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1193DEVELOPMENT AND DISEASE RESEARCH ARTICLE

INTRODUCTIONHuman embryonic stem cells (HESCs) are pluripotent cells derivedfrom the inner cell mass (ICM) of blastocyst-stage embryos(Reubinoff et al., 2000; Thomson et al., 1998). These cells have twodistinctive properties: an unlimited capacity for self-renewal andpluripotency (the ability to differentiate to each of the threeembryonic germ layers, endoderm, mesoderm and ectoderm).Pluripotency has been shown in vitro by inducing undifferentiatedES cells to form embryoid bodies (EBs) composed of cells from thethree germ layers (Itskovitz-Eldor et al., 2000; Schuldiner et al.,2000), and in vivo by injecting undifferentiated cells toimmunodeficient mice where they form tumors termed teratomas,composed of multiple differentiated cell types (Reubinoff et al.,2000; Thomson et al., 1998). Additionally, numerous studies havedescribed differentiation of HESCs to specific cell types usinggrowth factors (for reviews, see Schuldiner and Benvenisty, 2003).Thus, HESCs hold the promise to serve as a source of cells intransplantation medicine. As they mimic in vitro the onset of earlydevelopment they are also viewed as a promising model for earlyhuman development, during stages that are not accessible toresearch.

The propagation of HESCs requires the presence of feeder cells(e.g. mouse embryonic fibroblasts, MEFs) or addition of mediaconditioned by them (Xu et al., 2001). The factors secreted by theMEFs and required by HESCs are not fully characterized, althoughsome factors have been suggested to inhibit the differentiation ofcells (Amit et al., 2004; Sato et al., 2004; Xu et al., 2005). One suchfactor is bFGF, which has been suggested to enable HESCspropagation in the absence of conditioned media (Wang et al., 2005;Xu et al., 2005).

In addition, the intracellular factors that maintain pluripotencyand self-renewal of HESCs remain elusive. One factor known to beinvolved in maintaining pluripotency is OCT4, the downregulationof which in both mouse and human ES cells causes celldifferentiation (Matin et al., 2004; Niwa et al., 2000). However, sincethe derivation of HESCs, it has become apparent that knowledgeaccumulated on mouse embryonic stem cells (MESCs) cannotautomatically be deduced for HESCs. One fundamental differenceobserved so far is the role of LIF, which in MESCs substitutes theneed for support by feeder cells (Smith et al., 1988; Williams et al.,1988). In MESCs, LIF activates Stat3 signaling, which is sufficientto replace the requirement for feeder cells (Niwa et al., 1998). Bycontrast, in HESCs neither the addition of LIF nor the activation ofSTAT3 are able to release the cells from dependence on feeder cells(Daheron et al., 2004; Humphrey et al., 2004). BMP4 is anotherfactor shown to be involved in maintenance of self-renewal inMESCs, by inhibiting neural differentiation in serum-free media(Ying et al., 2003). However, when added to HESCs, BMP4 wasshown to actually promote differentiation to trophectoderm even inthe presence of MEF-conditioned media (Xu et al., 2002).Additional differences between these cells exist, among them aredifferences in expression of cell surface markers (Ginis et al., 2004)and a different differentiation potential, because HESCs, as opposedto MESCs, are capable of differentiating to trophoblast (Xu et al.,2002).

A gene recently shown to be fundamental in maintainingpluripotency in MESCs is Nanog (Chambers et al., 2003; Mitsui etal., 2003). Overexpression of Nanog releases the cells from LIFdependency, and prevents differentiation upon LIF withdrawal. Inaddition, its downregulation leads to differentiation to extra-embryonic endoderm. Nanog does not function via the Stat3pathway, but cooperates with it in maintaining ES cell identity. Itwas suggested that NANOG may have a key role in sustaining EScell identity also in HESCs, and that it may have taken over the roleof STAT3, which does not seem to be involved in HESC self-renewal.

Overexpression of NANOG in human ES cells enablesfeeder-free growth while inducing primitive ectodermfeaturesHenia Darr, Yoav Mayshar and Nissim Benvenisty*

Human embryonic stem cells (HESCs) are pluripotent cells derived from the ICM of blastocyst stage embryos. As the factors neededfor their growth are largely undefined, they are propagated on feeder cells or with conditioned media from feeder cells. This is incontrast to mouse embryonic stem cells (MESCs) where addition of leukemia inhibitory factor (LIF) replaces the need for a feederlayer. Recently, the transcription factor Nanog was suggested to allow LIF and feeder-free growth of MESCs. Here, we show thatNANOG overexpression in HESCs enables their propagation for multiple passages during which the cells remain pluripotent.NANOG overexpressing cells form colonies efficiently even at a very low density, an ability lost upon excision of the transgene. Cellsoverexpressing NANOG downregulate expression of markers specific to the ICM and acquire expression of a marker specific to theprimitive ectoderm (the consecutive pluripotent population in the embryo). Examination of global transcriptional changes uponNANOG overexpression by DNA microarray analysis reveals new markers suggested to discriminate between these populations.These results are significant in the understanding of self-renewal and pluripotency pathways in HESCs, and of their use formodeling early development in humans.

KEY WORDS: NANOG, Human embryonic stem cells, Self-renewal, Primitive ectoderm

Development 133, 1193-1201 (2006) doi:10.1242/dev.02286

Department of Genetics, Institute of Life Sciences, The Hebrew University, Jerusalem,Israel

*Author for correspondence (e-mail: nissimb@mail.ls.huji.ac.il)

Accepted 12 January 2006

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In this study, we examined the role of NANOG in HESC self-renewal and pluripotency. NANOG is used as a marker forundifferentiated HESCs but its role in these cells is not yet fullycharacterized. Although it has been shown that its downregulationleads to differentiation of HESCs (Zaehres et al., 2005), the result ofits overexpression has not yet been examined. We show thatoverexpression of NANOG enables the propagation of HESCs formultiple passages in the absence of feeder cells or conditioned media(CM). These cells grow as colonies derived from single cells evenin the absence of CM, and lose this ability when the transgene isexcised. Additionally, we show that NANOG expression in wild-typecells is upregulated during early differentiation, and that itsoverexpression in HESCs modifies the expression of marker genesto an expression pattern similar to that of primitive ectoderm cells.Using microarray analysis, we suggest new marker genes that maydistinguish between the ICM and primitive ectoderm cells in human.

MATERIALS AND METHODSCell cultureHuman ES cells (H9 and H13 cell lines) (Thomson et al., 1998) werecultured on Mitomycin-C treated mouse embryonic fibroblast (MEF) feederlayer (obtained from 13.5 day embryos) in 85% KnockOut DMEM medium(GIBCO-BRL), supplemented with 15% KnockOut SR (a serum-freeformulation) (GIBCO-BRL), 1 mM glutamine, 0.1 mM �-mercaptoethanol(Sigma), 1% nonessential amino acids stock (GIBCO-BRL), Penicillin (50units/ml), Streptomycin (50 �g/ml), ITSX100 (insulin-transferrin-selenium)in a 1:200 dilution (GIBCO-Invirogen Corporation), and 4 ng/ml basicfibroblast growth factor (bFGF, PeproTech). To obtain a feeder-free culture,the cells were plated on laminin (1 �g/cm2, Sigma) or gelatin (0.1%, Merck)coated plates and grown in media conditioned for at least 24 hours by MEFs.Differentiation in vitro into embryoid bodies (EBs) was performed bywithdrawal of bFGF from the growth media and allowing aggregation inpetri dishes. Differentiation in vivo into teratomas was performed byinjecting undifferentiated ES cells under the kidney capsule ofimmunodeficient mice and taken out for analysis 2 months later.

Transfections and clone establishmentWild-type ES cells were transfected using the calcium phosphate method asdescribed previously (Chen and Okayama, 1988). The cells were transfectedwith a plasmid described earlier (Chambers et al., 2003) that contains ahuman NANOG transgene followed by an IRES and a puromycin resistancegene. This transcriptional unit is located between two lox-P sites. FollowingCRE-recombinase excision there is removal of both the NANOG gene andpuromycin resistance, and the transcriptional activation of a GFP gene.NANOG overexpressing clones were established by puromycin selection(0.3 �g/ml, Sigma) following transfection. Revertant clones wereestablished by transfecting NANOG overexpressing clones with a CRE-recombinase plasmid. Following transfection, the cells were trypsinzed tosingle cells and seeded in low densities (1:1000 split). Cells expressing GFPwere selected, expanded and verified to have lost puromycin resistance.

Growth analysisCells were seeded in 96-well dishes coated with 0.1% gelatin in a density of2�104 cells per cm2 and medium was changed daily. Final cell densitieswere determined by fixating the cells with 0.5% glutardialdehyde (Sigma)and staining with Methylene Blue (Sigma) dissolved in 0.1 M boric acid (pH8.5). Color extraction was performed using 0.1 M hydrochloric acid, andstaining (which is proportional to cell number) was quantified by measuringabsorbance at 650 nm. Each experiment was performed in triplicate.

Colony forming assaysH9 and H13 Cells were trypsinized to a single cell suspension and seeded in12-well dishes to a density of 500 cells per cm2. After 8 days, the cells werefixed and assayed for alkaline phosphatase activity (86R kit, Sigma)according to the manufacturer’s instructions. Each experiment wasperformed in triplicate and at the end of the experiments the positivelystained colonies were counted.

RNA extraction and RT-PCR analysisRNA was extracted using TRI-reagent for total RNA isolation according tothe manufacturer’s instructions (Sigma). cDNA was synthesized usingrandom hexamer primers. Ampification was performed on the cDNA usingTakara Ex-Taq. PCR conditions include a first step of 3 minutes at 94°C, asecond step of 25-30 cycles of 30 seconds at 94°C, 45 seconds annealingstep at 58-64°C, 1 minute at 72°C and a final step of 7 minutes at 72°C.GAPDH was used as a housekeeping gene to evaluate and compare qualityof different cDNA samples. Primers and product sizes are listed in Table S1in the supplementary material. Final products were examined by gelelectrophoresis on 2% agarose ethidium bromide-stained gels. Real-time RT-PCR analysis was performed using Rotor-gene 2000 (Corbett Research,Sydney). The reaction was carried out according to the manufacturer’sprotocol using Absolute SYBR Green Rox Mix (from ABgene, usedaccording to the manufacturer’s recommendations) with PCR program asfollows: 95°C for 5 minutes; a second step of 35 cycles of 20 seconds at95°C, 15 seconds annealing step at 60°C, 25 seconds at 72°C and 15 secondsat 82°C; and a final step of 1 minute at 72°C.

Immunostaining and FACS analysisFor immunostaining cells were washed once with PBS and fixed with 4%paraformaldehyde. Blocking and permeabilization were performed with 2%BSA, 10% low-fat milk and 0.1% Triton-X in PBS. Staining with primarymouse anti-human OCT4 was performed for 1 hour (Santa CruzBiotechnology, used at a 1:50 dilution). As a secondary antibody, Cy3-conjugated goat anti-mouse IgG (H+L; Jackson ImmunoResearch, dilution1:200), was used. Nuclear staining was performed with Hoechst 33258(Sigma). FACS analysis for TRA-1-60 expression was performed aftertrypsinization of the cells. The cells were washed with 3% BSA in PBS with0.05% Sodium Azid, incubated with TRA-1-60 antibody (kind gift fromProf. Peter Andrews) for 1 hour, incubated with Cy3-conjugated rabbit anti-mouse IgM (Jackson Immunoresearch) and after washes analyzed using theFACSCalibur system (Becton Dickinson). Analysis was performed onCELLQUEST software (Becton Dickinson). Forward- and side-scatter plotswere used to exclude dead cells and debris from the histogram analysis.

Western blot analysisWestern blot analysis was performed according to standard protocols. ForNANOG detection a polyclonal rabbit antibody against human NANOGprotein (abcam) in 1:1000 dilution was incubated for 18 hours. A secondaryantibody (peroxidase conjugated Affinipure goat anti-rabbit IgG by JacksonImmunoresearch) was incubated for 1 hour in 1:10000 dilution. For loadingcontrol we used a mouse antibody against �-TUBULIN (Sigma).

DNA microarray analysisTotal RNA was extracted according to the manufacturers protocol(Affymetrix). When extracting RNA from undifferentiated ES cells, the cellswere grown for one passage on gelatin-coated plates with conditioned mediain order to avoid contamination by feeder cells. Hybridization to the U133ADNA microarray, washing and scanning were performed according to themanufacturer’s protocol, and expression patterns were compared betweensamples. Signals were normalized by dividing each probe by the averagevalue of the chip to avoid differences between different chips andexperiments.

RESULTSEstablishment of NANOG over-expressing clonesH9 HESCs were transfected with a plasmid harboring a humanNANOG transgene transcribed from a constitutive promoter andfollowed by a puromycin resistance gene (Chambers et al., 2003).These sequences are enclosed by lox-P sites, and upon CRE-recombinase excision they are removed, causing a GFP gene to beactivated (Chambers et al., 2003). NANOG overexpressing stableclones were isolated following selection with puromycin andexpression of the NANOG transgene was verified by RT-PCRanalysis using specific primers to the transgene (Fig. 1A, part I). Asthe puromycin resistant gene lies on the same transcriptional unit as

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the NANOG transgene, constant selection with puromycinguaranteed stable expression of the transgene over time. To asses theoverexpression level of the transgene in different clones, weperformed real-time RT-PCR analysis and observed that the totallevel of NANOG mRNA in the clones was significantly higher thanin wild-type cells (Fig. 1A, part II). Concurrently, elevated proteinlevels were observed in the clones by western blot (Fig. 1A, part III).It has previously been shown that Nanog overexpression releasesMESCs from LIF dependency (Chambers et al., 2003; Mitsui et al.,2003). However, regulation of self-renewal in HESCs is not identicalto that in MESCs. Thus, we set out to determine the role of NANOGin HESCs. HESCs are grown on mouse embryonic fibroblasts(MEFs) or in the presence of media conditioned by MEFs.Therefore, we first examined whether the requirement of cells forconditioned media (CM) could be replaced by overexpression ofNANOG transgene. When wild-type and NANOG overexpressingcells were grown in the presence of CM, both cell types showedsimilar growth rates (Fig. 1B, upper panel). However, when CM wasremoved, wild-type cells ceased to proliferate, while NANOGoverexpressing cells kept dividing (Fig. 1B, lower panel) eventhough their growth rate was markedly decreased. In the presence offeeder cells, overall colony morphology of the NANOGoverexpressing clones was very similar to that of wild-type cells,albeit being slightly flatter (see Fig. S1 in the supplementarymaterial). However, in the absence of CM, a striking difference isobserved, as early as a few days after CM withdrawal. While wild-type cells created only very small and partially differentiated

colonies, Nanog overexpressing clones created much largercolonies, a difference that was observed as early as 4 days after CMwithdrawal (see Fig. S1 in the supplementary material).

NANOG over-expressing clones can be seriallypassaged in the absence of conditioned mediawhile maintaining their undifferentiated identityTo verify that NANOG overexpressing cells may indeed proliferatewithout CM, the cells were grown for multiple passages in afeeder-free culture without CM. Under these conditions, wild-typecells ceased proliferating after a few passages and showedcompletely differentiated morphology (Fig. 2A, left panel).However, NANOG clones continued proliferating for more than 20passages without CM, with no apparent decline in growth rate ordoubling time throughout the passages (Fig. 2A, right panel).These cells remained responsive to CM and grew faster in itspresence than in its absence (data not shown). Verification of theundifferentiated identity was assessed using immunostaining forOCT4 (Fig. 2B) and FACS analysis using TRA-1-60 antibody.TRA-1-60 staining showed that the percentage of undifferentiatedcells (TRA-1-60 positive population) in the culture did notdecrease after the withdrawal of feeder cells (Fig. 2C). To furtherexamine whether the cells were still pluripotent, they were assayedfor their capacity to form teratomas after injection to SCID mice.Clone-derived teratomas arose at a comparable rate to thatobserved in wild-type cells and cells grown on feeder cells, andwere composed of different cell types from the three embryonic

1195RESEARCH ARTICLENANOG overexpression

Fig. 1. Establishment of NANOG overexpressing HESC clones and examination of their growth. (A, part I) Establishment of NANOGoverexpressing HESC clones was verified following selection with puromycin by RT-PCR. Amplification was performed using specific primers for theNANOG transgene. GAPDH was used as a positive control. (II) The level of NANOG overexpression was analyzed by real-time RT-PCR to examine thetotal NANOG mRNA level in the cells. The three clones examined showed significant elevation of the mRNA level (*P

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germ layers (Fig. 2D). Furthermore, cell type composition wasvery similar between the wild-type cell- and NANOGoverexpressing cell-derived teratomas.

NANOG over-expressing cells form colonies in theabsence of feedersAs NANOG overexpressing clones proliferate in the absence of CM,we examined whether the cells can also form colonies from singlecells under these conditions. Cells were trypsinized to single cells,

seeded at very low density, and the ability to form colonies wasassayed. Resulting colonies were verified as undifferentiated ES cellsby assaying the activity of the ES cell marker alkaline phosphatase(AP). When wild type and NANOG over-expressing cells were seededin the presence of CM, both were able to create a large number of AP-positive colonies (Fig. 3A). When cells were seeded in medium notconditioned by MEFs and not containing bFGF (basal media), thenumber of colonies formed by the wild-type cells was negligible,while NANOG overexpressing cells were capable of forming a

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Fig. 2. NANOGoverexpressing cells can beserially passaged in theabsence of feeders. (A) Wild-type and NANOGoverexpressing cells wereseeded on laminin-coatedplates at a density of 2�104

cells per cm2 and passaged inthe absence of conditionedmedia. Photos were taken ofthe third passage afterwithdrawal of conditionedmedia. Whereas wild-type cellsstopped proliferating andshowed differentiatedmorphology, NANOGoverexpressing cells continuedto proliferate for more than 15passages and formed colonieswith ES cell morphology.(B) OCT4 expression wasexamined on the 15th passageand was still positive in most ofthe cells. (C) TRA-1-60 stainingperformed 20 passages after CM withdrawal showed that the majority of the cells (more than 90%) remained undifferentiated. (I) MEF cellsonly, (II) wild-type cells grown on feeders, (III) clone grown on feeders, (IV) clone grown for 20 passages without feeders or CM. (D) After 15passages in the absence of CM, the cells still formed teratomas composed of multiple cell types following injection into immunodeficient mice.

Fig. 3. Formation of feeder-freecolonies is dependent on NANOGexpression. (A) Five-hundred cells percm2 from H9 and H13 cell lines wereseeded in 12-well plates coated withgelatin and FCS. After 8 days, the cellswere fixed and stained for alkalinephosphatase (AP) activity. Positivelystained colonies were counted. Theresults shown for H13 clones representthe average of three independentclones. Scale bars represent s.e.m.(*P

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considerably larger number of AP-positive colonies (Fig. 3A,B, APactivity shown as red staining). Therefore, NANOG is capable ofmaintaining ES cells undifferentiated independently of CM, eventhough the frequency of colony formation is lower than with CM. Toconfirm that the effect observed upon NANOG overexpression wasnot unique to one cell line, NANOG overexpressing clones wereestablished in another cell line, H13. As in H9 cells, H13 cells wereable to create colonies in the absence of CM only when transfectedwith a NANOG overexpression vector (Fig. 3A,B).

To verify that the phenotype observed is dependent onoverexpression of NANOG and not on a specific sub-clone, weestablished revertant clones from NANOG overexpressing cells.Through application of CRE-recombinase, we removed the NANOGtransgene, and collected GFP positive colonies (see Materials andmethods). The assay was then repeated for the parental clonesoverexpressing NANOG, and three revertant clones. The results forthe revertant clones were similar to those of wild-type cells, andgrowth in basal media was virtually abolished (Fig. 2C,D). Thisconfirms that the phenotype observed indeed results from theoverexpression of NANOG.

NANOG is upregulated upon early differentiationIn mouse, Nanog acts a stage later than Oct4 during normalembryonic development. Oct4 inhibits differentiation to trophoblastand its knockout in MESCs triggers differentiation to trophoblast(Niwa et al., 2000). Nanog acts to inhibit differentiation to primitiveendoderm and its knockout in MESCs triggers differentiation toprimitive endoderm (Mitsui et al., 2003). We examined whether thistemporal difference is observed in a human system by assaying theexpression pattern of both OCT4 and NANOG genes during thedifferentiation of HESCs. Four time points along their differentiationwere assayed: ES cells, the pluripotent undifferentiated stage; 2-day-old embryoid bodies (EBs), the early stage of differentiation; 10-day-old EBs, which represent mid differentiation; and 30-day-oldEBs, which represent late differentiation (Dvash et al., 2004; Leahyet al., 1999). OCT4 expression is high in ES cells, 2- and 10-day-oldEBs, and drops drastically in 30-day-old EBs (Fig. 4B). However,the expression pattern of NANOG is different. It is expressed in EScells, but its level increases tenfold in 2-day-old EBs, remains highin 10-day-old EBs and drops in 30-day-old EBs (Fig. 4A,B). Thisimplies that NANOG may actually be expressed at the highest levelin early differentiating cells and not in undifferentiated cells.

Overexpression of NANOG alters the markergenes expressed by HESCsFollowing its formation, the ICM differentiates to primitiveendoderm and primitive ectoderm. We hypothesized that NANOGmay be expressed at higher levels in primitive ectoderm than in theICM in human. There are several known marker genes in the mousethat distinguish ICM from primitive ectoderm (Rathjen and Rathjen,2003). Rex1 (Zfp42 – Mouse Genome Informatics), Gbx2 and Crtr1(Tcfcp2l1 – Mouse Genome Informatics) are enriched in ICM, andFgf5 and PRCE are absent from ES cells and ICM, and enriched inprimitive ectoderm (Fig. 5A). When compared with wild-type cells,in NANOG overexpressing cells there is a significant downregulationof REX1 (fourfold) and GBX2 (twofold) and upregulation of FGF5(ninefold) (Fig. 5B,C; see Fig. S2 in the supplementary material).No change in the expression pattern of CRTR1 and PRCE wasobserved (data not shown). The magnitude of the change seemed tocorrelate with the level of overexpression of NANOG, with thehighest overexpression (as assessed by the results shown in Fig. 1)leading to the most significant changes in mRNA levels (clone 9, see

Fig. S2 in the supplementary material) and the clone with the lowestNANOG expression levels showing less significant changes, andonly in two of the markers examined (clone 4, see Fig. S2 in thesupplementary material). To examine if the same phenomenon existsin MESCs, we looked at the expression pattern of markers known todistinguish ICM from primitive ectoderm in wild-type and Nanogoverexpressing MESCs. MESCs overexpressing Nanog wereobtained from the laboratories that demonstrated the effect of Nanogon MESCs (Chambers et al., 2003; Mitsui et al., 2003). However, nochange was observed upon overexpression of Nanog in any of themarker genes examined, in the various cell lines (Fig. 5D).

In mouse, it is known that primitive ectoderm like (EPL) cells arecapable of reverting back to ES cells (as defined by marker geneexpression pattern) (see Rathjen and Rathjen, 2003). We thereforeexamined whether this is also the case in HESCs. The expression ofthe differential markers was examined in the revertant clones derivedfrom NANOG overexpressing clones. However, the expression of theexamined genes remained similar to that of the overexpressingclones, and no reversion to the expression pattern of wild-type cellswas observed (Fig. 5C).

Transcriptome analysis of NANOG over-expressingcells reveals an increase in similarity to earlydifferentiating cellsDNA microarray analysis was performed on RNA extracted fromwild-type and NANOG overexpressing ES cells, and three stages ofdifferentiating embryoid bodies (EBs). Dendogram analysis, whichclusters samples according to their degree of similarity, shows thatupon overexpression of NANOG in HESCs, the similarity to earlydifferentiating cells increases (Fig. 6A). However, the cells do notappear differentiated as they cluster apart from the samples of 2- and

1197RESEARCH ARTICLENANOG overexpression

Fig. 4. NANOG is upregulated upon early differentiation. (A) Theexpression pattern of NANOG during differentiation was examined byreal-time RT-PCR. Four stages of in vitro differentiation were examined:ES, EB2d, EB10d and EB30d. Shown is the abundance of NANOGtranscript normalized to GAPDH levels per sample. Error bars represents.e.m. (B) The expression pattern revealed by real-time RT-PCR wasverified by semi-quantitative RT-PCR under non-saturating conditions.The temporal expression pattern of NANOG was compared with that ofOCT4, which, unlike NANOG, does not show elevation of expressionduring differentiation. NTC (no templates control) was used as anegative control. GAPDH was used as positive control.

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10-day-old EBs. Four groups of genes differentially expressedbetween NANOG overexpressing and wild-type cells were identified(Fig. 6B).

(1) Genes upregulated in both NANOG cells and in 2-day-oldEBs. These may be new markers of primitive ectoderm. 2 genesbelong to this group (Fig. 6C). ALPPL2 (alkaline phosphatase,placental-like 2), a gene expressed in certain germ cell tumors andHSPA1A (heat shock protein 70 kDa protein 1A), which encodes achaperone protein, also involved in the inhibition of apoptosis.

(2) Genes upregulated in NANOG overexpressing cells comparedwith ES cells and 2-day-old EBs. These may representtranscriptional targets of NANOG.

(3) Genes downregulated in NANOG overexpressing cells and 2-day-old EBs compared with wild-type ES cells (Fig. 6D). These maybe ICM (and therefore ES cell) markers that are downregulated uponthe transition to primitive ectoderm. Among these genes are LECTIN(LGALS1), a known marker of HESCs (Dvash et al., 2004) that isabsent from all samples but ES cells, and HOXA1 (homeo box A1),a transcription factor involved in development. Among the genesdownregulated in NANOG overexpressing cells but to a smallerextent are genes suggested to be downstream targets of HOXA1(Shen et al., 2000), such as GBX2 and BMP2.

(4) Genes downregulated in NANOG cells compared with both2-day-old EBs and wild-type ES cells. These genes may also betargets of NANOG, which has been suggested to be both atranscriptional activator and repressor. In addition, genes reportedto be upregulated upon knock-down of Nanog in MESCs (Mitsuiet al., 2003) are downregulated upon overexpression of NANOG inHESCs. These include parietal endoderm markers (like LAMB1,downregulated by 3.5-fold) and visceral endoderm markers (likeBMP2, downregulated by 14-fold). It has been suggested thatNANOG represses expression of primitive endoderm genes inMESCs, and it seems that the same effect is observed in HESCs.

The transition to EPL cells from MESCs has been shown tofollow treatment with MEDII medium (a medium conditioned bythe human hepatocellularcarcinoma cell line, Hep G2). This

medium has been shown in MESCs to lead to a change in themarker gene pattern expression from that of ICM to that ofprimitive ectoderm. The treatment of HESCs with MEDII (Calhounet al., 2004) has also shown that the resultant cells are pluripotentin nature though with an early differentiated phenotype. However,unlike MESCs, which show transition to EPL cells, HESCs treatedwith MEDII displayed similar gene expression to primitive streakcells and nascent mesoderm cells, through activation of theTGF�1/NODAL pathway. We therefore examined NANOGoverexpressing cells to asses whether they show similar changes ingene expression as has been shown after treatment with MEDII.Our NANOG overexpressing cells did not fully mimic the effect ofMEDII media, as reported for HESCs. Thus, three of the markers[CRIPTO (TDGF1 – Human Gene Nomenclature Database), FSTand TBX1] examined in the report on MEDII treatment showed asimilar response in our clones, whereas two showed no effect[GATA6 and ZNF1A1 (ZNFN1A1 – Human Gene NomenclatureDatabase)], and one had an opposite effect [LEFTYA (LEFTY2 –Human Gene Nomenclature Database), see Fig. S3 in thesupplementary material].

DISCUSSIONThe pathways underlying pluripotency and self-renewal in HESCsare largely unknown. Understanding these pathways is hamperedby the fact that although much knowledge has been accumulatedconcerning them in MESCs, much of it does not prove valid forHESCs. In this work, we show that overexpression of NANOG inHESCs enables their feeder-free growth. Single cells over-expressing NANOG are capable of forming colonies in the absenceof CM, a capability lost upon excision of the transgene. AlthoughNANOG overexpressing HESCs are capable of growing formultiple generations in the absence of CM, their growth is slowed.Therefore, it would seem that an additional pathway is activated bythe addition of CM. After the discovery of the role of Nanog inMESCs, it has been suggested that the lack of effect by LIF onHESCs results from high enough levels of NANOG, so that LIF

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Fig. 5. NANOG overexpressingcells adopt markers of primitiveectoderm cells. (A) The ICM (innercell mass) and epiblast (primitiveectoderm) can be distinguished bythe differential expression of a subsetof marker genes. ICM has a highexpression of Rex1 and Gbx2, andlow expression of Fgf5. Epiblast haslow expression of Rex1 and Gbx2,and upregulation of Fgf5. (B) Real-time RT-PCR analysis was performedon markers that distinguish betweenICM and primitive ectoderm cells.Expression was examined on wild-type cells and on NANOGoverexpressing clones (shown as theaverage of three separate clones).Although GAPDH and OCT4 did notshow a significant change, REX1 andGBX2 were significantlydownregulated, and FGF5 wassignificantly upregulated following NANOG overexpression (*P�0.05). (C) Semi-quantitative RT-PCR analysis was executed on the markers describedin B. Expression was examined in wild-type cells, NANOG overexpressing clones and revertant clones from which the transgene was excised. Shownare representative pictures of the examined clones. (D) RT-PCR analysis was performed on Nanog overexpressing MESCs. A comparison wasperformed between two separate MESC lines (E14Tg2a and RF8) and clones overexpressing Nanog derived from them.

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signaling is dispensable. However, it seems that, as in mouse,human NANOG cannot fully compensate for lack of exogenousfactors supplied by the CM.

We further show that NANOG is upregulated upon earlydifferentiation of HESCs. This implies the possible involvement ofNANOG in early differentiation. In mouse, Nanog seems to act onestage later in development than Oct4. Oct4 expression is essentialfor the development of the ICM (Nichols et al., 1998), whereasNanog expression is essential for the development of primitiveectoderm. Mouse embryos mutated in the Oct4 gene develop up tothe blastocyst stage, but their ICM is restricted to the trophoblastlineage (Nichols et al., 1998). Mouse embryos mutated in the Nanoggene also develop up to the blastocyst stage, but when cultured invitro, their ICM creates only primitive endoderm cells and noprimitive ectoderm (Mitsui et al., 2003). This indicates that in bothhuman and mouse, NANOG may promote the transition from ICMto primitive ectoderm. However, the idea that NANOG can activelypromote this transition, not only by preventing the transition toprimitive endoderm but by actually driving the transition to primitiveectoderm, has not been suggested or shown yet, and this is the firstreport of such a function. Indeed, a change in marker geneexpression occurs upon overexpression of NANOG in HESCs from

that characteristic of ICM to that characteristic of primitiveectoderm. A protocol for differentiation to primitive ectoderm wasestablished for MESCs (Rathjen et al., 1999). These cells, termedearly primitive ectoderm-like (EPL) cells have been defined in themouse by three characteristics: different expression of a subset ofmarker genes that distinguish ICM from epiblast; different cytokinedependency, which includes decreased dependency on LIF andcreation of undifferentiated colonies in its absence; and failure toform chimeras upon injection into a blastocyst. We have shown thefirst two requirements in HESCs overexpressing NANOG, while thethird cannot be examined in humans. Although we did not observea change in all markers known to distinguish ES cells from EPL cellsin mouse, this may result from inherent differences between the twospecies, known to exist for several markers. For example, PRCE,which in mouse is expressed mainly in epiblast and not in ICM orES cells, is expressed in wild-type HESCs. As human primitiveectoderm cells are not available for research, nothing is known aboutthe in vivo expression pattern of the examined genes, and ouranalysis was based on the data established in mouse. However,although the population of NANOG overexpressing cells isundoubtedly a pluripotent population, a divergence from the normalmarker gene expression pattern of ES cells does occur. NANOG

1199RESEARCH ARTICLENANOG overexpression

Fig. 6. Transcriptome analysis of NANOG overexpressing cells. DNA microarray analysis was performed on RNA extracted from NANOGoverexpressing cells, wild-type ES cells, EB2d, EB10d and EB30d. Analysis was performed using the U133A chip from Affymetrix, each experimentwas performed in triplicate and each DNA microarray was normalized to an average value of 100. (A) Dendogram analysis reveals that uponoverexpression of NANOG, the similarity of the cells to cells of EB2d increases. (B) Differentially expressed genes were divided into four groups:(I) genes upregulated in NANOG overexpressing cells and in EB2d compared with ES cells; (II) genes upregulated in NANOG overexpressing cellscompared with both ES cells and EB2d; (III) genes downregulated in both NANOG overexpressing cells and in EB2d compared with ES cells; and (IV)genes downregulated in NANOG overexpressing cells compared with both ES cells and EB2d. (C) Shown are genes upregulated in NANOGoverexpressing cells in comparison with wild-type ES cells. All the genes shown are at least fourfold upregulated in NANOG overexpressing cells.(D) Shown are genes downregulated in NANOG overexpressing cells in comparison with wild-type ES cells. All the genes shown are downregulatedby at least 25-fold in NANOG overexpressing cells.

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overexpressing cells also have different cytokine dependency, asobserved by the ability to grow in the absence of CM, anothercharacteristic of mouse primitive ectoderm cells.

When expression of ICM-specific marker genes was examined inwild-type and in Nanog overexpressing MESCs, no change wasobserved in either of the cell lines examined. One possibleexplanation is species differences between mouse and human.Another possibility is that this is a result of culture conditions or invitro effects. For example, LIF has been shown to inhibit thetransition from ICM to primitive ectoderm, and the appearance ofprimitive ectoderm markers, such as Fgf5 (Shen and Leder, 1992).

Another feature of EPL cells in mouse is that the primitiveectoderm gene expression pattern can be reverted back to an ICMexpression pattern. In MESCs converted to EPL cells, once MEDIIis removed and LIF is returned to the medium, genes that weredifferentially expressed are reverted back to levels distinctive toICM. However, in HESC revertant clones (after the removal of theNANOG transgene), although the growth characteristics arerestored to those of wild-type cells, the expression of REX1, GBX2and FGF5 remains similar to that in the parental clones. Thisdifference can be attributed to several differences between theexperimental systems. One of these differences may be thedifference between mouse and human ES cells, which could meanthat although in mouse this transition is reversible, in human cellsit is not. However, it could also be the result of the different time-frames used in each experiment. While in the mouse system themethod to derive EPL cells used treatment with MEDII for only afew days (these cells have been reported to undergo crisis after 8days, see Rathjen and Rathjen, 2003), in our hands the cells werepassaged several times (at least five) before deriving the revertantclones. Therefore, the human mouse discrepancy may not originatefrom lack of the ability of human cells to revert from primitiveectoderm to ICM, but it may be that the longer time frame fixed thefate of the cells as primitive ectoderm cells, not permitting return toan ICM state.

When NANOG overexpression in HESCs is compared with whatis published on HESCs treated with MEDII (Calhoun et al., 2004),two points seem noteworthy. First, in both cases the cells remainpluripotent and express markers of pluripotent cells; second, in bothcases there seems to be an activation of the TGF�1/NODALpathway. Upregulation of CRIPTO (a co-receptor/ligand ofNODAL) and downregulation of FST (which may function as anactivin-binding inhibiting protein and therefore an inhibitior of thispathway) were observed in both cases. However, in contrast totreatment with MEDII, in our cells LEFTYA was actuallyupregulated. Still, LEFTYA is a transcriptional target of NODAL andtherefore this is consistent with activation of the pathway. Followingtreatment with MEDII no change was reported regarding theprimitive ectoderm markers, REX1, GBX2 and FGF5, and somemesoderm markers shown to be upregulated upon MEDII treatment,did not change upon overexpression of NANOG. Treatment withMEDII results in the addition of multiple factors to the cells, factorswhich may work together or apart to direct cell fate to more than onedirection. By contrast, overexpression of NANOG probably createsa more unified cell culture, as only the expression of a single gene isaltered compared with wild-type cells. Therefore, this report is thefirst to describe the transition from ICM to primitive ectoderminduced by the genetic manipulation of a specific gene.

Using microarray analysis, we searched for genes differentiallyexpressed between wild-type and NANOG overexpressing HESCs.NANOG has been suggested both to repress differentiation toprimitive endoderm and to actively maintain pluripotency. As

parietal endoderm markers are among the genes downregulatedupon overexpression of NANOG, it is likely that human NANOGalso inhibits differentiation to primitive endoderm. The largernumber of genes downregulated by NANOG overexpression thanthose upregulated might suggest that a significant portion of theeffects of NANOG may come from repressing differentiation intoprimitive endoderm. Genes upregulated after NANOGoverexpression may have importance in future research as they maycontribute to the release from CM dependency. One pressing issuein current ES cell research is the establishment of feeder-freecultures. Therefore, pathways that govern HESC self-renewal andpluripotency have to be elucidated. Future research of the group ofgenes upregulated in NANOG over-expressing clones may facilitateand optimize this goal, as understanding how NANOG enablesfeeder free growth may improve the culture obtained and define therequirements of the cells. A clue to a possible mechanism of therelease from conditioned media dependency comes from the factthat LECTIN1, one of the genes downregulated upon overexpressionof NANOG, is involved in promotion of apoptosis (Yang and Liu,2003). Similarly, HSPA1A, one of the genes upregulated by NANOG,is known to inhibit apoptosis (Gabai et al., 2002). Therefore, the totaleffect observed upon NANOG overexpression may result from twoactivities: the inhibition of differentiation and an increase in cellsurvival that results from downregulation of mechanisms thatpromote apoptosis.

Finally, using microarrays, new genes expressed differentiallybetween ICM and primitive ectoderm in humans are suggested.Genes expressed differentially between these two closely relatedpopulations can be used to identify the primitive ectodermpopulation. It is likely that at least some of these genes have a rolein the transition from one cell population to the other. Mostimportantly, the recapitulation of the first differentiation step of theICM in vitro shows that early stages of human development, notaccessible otherwise to research, may indeed be mimicked inculture.

We thank Dr Ian Chambers and Prof. Austin Smith for their assistance withestablishing part of the experiments described and for kindly supplying us withthe NANOG expression vector. We also thank Prof. Shinya Yamanaka for kindlysupplying us with mRNA from wild-type and Nanog overexpressing RF8MESCs. This research was partially supported by funds from the Israel ScienceFoundation and by an NIH grant.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/cgi/content/full/133/6/1193/DC1

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