T-cell factor 3 (Tcf3) deletion increases somatic cellreprogramming by inducing epigenome modificationsFrederic Lluisa,1, Luigi Ombratoa,1, Elisa Pedonea, Stefano Pepea, Bradley J. Merrillb, and Maria Pia Cosmaa,c,d,2

aCentre de Regulació Genòmica, Universitat Pompeu Fabra, 08003 Barcelona, Spain; bDepartment of Biochemistry and Molecular Genetics, University ofIllinois, Chicago, IL 60607; cInstitució Catalana de Recerca i Estudis Avançats, 08003 Barcelona, Spain; and dInstitute of Genetics and Biophysics, ConsiglioNazionale delle Ricerche, 80134 Naples, Italy

Edited by Terry Magnuson, University of North Carolina, Chapel Hill, NC, and accepted by the Editorial Board June 10, 2011 (received for review November23, 2010)

The heterochromatin barrier must be overcome to generate in-duced pluripotent stem cells and cell fusion-mediated reprog-rammed hybrids. Here, we show that the absence of T-cell factor 3(Tcf3), a repressor of β-catenin target genes, strikingly and rapidlyenhances the efficiency of neural precursor cell (NPC) reprogram-ming. Remarkably, Tcf3−/− ES cells showed a genome-wide in-crease in AcH3 and decrease in H3K9me3 and can reprogramNPCs after fusion greatly. In addition, during reprogramming ofNPCs into induced pluripotent stem cells, the silencing of Tcf3 in-creased AcH3 and decreased the number of H3K9me3-positiveheterochromatin foci early and long before reactivation of theendogenous stem cell genes. In conclusion, our data suggest thatTcf3 functions as a repressor of the reprogramming potential ofsomatic cells.

Wnt pathway | induced pluripotent stem cell generation

Overexpression of defined factors and modulation of signalingpathways can induce somatic cell reprogramming of differ-

entiated cells. The process that leads to the generation of in-duced pluripotent stem cells (iPSCs) usually takes several weeksto complete, and it is normally very inefficient (1). Somatic cellreprogramming can also be achieved via their fusion with stemcells (2). We have shown that Wnt3a-mediated activation of theWnt/β-catenin pathway enhances cell fusion-mediated reprog-ramming of a variety of somatic cells (3). In addition, the numberof iPSC clones was increased when mouse embryonic fibroblasts(MEFs) infected with retroviruses expressing Oct4, Sox2, andKlf4 were cultured in Wnt3a-conditioned medium (4). Likewise,a large number of iPSCs were obtained when human primarykeratinocytes were transduced with Oct4 plus Klf4 (OK) andcultured in the presence of CHIR99021 [a glycogen synthasekinase-3 (GSK-3) inhibitor that activates the Wnt pathway] (5).One effect of activation of the Wnt canonical pathway is the

inactivation of the destruction complex APC/GSK3/Axin. Asa consequence, β-catenin is not phosphorylated by GSK3; instead,it translocates into the nucleus, where it binds to target promotersthrough its interactions with the Tcf proteins. Tcf1 (Tcf7), Lef-1,Tcf3 (Tcf7l1), and Tcf4 (Tcf7l2) form a family of transcriptionfactors that modulate transcription of genes by recruiting chro-matin remodeling and histone-modifying complexes to their targetgenes (6, 7). Tcf3 is the most frequently expressed of the Tcfisoforms in ES cells (8) (see Fig. S3A), and it coregulates specificclasses of target genes by associating with their promoter regions,along with Oct4, Nanog, and Sox2 (9). Tcf3 has a dual function:It can repress β-catenin target genes by recruiting corepressorfactors, and it can activate the same or different classes of genesby interacting with β-catenin and by recruiting different sets ofcofactors (6). Interestingly, in gastrulating Xenopus embryos andmammalian cells, on Wnt signaling activation, Tcf3 was shown tobe phosphorylated by homeodomain-interacting protein kinase 2(HIPK2) and to dissociate from target promoters. This suggestedan alternative model for Tcf3 activity that recognizes Tcf3 to bemainly a repressor, which, once phosphorylated, can leave Wnt-target genes, which, in turn, become derepressed and transcrip-tionally active (10).

We have shown that constitutive activation of the Wnt path-way in GSK3−/− ES cells leads to a block in the reprogrammingactivity of somatic cells after fusion. This was attributable to veryhigh levels of active β-catenin in the nucleus of GSK3−/− ES cells;indeed, ES cell clones expressing high levels of β-catenin alsocannot reprogram somatic cells after fusion. In contrast, ES cellclones expressing low levels of β-catenin showed high reprog-ramming capacities (3).We thus investigated whether deletion of Tcf3, and therefore

derepression of β-catenin target genes, can enhance the reprog-ramming activity. Here, we show that Tcf3 represses Oct4 plusKlf4 (OK)-induced reprogramming of neural precursor cells(NPCs). Deletion of Tcf3 enhances both cell fusion-mediated anddirect reprogramming. Furthermore, we show that the increasedreprogramming efficiency is largely attributable to genome-wideepigenome modifications that occur before the endogenous stemcell genes are reactivated in the iPSC clones.

ResultsThe deletion of Tcf3 derepresses the transcription of β-catenin–dependent genes (8, 9, 11). To determine whether the deletion ofTcf3 in ES cells can enhance cell fusion-mediated reprogram-ming, we cocultured somatic NPCs carrying the Oct4-Puro-GFPtransgene (puromycin resistance and GFP, under the control ofthe Oct4 promoter) with WT ES cells or Tcf3−/− ES cells (Fig.S1A). The cells fused spontaneously without addition of poly-ethylene glycol (12), and deletion of Tcf3 did not increase theefficiency of fusion (Fig. S1B). WT and Tcf3−/− cells were notresistant to puromycin selection (Fig. S1C), whereas reprog-rammed clones were selected by Oct4 reactivation by addingpuromycin to the hybrids. The puromycin-resistant clones weretetraploids (Fig. S2A), were GFP-positive (Fig. S2B), expressedpluripotent markers, and silenced neural markers (Fig. S2 C andD). They were stained for alkaline phosphatase (ALP) expres-sion, a stem cell marker (13), and counted. The reprogrammingof NPCs was 300-fold higher after fusion with Tcf3−/− ES cellswith respect to fusion with WT ES cells (Fig. 1A). This effect wasnot attributable to the activities of the other Tcf family members(i.e., Tcf1, Lef-1, Tcf4) because they were expressed at the samelevels in WT and Tcf3−/− ES cells (Fig. S3B). Also, it was notattributable to an increase of stabilized β-catenin in these cells(Fig. S4B, compare with NO BIO samples). On average, 1,200reprogrammed clones were obtained after coculturing 1 × 106

cells of each of the two cell types.

Author contributions: F.L., L.O., and M.P.C. designed research; F.L., L.O., E.P., and S.P.performed research; B.J.M. contributed new reagents/analytic tools; F.L., L.O., and M.P.C.analyzed data; and M.P.C. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. T.M. is a guest editor invited by the EditorialBoard.1F.L. and L.O. contributed equally to this work.2To whom correspondence should be addressed. E-mail: pia.cosma@crg.es.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1017402108/-/DCSupplemental.

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Interestingly, the reprogramming efficiency did not increasefurther when NPCs were cocultured with Tcf3−/− ES cells thathad been pretreated with the Gsk3 inhibitor BIO (14) for dif-ferent times at a concentration of 1 μM (Fig. 1B). Furthermore,increasing the BIO concentration resulted in increased β-cateninnuclear accumulation and a consequent increase in TCF/LEF-TopFlash activity, although there was a decrease in the reprog-ramming efficiency after fusion (Fig. S4 B–D). In contrast, and aswe have previously shown, WT ES cells enhanced the reprog-ramming of NPCs, compared with the controls, when they werepretreated for 24 h with BIO before fusion, although they did notenhance reprogramming when they were pretreated for 12 or 48 h(Fig. 1B). We have shown previously that this time-dependentreprogramming is attributable to nuclear accumulation ofβ-catenin at the 24-h time point up to a specific threshold level(3) (Fig. S4A). Furthermore, high β-catenin accumulation inβ-catenin–expressing ES cell clones and in the GSK3−/− ES cellsimpaired reprogramming activity after fusion (3, 12), and thiswas not attributable to a transcriptional increase in Tcf3 in thesecells (Fig. S5A) but, rather, to the activation of the Axin2-dependent negative feedback loop (3).Next, to confirm the essential role of Tcf3 in the reprogram-

ming process, we generated Tcf3−/− ES clones that express a WTTcf3 or a truncated Tcf3 form that cannot interact with β-catenin(7) (called Tcf3−/−WTcf3 and Tcf3−/−ΔΝTcf3, respectively) (Fig.S5B). Both Tcf3−/−WTcf3 and Tcf3−/−ΔΝTcf3 clones did notreprogram NPCs after fusion (Fig. 1C). This effect was reversedby pretreatment of the Tcf3−/−WTcf3 clones with BIO for 24 hbut not when Tcf3−/−ΔΝTcf3 clones were pretreated with BIObefore fusion (Fig. 1D).

All in all, these data clearly show that deletion of the Tcf3repressor can allow ES cells to reprogram somatic cells with highefficiency and, furthermore, that this process is not attributableto an increased accumulation of nuclear β-catenin; rather, highlevels of β-catenin block reprogramming activity even in absenceof the Tcf3 repressor.Next, we examined whether the reprogramming process was

more rapid in the absence of Tcf3. We usually started the pu-romycin selection of reprogrammed clones 72 h after the co-culturing of the cells. This allows sufficient time for the hybridsto be reprogrammed and to survive the puromycin selection afterreactivation of the Oct4 promoter (15, 16). When we appliedpuromycin selection 24 or 48 h after the coculturing of WT EScells and NPCs, we could not select any viable clones because thepuromycin killed all the cells before reactivation of the Oct4promoter. Surprisingly, we were able to select a very large numberof clones (340 GFP+ and puromycin-resistant colonies) by addingthe puromycin only 24 h after the coculturing of Tcf3−/− EScells and NPCs. The number of colonies increased even furtherwhen puromycin was applied 48 and 72 h after the coculturing(with 635 and 3,125 clones selected, respectively; Fig. 1E). Thesedata show that as well as being more efficient, the reprogram-ming of somatic cells is more rapid when Tcf3 is deleted, and thismight be attributable to constitutive derepression of specificTcf3-targeted genes that can efficiently activate reprogrammingof somatic cells in trans after fusion.Because Tcf3 can be released from target promoters after its

phosphorylation (10, 17), we investigated whether β-catenin canactivate target genes when it is in a complex with a different Tcfprotein, such as Tcf1, which is also highly expressed in ES cells(Fig. S3A). Interestingly, the silencing of Tcf1 in Tcf3−/− ES cells

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Fig. 1. Deletion of Tcf3 in ES cells increases reprogrammingof NPCs after cell fusion. (A) Bright fields of hybrid coloniesformed between WT (wt) ES cells plus NPCs and Tcf3−/− EScells plus NPCs stained for ALP expression are shown.Quantification of reprogramming efficiency (fold increasesin colony numbers) is shown in the graph. WT ES cells andTcf3−/− ES cells have the same genetic background. (B) EScells (WT and Tcf3−/−) were pretreated with 1 μM BIO for theindicated times and then cocultured with NPCs-Oct4-puro.(Right) Representative growth plates with quantification ofreprogramming efficiency (fold increases in colony num-bers) of the cocultured cells (mean ± SEM, n = 3). (C)Quantification of reprogramming efficiency (fold increasesin colony numbers) of different Tcf3−/− ES cell clonesexpressing a truncated Tcf3 (Tcf3−/−ΔΝTcf3) form or WT Tcf3(Tcf3−/−WTcf3) cocultured with NPCs-Oct4-puro (mean ±SEM, n = 3). (D) Different Tcf3−/−ΔNTcf3 and Tcf3−/−WTcf3ES cell clones were pretreated with BIO for the indicatedtimes and then cocultured with NPCs-Oct4-puro. Reprog-ramming efficiency (fold increases in colony numbers) of thecocultured cells is shown (mean ± SEM, n = 3). (E) ES cells(WT and Tcf3−/−) and NPCs-Oct4-puro were cocultured, andpuromycin selection was applied at the indicated times. Thetotal number of reprogrammed colonies in three experi-ments is indicated.

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(Fig. S6 A and B) completely abrogated their reprogrammingactivity after fusion (Fig. S6C). These results lead us to concludethat enhancement of reprogramming after derepression of Tcf3target genes might be coupled to β-catenin/Tcf1-dependent ac-tivation of target promoters.Next, we investigated whether silencing of Tcf3 increases the

efficiency of iPSC generation. NPCs transduced with OK cangenerate iPSCs, although with a very low efficiency (18–20).Here, NPCs were infected with retroviruses carrying OK as wellas with a retrovirus carrying the shRNA for Tcf3 or a controlshRNA (OKshTcf3 and OKshCtrl). The cells were efficientlyinfected (Fig. S7A), and OK was efficiently expressed, whereasTcf3 was efficiently silenced (Fig. S7 B and C). Cells were cul-tured for 3, 5, and 7 d in NPC medium (the 3-, 5- and 7-d timepoints), and the infected NPCs were then counted, replated inequal numbers in ES cell medium, and cultured for severalweeks. In the plates in which NPCs were infected withOKshTcf3, ALP+ clones emerged in high numbers and veryearly: only 15 d after the infections, at the 7-d time point (Fig. 2Aand Fig. S8A). Then, 18 d postinfection, the clones started toexpress GFP stably (7-d time point; a total of 122 GFP+ clones in4 different experiments; mean: 31 ± 13 clones, n = 4) (Fig. 2 Aand B). In the controls (NPCs-OKshCtrl) at the 7-d time point,the clones emerged later, at 22 d postinfection, and they startedto express GFP 28 d postinfection. In addition, the number ofclones was much lower (7-d time point; 11 GFP+ clones in 4experiments; mean: 3 ± 3 clones, n = 4). The silencing of Tcf3also increased the efficiency of reprogramming at the 3- and 5-d time points compared with the respective control time pointsbecause of an increased number of ALP+ and GFP+ clones andreduced timing of reprogramming (Fig. 2A and B and Fig. S8A).Both OKshTcf3-iPSCs and OKshCtrl-iPSCs showed an ES cellmorphology; expressed Oct4-driven GFP; and also showedreactivation of endogenous Nanog, SSEA-1, Gdf3, and Fgf4, aswell as silencing of the NPC markers Olig2 and Blbp (Fig. 2 Cand D and Fig. S8B). Interestingly, the overexpression of Oct4transgene was progressively silenced at the 3- and 7-d time points

(Fig. S8C). In contrast, some puromycin-selected iPSC coloniesshowed efficient reactivation of endogenous Oct4 and Nanog,which was comparable to expression in ES cells, and they showedsilencing of the transgenes (Fig. S8 D and E). Finally, the cloneswere pluripotent because they could differentiate in tissues ofthe three germ layers when injected s.c. into nude mice (epi-dermis, neural tissue, cartilage, muscle, and gut-like epithelium)(Fig. S8F). All in all, these data show that NPC-derived iPSCscan be generated in large numbers and in a timely manner bysilencing Tcf3.We then investigated the molecular mechanisms by which Tcf3

deletion increased the efficiency of both fusion-mediated anddirect reprogramming. We analyzed the levels of expression ofthe Oct4, Nanog, and Sox2 genes in Tcf3−/− ES cells. Thesegenes were previously shown to be dependent on Tcf3 for theirtranscription (9, 21). In accordance with previous work (11), wedid not see an increase in Oct4 and Sox2 here; however, therewas a small increase in Nanog expression in Tcf3−/− ES cells withrespect to the WT cells (about a twofold increase) (Fig. 3A). Todetermine whether Tcf3−/− ESCs were more powerful in theirreprogramming of somatic cells because of the increased Nanogexpression, we increased the Nanog levels in WT ES cells toa level comparable to that measured in Tcf3−/− ESCs. In addi-tion, we increased the Nanog levels in Tcf3−/− ES cells evenfurther (Fig. S9A). Fusion-mediated reprogramming of NPCswith Tcf3−/− ES cells both without and with Nanog over-expression enhanced reprogramming of the somatic cells 300-fold over the control (Fig. 3B). In contrast, the twofold increasein Nanog in WT ES cells provided only a threefold enhancementof reprogramming after fusion compared with the control (Fig.3B). These data ruled out a role for Nanog in increased re-programming in the absence of Tcf3.Tcf3 recruits some repressive cofactors, such as Groucho, TLE-

1, and histone deacetylase, to their target genes (22); as a result, itinduces heterochromatin formation and global repression oftranscription. We then asked whether activation of Wnt signalingcan modify the epigenome profile of ES cells. WT ES cells were

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Fig. 2. Generation of iPSCs is increased byTcf3 silencing. (A) Experimental schemeindicating iPSC generation. NPCs-Oct4-puro were infected with OKshCtrl orOKshTcf3. Cells were maintained in NPCmedium for 3, 5, or 7 d. Cells were thentrypsinized and counted, and 70,000 cellswere replated in ES cell medium. GFP+ andGFP− iPSCs appeared at the indicatedtimes. Puromycin selection was applied atthe indicated times. (B) Number of GFP+

iPS colonies obtained after infection withOKshCtrl or with OKshTcf3 in a totalof four experiments. The colonies werecounted at day 35 after infection. Repre-sentative images of GFP expression (C) andimmunofluorescence for Nanog nuclearstaining and SSEA-1 surface localization(D) in iPSCs.

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treated for 12, 24, and 48 h with BIO, followed by a genome-wideanalysis of their epigenome. Only the 24-h–treated ES cells thatwere also able to reprogram somatic cells after fusion (Fig. 1B)showed a marked increase in acetylated H3, a marked decreasein H3K9me3, and a tendency to increase in H3K4me3, comparedwith untreated ES cells and ES cells that were BIO-treated for 12and 48 h. There was no variation in H3K27me3 (Fig. 3C and Fig.S9B). The global modifications of these histone lysines indicatedthat the state of the chromatin was largely open and the presenceof a substantial fraction of transcriptionally active euchromatin.Furthermore, and importantly, activation of the Wnt/β-cateninpathway for a specific time (24 h) can induce massive histonemodifications in ES cells. We then analyzed the Tcf3−/− ES cellsand found that these cells also showed a similar genome-wideepigenome profile (Fig. 3 C and D). Thus, we analyzed someTcf3 target and nontarget genes by ChIP in WT and Tcf3−/−ESCs. We found an increase of AcH3 and a decrease ofH3K9Me3 in a subset of Tcf3 target genes but also in genes thatwere non-Tcf3 targets, which confirmed that chromatin in Tcf3−/−ESCs is genome-wide open and transcriptionally active (Fig. S9C).Remarkably, the open state of the chromatin in Tcf3−/− ES

cells also enhanced the binding of Oct4 to specific promoters of

target genes, such as Lefty2, Hoxb1, Tbx3, Nanog, Trp53b1, andDppa3, as measured by ChIP (Fig. 3E). These promoters werechosen because they have already been shown to be commontargets of Tcf3 and Oct4 (9, 21). All these data demonstrate thatin ES cells, deletion of Tcf3 or activation of the Wnt pathway fora specific time leads to the establishment of an open chromatinstate and a general chromatin derepression that enhances thereprogramming efficiency.Finally, to demonstrate further that the silencing of Tcf3 causes

a modification of the epigenome that might be essential in theaugmentation of the reprogramming process, we analyzed his-tone modifications during iPSC generation. NPCs were infectedwith OKshTcf3 or OKshCtrl. Then, 3, 5, and 7 d postinfection,they were replated in ES cell medium, cultured for a further 24 h,and analyzed for modifications of AcH3 and H3K9me3 by im-munofluorescence and Western blotting (Fig. 4A). Only the cellsin which Tcf3 was silenced showed a high level of AcH3 at the 5-d and 7-d time points (Fig. 4B), along with a decreased numberof H3K9me3 heterochromatin foci at all time points, comparedwith cells infected with OKshCtrl (Fig. 4C). This was also con-firmed by Western blotting using 5-d and 7-d total extracts (Fig.4D and Fig. S9D). The numbers of heterochromatic foci inOKshCtrl- and OKshTcf3-infected NPCs were counted, and thecurves from the experimental data were fitted. At all three timepoints, we observed a reduction in the number of heterochro-matin foci in the OKshTcf3-infected NPCs with respect to thecontrols (OKshCtrl-infected NPCs) (Fig. 4C). This clearly showsthat Tcf3 maintains the heterochromatin state and that its de-letion leads to the opening of the chromatin. Interestingly, al-though there were epigenome modifications, these OKshCtrl-and OKshTcf3-infected NPCs did not show reactivation of stemcell genes, such as Oct4, Rex1, Fbx15, and Eras, up to 8 d post-infection. Only Nanog was partially reactivated in these infectedNPCs (Fig. S9E). Reactivation of Oct4 and Nanog was seen onlyin puromycin-selected iPSC clones (Fig. S8 D and E). Finally,because previous studies have shown that reprogramming canresult in large changes in nuclear volume (23), we estimated thenuclear areas of these infected NPCs. At the 7-d time point,OKshTcf3-infected NPCs and OKshCtrl-infected NPCs were cul-tured for 24, 48, and 72 h in ES cell medium and the areas of theirnuclei were measured. The nuclei infected with OKshTcf3 showeda significantly greater calculated volume increase compared withthe OKshCtrl-infected nuclei (Fig. 4E). These data indicate thatsilencing of Tcf3 induces epigenome modifications, formation ofeuchromatin, and nuclear volume increases in cells undergoingreprogramming. These events occurred very early and beforereexpression of the endogenous stem cell genes.

DiscussionWe have shown here that cell reprogramming can be inducedwith high efficiency by deletion of the repressor Tcf3. This Tcf3ablation led to a massive genome-wide modification of the epi-genome: an increase in AcH3, a slight increase in H3K4me3, anda decrease in H3K9me3. These modifications established anactive transcriptional state of the chromatin that favored theenhancement of reprogramming (Fig. S9F). Indeed, the openstate of the chromatin was also confirmed by the efficient bindingof the endogenous Oct4 to target promoters in the Tcf3−/− EScells. Interestingly, we did not observe changes in H3K27me3,which was also recently shown to be an essential marker for ef-ficient reprogramming of human B cells (23, 24).WT ES cells can reprogram somatic cells after fusion (13);

however, this process is particularly inefficient. Here, we haveshown that derepression of β-catenin target genes in Tcf3−/− EScells can switch on a cascade of events that finally promotes theefficient reprogramming of somatic cells after their fusion. Wepreviously showed that only a specific level of β-catenin accu-mulation in the nucleus of ES cells can enhance fusion-mediatedreprogramming (3). To control gene expression, β-catenin mustassociate with the Tcf factors (6), and these, in turn, switch therecruitment of the corepressors to that of the coactivators of the

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Fig. 3. Deletion of Tcf3 leads to genome-wide epigenome modifications inES cells. (A) Quantitative PCR analysis of Oct4, Sox2, and Nanog expression inWT (wt) and Tcf3−/− ES cells. (B) Quantification of reprogramming efficiency(fold increases in colony numbers) of ES cells (WT ES cells, Tcf3−/− ES cells,Nanog overexpressing Tcf3−/− ES cells, Nanog overexpressing WT ES cells)cocultured with NPCs-Oct4-puro (mean ± SEM, n = 3). (C) Western blottingquantification of histone modifications in untreated or BIO-treated WT EScells and Tcf3−/− ES cells. All values are normalized relative to total H3 (t test:*P < 0.05; **P < 0.01; mean ± SEM, n = 3). (D) Representative Western blotanalysis of histone modifications of protein extracts from WT and Tcf3−/− EScells. (E) Quantitative ChIP assay for Oct4 target genes in WT and Tcf3−/− EScells. Lefty2, Hoxb1, Tbx3, Nanog, Trp53b1, and Dppa3 are all Tcf3 and Oct4target genes. Diablo and IGX1A are control nontarget genes. The fold en-richment for each promoter region vs. IgG control immunoprecipitation isshown, after normalization for input DNA (t test: *P < 0.05; mean ± SEM, n = 3).

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target genes (22). Here, we show that deletion of Tcf3 stronglyincreases the efficiency of reprogramming, most likely throughthe constitutive release of the corepressors from the target genesthat encode for reprogrammers (reprogramming factor-encodinggenes). Interestingly, this appears not to be independent ofβ-catenin; rather, it appears to be coupled with the activity of theWnt signaling pathway. In the absence of Tcf3, β-catenin appearsto activate reprogramming in a complex with a Tcf activator,such as Tcf1. On the other hand, we cannot exclude a possibleTcf-independent β-catenin stabilization on target promoters (25).Reprogrammed clones can be isolated not only in extremely

large numbers but, importantly, by applying puromycin selectionvery early (i.e., 24 h after fusion). This indicates that the ex-pression of the reprogrammers is already active in the Tcf3−/− EScells and that these factors can act immediately after fusion withNPCs in trans. Reprogramming of human B-cell nuclei or humanfibroblasts was also very rapid in the case of heterokaryon for-mation with mouse ES cells (23, 24). Importantly, our data showthat silencing of Tcf3 is also valuable for the efficient derivation

of iPSCs, which can be generated in large numbers and also ina short time (Fig. S9F).Our observations indicate that deletion of Tcf3 strongly

enhances reprogramming by modifying the epigenome and thatthis, in turn, leads to the expression of essential reprogrammersthat can be reactivated by both stem and somatic cells. It is alsoworth noting that the largest number of iPSCs was selected whenthe cells were cultured in NPC medium for 7 d after beinginfected (i.e., under culture conditions that induce ES cell dif-ferentiation). We prolonged this time to 9 d, but we did not seeany further improvement in the reprogramming efficiency (Fig.2B). Because histone modifications, such as an increase in AcH3and a decrease in H3K9me3 foci, had already started 5 d post-infection, 7 d appeared to be the right time for the reprogram-mers to be expressed by the NPC genome. Indeed, we also sawincreases in the nucleus volume, which might well be associatedwith high transcriptional activity. Thus, after these 7 d in NPCmedium, when the cells were cultured for some additional days

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Fig. 4. NPC epigenome modifications induced byshTcf3. (A) Experimental scheme: NPCs-Oct4-purowere infected with OKshTcf3 or OKshCtrl. After 3,5, and 7 d in NPC medium, the cells were trypsinizedand replated in ES cell medium for an additional24 h. AcH3 and H3K9me3 staining was then per-formed. (B) Representative images and quantifica-tion (Lower) of total H3 acetylation levels in NPCsinfected with OKshCtrl or OKshTcf3. Intensity ofAcH3 was evaluated using ImageJ software (mean± SEM, n = 3; 100 cells in total were analyzed foreach sample). (C) Representative images and quan-tification (Lower) of H3K9me3 heterochromatinfoci. H3K9me3 staining was analyzed by immuno-fluorescence. The number of immunopositiveH3K9me3 foci in the nuclei was counted (total of 70nuclei were analyzed for each sample). Smoothingsplines were fitted to the experimental data usingthe curve-fitting toolbox implemented in MATLABR2009a (Mathworks). (D) Representative Westernblot analysis of histone modifications of proteinextracts from NPCs-Oct4-puro infected with OKshTcf3or OKshCtrl. (E) Nucleus areas of OKshCtrl- andof OKshTcf3-infected NPCs. The cells were infectedwith OKshCtrl or OKshTcf3 and maintained in NPCmedium for 7 d. Nucleus area was analyzed 24, 48,and 72 h after the shift to ES cell medium (mean ±SEM, n = 3; 100 cells were analyzed for each treat-ment time point and experiment; two-sample Wil-coxon test was used to calculate the P value).

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in ES cell medium, they were ready to complete their reprog-ramming and could be selected in large numbers.In the OKshTcf3-infected NPCs, the transgenes were silenced

with high efficiency and the endogenous stem cell genes werereactivated later on, a long time after their epigenome wasmodified (Fig. S9F). This indicates that the epigenome mod-ifications, such as the increase in AcH3 and decrease inH3K9me3 heterochromatin foci, are epistatic to the stem cellgene reactivation and transgene silencing. Interestingly, the BAFcomplex components that modify the epigenome of MEFs fa-cilitated Oct4 transgene binding to the target promoters duringreprogramming very soon after infection (26), whereas we didnot see reactivation of the endogenous Oct4, Rex1, Fbx15, orEras for up to 8 d postinfection.Tcf3−/− ES cells have been shown to differentiate poorly

in vitro and in vivo, although they form embryoid bodies andteratomas (8); this is in apparent contrast to our present obser-vation that deletion of Tcf3 induces extraordinary reprogram-ming. Evidently, this means that pluripotency can be dissociatedfrom reprogramming potential and that different sets of genescontrol these two developmental fates. It will be very interestingin the future to dissect out these divergent pathways and genesthat can push cells to embark on these two different states.

Materials and MethodsCells. NPCs-Oct4-puro were isolated from HP165 mice, and they carry theregulatory sequences of the mouse Oct4 gene driving GFP and puromycin-resistance genes. The NPCs-Oct4-puro were a gift from A. Smith (WellcomeTrust Centre for Stem Cell Research, University of Cambridge, Cambridge,United Kingdom) and were cultured as previously described (27). WT andTcf3−/− ES cells have the same genetic background and were produced aspreviously described (28). ES cells were cultured on gelatin in KO DMEMsupplemented with 20% FBS (HyClone), 1× nonessential amino acids, 1×GlutaMax (Invitrogen), 1× penicillin/streptomycin, 1× 2-mercaptoethanol,and 1,000 U/mL LIF ESGRO (Chemicon). BIO was added to a concentration of1 μM. The 293T cells were cultured in DMEM supplemented with 10% FBS, 1×GlutaMax, and 1× penicillin/ streptomycin.

Cell Hybrids. For ES cell plus NPC cocultures, 1.0 × 106 ES cells were platedonto preplated 1.0 × 106 NPCs, first for 2 h in NPC medium and then for 2 hin ES cell medium. The cells were then trypsinized and plated at 1/5 intogelatin plus laminin-treated p100 dishes in ES cell medium, without or with1 mM BIO (Calbiochem), for different times. After 72 h, puromycin wasadded to the ES cell medium for hybrid selection.

Retroviral Infection and iPSC Generation. Retroviral infection was performedas described previously (18), with minor modifications. pMX-based retroviralvectors (Addgene) encoding mouse complementary cDNA of Oct4 and Klf4and pSUPER-shTcf3 were separately cotransfected with packaging helperplasmids into 293T cells using CalPhos mammalian transfection kits (Clon-tech). After 24 h, the medium was changed for fresh medium; 24, 48, and72 h later, the virus supernatants were collected, and filtered through 0.45-μm filters. The virus was concentrated by ultracentrifugation. NPCs wereseeded at a density of 7 × 104 cells per well in six-well plates and incubatedwith the concentrated virus for Oct4, Klf4, and shCtrl (1:1:1) or for Oct4, Klf4,and shTcf3 (1:1:1), supplemented with 6 μg/mL polybrene (Sigma) for 24 h inNPC medium. The transduction efficiencies were calculated with the pBABE-GFP control virus (by FACS) and quantitative real-time PCR. Subsequently,the medium was changed for fresh NPC medium, and the cells were culturedfor a total of 3, 5, or 7 d. The cells were then trypsinized and counted, and7 × 104 cells were replated in gelatin plus laminin p60 dishes with ES cellmedium. After several weeks, puromycin (0.5 μg/mL) was added for 7–10 d.Resistant clones were picked and replated onto MEFs. The colonies wereselected for expansion.

Other Methods. Details of other procedures are provided in SI Materialsand Methods.

ACKNOWLEDGMENTS. We thank S. Casola, L. di Croce, T. Graf, and G. Testafor suggestions and critical reading of the manuscript; L. Marucci,F. Mancuso, and G. Roma for statistical support; and J. Frade, F. Aulicino,L. Stojic, and U. Di Vicino for technical support. F.L. is funded by CP10/00445Project “Miguel Servet” of the Instituto de Salud Carlos III. We are gratefulfor support from European Research Council Grant 242630-RERE (to M.P.C.)and from HFSP Grant RGP0011/2010 (to M.P.C.).

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