (E): Smooth muscle. (F): Ciliated epithelium. (G): Gutepithelium. (H): SOCS-3–null embryonic stem cells and WT ES cells (not shown) also gave rise to trophoblast tissue. Abbreviations: SOCS-3,suppressor of cytokine signalling 3; WT, Wild-type; LIF, Leukemia inhibitory factor; SOCS-3–null ES cells maintained in LIF; D6 EB, in day 6embryoid bodies; ne, neuroepithelium; c, cartilage; ce, ciliated epithelium; ge, gut epithelium; tgc, trophoblast giant cells. Magnification: �200.

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cies from SOCS-3–null ES cell lines (data not shown). RNA wasprepared from adherent cultures 5 days after LIF withdrawal andfrom embryoid bodies at day 6, and RT-PCR was performed.Differentiated WT and SOCS-3–null ES cell cultures and em-bryoid bodies expressed markers of mesoderm, ectoderm, andendoderm (Fig. 3B). To evaluate the differentiation potential ofthe SOCS-3–null ES cell lines in vivo, we injected ES cells s.c.into nude mice to induce the formation of teratomas. Thesebenign tumors contain well differentiated tissues of ectodermal,mesodermal, and endodermal origin [49]. Differentiation pro-files of the resulting teratomas were assessed by histologicalexamination. Both WT and SOCS-3–null teratomas had a het-erogeneous differentiation profile, with cells of ectodermal lin-eage (neuronal structures), mesodermal lineage (smooth muscle,cartilage), and endodermal lineage (goblet cells, respiratoryepithelium) (Fig. 3C–3G). Both WT and SOCS-3–null ES cell–derived teratomas also contained trophoblast cells (Fig. 3H).When injected into genetically marked blastocysts, the SOCS-3–null ES cells contributed to all tissues of chimera embryos(data not shown). Together, the results indicate that the absenceof SOCS-3 does not affect the capacity of ES cells to differen-tiate into tissues derived from all three germ layers.

Greater STAT-1, STAT-3, and SHP-2 Activation inthe Absence of SOCS-3Western blots were performed using lysates prepared from feed-er-independent WT and null cell lines cultured in ES cell me-

dium with LIF (steady state) and from cultures that had beencytokine and serum starved for 4 hours prior to readdition of LIF(1000 units/ml) (Fig. 4). SOCS-3 was present in WT ES cells insteady state and was detected at 30 minutes after LIF stimula-tion. Steady-state levels of phosphorylated STAT-1 and STAT-3were higher in the SOCS-3–null ES cell lines (Fig. 4). Afterstarvation and LIF addition, STAT-3 phosphorylation in WTcells was maximal between 5 and 30 minutes, but by 1 hour hadreturned to the level seen during routine culture. In contrast,SOCS-3–null ES cells showed a blunted response to readditionof LIF, exhibiting sustained STAT-3 phosphorylation. After LIFstimulation, ongoing STAT-1 phosphorylation was observed inthe SOCS-3–null ES cells.

SHP-2 was immunoprecipitated from cellular extracts ofWT and SOCS-3–null ES cells, and SHP-2 phosphorylationwas analyzed by Western blotting. Strikingly, in steady-stateculture conditions, SHP-2 phosphorylation was detectable inSOCS-3–null ES cells but not in WT cells. LIF stimulation ofWT cells induced SHP-2 phosphorylation that was rapidlyattenuated, whereas in SOCS-3–null ES cells, there was asustained induction of SHP-2 phosphorylation. The absenceof SOCS-3 in ES cells also resulted in greater steady-stateMAPK activation. Phosphorylated ERK-1 and ERK-2 pro-teins were present in greater amounts in steady-state SOCS-3–null ES cells, and in LIF readdition experiments, phos-phorylation of ERK-1/2 lasted longer than with WT cells

Figure 4. LIF signalling is deregulated SOCS-3–null ES cells. Feeder-independent cell lines were grown to near confluence over 48 hours in standardES cell culture medium, after which they were washed and placed into embryonic stem cell medium containing 0.5% serum without LIF for 4 hours.After starvation, 1000 units/ml LIF was added, and cell lysates were prepared at the indicated times thereafter. Lysates were separated by SDS-PAGE,blotted, and probed with antibodies specific to SOCS-3 or to pSTAT-3, pSTAT-1, or pERK-1/2. Membranes were stripped and reprobed withantibodies to total STAT-3, STAT-1, and ERK-1/2. Total SH2 domain–containing SHP-2 was immunoprecipitated from cell lysates, separated bySDS-PAGE, and probed with antibodies specific to phosphorylated SHP-2 (pSHP-2) or total SHP2. �LIF indicates cells maintained in standardculture conditions throughout, �LIF indicates cells harvested after 4 hours of starvation without restimulation. Histograms show densitometricquantitation of mean and standard deviation of the signal from three Western blots. The phosphorylation level is reported as a percentage relative tototal STAT-1, STAT-3, SHP-2, or ERK-1/2 protein. Abbreviations: LIF, Leukemia inhibitory factor; SOCS-3, suppressor of cytokine signalling 3;pSTAT, phosphorylated signal transducer and activator of transcription; pERK-1/2, phosphorylated forms of extracellular signal–related kinase 1/2;ERK; extracellular signal–regulated kinase; SHP-2, cytoplasmic SH2-containing protein tyrosine phosphatase 2; WT, wild type.

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(Fig. 4). In keeping with previous observations, ERK-2was the predominant phosphorylated ERK detected in EScells.

Addition of Ras–MAPK Inhibitors Rescues theSOCS-3–Null ES Cell Differentiation PhenotypeSHP-2–Ras–ERK-1/2–MAPK signalling provides a differentia-tive signal in ES cells. To establish if the upregulation of thispathway in SOCS-3–null ES cells was responsible for theirphenotype, WT and null ES cell lines were treated with theMEK inhibitors PD98059 or U0126 [50, 51]. Feeder-indepen-dent WT and null ES cells were cultured in the presence ofinhibitor or carrier (DMSO) and assessed for the presence ofdifferentiating colonies. The SOCS-3–null ES cell differentia-tion phenotype was completely reversed by the addition ofeither inhibitor to the culture medium (Fig. 5A–5H). This wasconfirmed by RT-PCR analysis, which showed that treatmentwith MEK inhibitors reduced Gata4 expression in the SOCS-3–null lines to a level similar to that observed in WT cells (Fig.5I). Immunoblotting of lysates from treated cells showed lessERK-1/2 phosphorylation in the SOCS-3–null ES cells aftertreatment with inhibitor (Fig. 5J). To assess whether MEKinhibition affected proliferation and clonogenicity, PD98059- orU0126-treated WT and SOCS-3–null cells were plated at lowdensity; the number of cells per well was counted daily and thecolony number and morphology on day 3 after plating wasscored (Fig. 5K, 5L). Addition of U0126 or PD98059 (notshown) reduced proliferation and clonogenicity in the WT celllines. This effect was observed even at the lowest concentrationof either inhibitor that was sufficient to inhibit ERK-/2 phos-phorylation (not shown). In contrast to the effect on the differ-entiation phenotype, the lower proliferative capacity and clono-genicity of the SOCS-3–null lines was not rescued by inhibitionof Ras–ERK-1/2–MAPK signalling.

DISCUSSIONTo ascertain the role of SOCS-3 in ES cells we generated SOCS-3–null ES cell lines. Null cell lines could not be derived by genetargeting but were successfully isolated from preimplantation blas-tocysts. Our inability to derive doubly targeted clones by genetargeting likely reflects the lower proliferation capacity and clono-genicity of SOCS-3–null ES cells. SOCS-3–null ES cell linesexhibited less self-renewal and a greater propensity to differentiateinto endoderm. Undifferentiated SOCS-3–null ES cells expressedstem cell markers and retained the capacity to differentiate intotissues of ectodermal, mesodermal, and endodermal origin in vitroand in vivo. Strikingly, the differentiative phenotype could berescued by inhibition of MAPK signalling.

The ablation of SOCS-3 resulted in greater intensity andduration of activation of both the JAK–STAT and the Ras–ERK-1/2–MAPK signalling cascades both in the steady stateand in response to LIF signalling. The null cells exhibitedhigher STAT-3 activation in steady-state cultures and sus-tained activation of STAT-3 after starvation and LIF readdi-tion. Unlike WT cells, STAT-1 was constitutively phosphor-ylated. Phosphorylated SHP-2 was not detectable in WT EScells maintained in LIF and serum, but in the absence ofSOCS-3, phosphorylated SHP-2 was readily detectable, pre-sumably as a result of a lack of competition from SOCS-3 forbinding to Y757 of gp130. After cytokine stimulation, greater

and sustained SHP-2 activation was observed in the nullcells, whereas in WT cells, SHP-2 phosphorylation waned asSOCS-3 was upregulated. Downstream of SHP-2, ERK-1/2phosphorylation was greater in the null cells. In other sys-tems, SOCS-3 has been shown to positively regulate MAPKactivation. In human T cells, SOCS-3 is tyrosine phosphor-ylated in response to multiple stimuli and binds to andinactivates Ras/GTPase-activating protein (GAP), leading toRas–ERK-1/2–MAPK activation [52]. It is not knownwhether a similar mechanism operates in murine ES cells.

STAT and SHP-2–Ras–ERK signalling via gp130 are keyregulators of cellular homeostasis. In vitro, the simultaneousactivation of Ras–ERK–MAPK and STAT-1/3 has been re-peatedly shown to generate opposing signals, the balance ofwhich determines the biological outcome and, in vivo, bal-anced activation of the two signalling cascades is required toprevent disease [53–56]. In murine ES cells, opposing signalsvia the JAK–STAT and Ras–ERK–MAPK pathways contrib-ute to the regulation of self-renewal and differentiation [57,58]. STAT-3 is a critical mediator of LIF-induced signallingpathways that regulate ES cell self-renewal. Disruption ofSTAT-3 activation abrogates the ability of LIF to maintainself-renewal of ES cells while constitutive expression ofactivated STAT-3 prevents differentiation of ES cells afterwithdrawal of LIF [5–7]. Conversely, activation of Ras–ERK–MAPK signalling results in ES cell differentiation.Overexpression of a mutant Ras that stimulates the ERK-1/2–MAPK pathway activates expression of endoderm tran-scription factors in ES cells and embryoid bodies, and ex-pression of dominant negative Ras suppresses expression ofendoderm transcription factors [59, 60]. When Ras–ERK–MAPK signalling in ES cells is attenuated, the self-renewalresponse is enhanced [10]. Furthermore, genetic disruption ofGrb2 or Shp2 in ES cells enhances self-renewal and impairsdifferentiation [11, 59]. Inhibition of ERK-1/2–MAPK sig-nalling does not replace the requirement for STAT-3 signal-ling, but rather works synergistically with it.

In SOCS-3–null ES cells, constitutive upregulation of Ras–ERK-1/2–MAPK signalling provides a differentiative signal.This is opposed by augmented self-renewal signals via phos-phorylated STAT-3. However, increased STAT-3 activation isnot sufficient to prevent differentiation when Ras–ERK-1/2–MAPK signalling is activated. We hypothesize that this mixtureof self-renewal and differentiative signals drives the phenotypeobserved in the SOCS-3–null ES cell cultures and that alter-ations in the thresholds of self-renewal and differentiative sig-nals in individual cells result in the partial differentiation phe-notype that can be reversed by pharmacological inhibition ofMEK. During continuous subculturing, the undifferentiatedcells in SOCS-3–null ES cell cultures would be expected to havea preferential replating advantage over the more differentiatedcells, thus supporting their long-term dominance [61].

ES cell proliferation was markedly lower in the absence ofSOCS-3 despite high levels of steady-state STAT-3 phosphoryla-tion. The role of STAT-3 activation in ES cell proliferation isunclear. While it is well established that less STAT-3 activationleads to abrogation of ES cell self-renewal, it has not been shownto affect proliferation [5, 6], and greater STAT-3 activation maynegatively affect proliferation [10]. The lower proliferation ob-served in the SOCS-3–null ES cell lines may be in part a result of

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dysregulated STAT-3 activation, but it is likely that alterations inother, as yet unidentified, signals also affect proliferation.

LIF is only able to sustain ES cells in the presence of serum,suggesting that additional factors are required. Recently, evidencehas emerged that bone morphogenetic proteins (BMPs) may act incombination with LIF to sustain self-renewal of murine ES cells by

inducing the expression of inhibitors of differentiation (Id) genes[62]. In addition, the Wnt signalling pathway appears to sustain EScell pluripotency [63]. Intriguingly, the homeodomain proteinNanog has been shown to maintain ES cell self-renewal, inde-pendently of the LIF–STAT-3 and BMP-4 pathways, although acombination of LIF signalling and Nanog expression promotes

Figure 5. Treatment of SOCS-3–null embryonic stem (ES) cells with mitogen-activated protein kinase/ERK kinase (MEK) inhibitors preventsdifferentiation into endoderm. (A–H): Feeder-independent wild-type (WT) and SOCS-3–null ES cell lines were trypsinized and replated in ES cellmedium (�) with or without the MEK inhibitors PD98059 or U0126 or the carrier DMSO. (I): Reverse transcription-polymerase chain reactionanalysis of gene expression in WT and SOCS-3–null lines after treatment with U0126 shows that Gata4 expression in treated SOCS-3–null lines, butnot in controls, is reduced to near WT levels. (J): Cell lysates prepared from WT and SOCS-3–null ES cell lines prior to and after treatment withU0126 were separated by SDS-PAGE, blotted, and probed with antibodies specific to phosphorylated forms of ERK-1/2 (pERK-1/2). Membraneswere stripped and reprobed with antibodies to total ERK-1/2. Note the difference in the amount of total lysate loaded in the WT and SOCS-3–nulllanes. (K, L): WT and SOCS-3–null ES cells were treated with MEK inhibitors and then plated at low density. W, wild-type cells; N, SOCS-3–nullES cells. Cell (K) and colony (L) counts were performed daily to assess proliferation and response to leukemia inhibitory factor (LIF) withdrawal.Data shown in (L) are the mean triplicate wells from a representative experiment. Similar results were obtained with two WT and four SOCS-3–nullES cell lines. Abbreviations: SOCS-3, suppressor of cytokine signalling 3; DMSO, dimethylsulfoxide; W and WT, wild type; ERK, extracellularsignal–regulated kinase; N, SOCS-3 null. Scale bars � 25 �m.

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maximal self-renewal efficiency [62, 64, 65]. The analysis of theSOCS-3–null ES cell phenotype reported here was conducted usingES cell media supplemented with FCS. In future experiments, itwill be important to dissect the contribution of individual factors tothe SOCS-3–null phenotype.

The in vivo relevance of the LIF signalling pathway forearly embryo development is uncertain. During embryogenesis,the inner cell mass (ICM) exists transiently and does not act asa long-term stem cell compartment. It is not clear whether apopulation equivalent to the ES cell ever exists in vivo. NeitherLIF, LIFR-�, nor gp130 mutants show defects in the develop-ment of the ICM or early epiblast [66–69]. Similarly, SOCS-3–null blastocysts develop normally into the multilayered em-bryo [19, 20]. The LIF pathway is, however, required forsurvival of the ICM during implantation delay (diapause), and itwill be interesting to ascertain whether this process is affected inSOCS-3–null blastocysts [70].

In summary, activation of cytokine signalling pathways inmurine ES cells alters cell fate and potency. Results of SOCS-3overexpression in ES cells, together with the data presented hereon the effects of SOCS-3 ablation in ES cells, point to a key role

for SOCS-3 in regulating signals emanating from the LIFR-�–gp130 receptor complex in ES cells. In doing so, SOCS-3regulates ES cell self-renewal and insulates ES cells from thefunctional consequence of lineage priming via the Ras–ERK–MAPK signalling pathway.

ACKNOWLEDGMENTSWe thank Lucille Vollaire for karyotyping, Ruili Li for blastocystinjections, and Janelle Lochland for genotyping. We also thankTim Thomas for genotyping the ES cell lines—derived de novofrom SOCS-3 heterozygous intercrosses. We are grateful to Prof.Terry Speed and Dr. Gordon Smyth for discussion regarding anal-ysis of the microarray data and Profs. Nicos Nicola and DougHilton for comments on the manuscript. The project was supportedby the National Health and Medical Research Council of Australiaprogram grant 257500, NIH grant CA22556 and Zenyth Therapeu-tics Limited. A.F. and K.B. contributed equally to this work.

DISCLOSURESThe authors indicate no potential conflicts of interest.

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