lineage conversion of murine extraembryonic trophoblast stem cells to pluripotent stem cells

MOLECULAR AND CELLULAR BIOLOGY, Apr. 2011, p. 1748–1756 Vol. 31, No. 80270-7306/11/$12.00 doi:10.1128/MCB.01047-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Lineage Conversion of Murine Extraembryonic TrophoblastStem Cells to Pluripotent Stem Cells�†

Peter Kuckenberg,1 Michael Peitz,4 Caroline Kubaczka,1 Astrid Becker,3 Angela Egert,1Eva Wardelmann,2 Andreas Zimmer,3 Oliver Brustle,4 and Hubert Schorle1*

Department of Developmental Pathology, Institute of Pathology, University of Bonn Medical School, Sigmund-Freud-Str. 25, 53127 Bonn,Germany1; Institute of Pathology, University of Bonn Medical School, Sigmund-Freud-Str. 25, 53127 Bonn, Germany2; Institute of

Molecular Psychiatry, Life and Brain Center, University of Bonn, 53127 Bonn, Germany3; and Institute of ReconstructiveNeurobiology, Life and Brain Center, University of Bonn, 53127 Bonn, Germany4

Received 8 September 2010/Returned for modification 8 October 2010/Accepted 27 January 2011

In mammals, the first cell fate decision is initialized by cell polarization at the 8- to 16-cell stage of thepreimplantation embryo. At this stage, outside cells adopt a trophectoderm (TE) fate, whereas the inside cellpopulation gives rise to the inner cell mass (ICM). Prior to implantation, transcriptional interaction networksand epigenetic modifications divide the extraembryonic and embryonic fate irrevocably. Here, we report thatextraembryonic trophoblast stem cell (TSC) lines are converted to induced pluripotent stem cells (TSC-iPSCs)by overexpressing Oct4, Sox2, Klf4, and cMyc. Methylation studies and gene array analyses indicated thatTSC-iPSCs had adopted a pluripotent potential. The rate of conversion was lower than those of somaticreprogramming experiments, probably due to the unique genetic network controlling extraembryonic lineagefixation. Both in vitro and in vivo, TSC-iPSCs differentiated into tissues representing all three embryonic germlayers, indicating that somatic cell fate could be induced. Finally, TSC-iPSCs chimerized the embryo properand contributed to the germ line of mice, indicating that these cells had acquired full somatic differentiationpotential. These results lead to a better understanding of the molecular processes that govern the first lineagedecision in mammals.

Life starts with a single totipotent cell, followed by severalcleavages and the specification of the earliest cell lineages atthe blastocyst stage. The trophectoderm (TE) represents theextraembryonic trophoblast lineage and envelops the inner cellmass (ICM), which gives rise to the embryonic tissues and theprimitive endoderm. Embryonic stem cells (ESCs) derivedfrom the ICM are pluripotent because they differentiate intoall embryonic tissues, including the germ line, but have lost theability to form trophoblast derivatives (10). Likewise, tropho-blast stem cells (TSCs) derived from the TE layer exclusivelyrecapitulate the developmental potency of their parental lin-eage and contribute to all trophoblast cell types of the concep-tus (40, 50).

The first cell fate decision is initiated by cell polarization atthe 8- to 16-cell stage, where inner and outer cells are differ-entiated from each other. Outer cells adopt the trophoblastfate, whereas the inner cell population gives rise to the ICM.Differentiation is established by the expression of lineage-spe-cific transcription networks and fixed by epigenetic asymme-tries at the late blastocyst stage (4, 42, 43, 54).

The caudal-related homeobox 2 factor, Cdx2, is regarded asone key transcription factor for TE specification and mainte-nance. Cdx2 is expressed in the outer blastomeres and later in

TE (7, 32). Cdx2-null embryos lose trophectoderm integrity,and TSCs cannot be derived (48). The ectopic expression ofCdx2 in ESCs leads to lineage conversion to the extraembry-onic trophoblast fate by the direct downregulation of pluripo-tency-associated transcription factors, such as Oct4 and Nanog,and the activation of the trophectodermal transcription net-work (5, 35). Oct4 (Pou5F1), a Pou-domain transcription fac-tor, is essential for the maintenance of the ICM and the plu-ripotent state of ESCs. Oct4 also represses Cdx2 and, thus, TEformation (33, 34, 37). In addition to Cdx2, the specificationand maintenance of TSCs are regulated by other transcriptionfactors, such as Eomes, Gata3, Ets2, or Tcfap2c, acting down-stream or in parallel to Cdx2 (26, 35, 37, 44, 53). Nevertheless,a common feature shared by all key TE transcription factors isthe failure to generate stable TSC lines from the respectivenull embryos (8, 26, 41, 44, 48, 53). Additionally, after overex-pression in ESCs, Gata3, Ets2 Tcfap2c, Cdx2, Eomes, Elf5, andTead4 are able to break the embryonic lineage barrier andinduce a trophoblast-like cell fate (26, 31, 32, 35–37, 39, 51).Extraembryonic lineage identity is maintained by unique epi-genetic marks, most notably promoter DNA methylation andaltered histone modifications (11, 14–16, 43). The TE-specificdemethylation of the Elf5 promoter leads to a positive-feed-back loop with Cdx2 and Eomes, resulting in the reinforcementof the trophoblast-specific transcription factor network (31). Inextraembryonic tissues, the lineage identity also is reflected inaltered histone modifications compared to that of somaticcells. In ESCs, developmental regulators bear a bivalent re-pressive mark by H3K4me3/H3K27me3, whereas in TSCs thebivalent repressive marks seem not to be linked to the differ-entiation state (43). The establishment and maintenance of the

* Corresponding author. Mailing address: Department of Develop-mental Pathology, Institute of Pathology, University of Bonn MedicalSchool, Sigmund-Freud-Str. 25, 53127 Bonn, Germany. Phone: 49-228-28716342. Fax: 49-228-28719757. E-mail: [email protected].

† Supplemental material for this article may be found at http://mcb.asm.org/.

� Published ahead of print on 7 February 2011.

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ICM depends on mutually interacting transcription factors:Oct4, Nanog, Sall4, and Sox2. In addition, these four factorsmaintain ESC pluripotency (17, 33). Similarly to the transcrip-tion factor network in TSCs, the pluripotency factors form aself-reinforcing transcriptional network establishing embryoniclineage identity and pluripotency (3). The deficiency of eitherof these factors results in the induction of the trophectodermalcell fate in ESCs (39). Thus, embryonic and extraembryoniclineages arising from totipotent blastomeres are clearly sepa-rated by differential gene expression and unique epigeneticsignatures, which efficiently suppress ICM-to-TE conversionand vice versa during developmental progression.

In cells of all three germ layers of the embryonic lineage, theunidirectional process of differentiation can be reversed by theectopic expression of Oct4, Sox2, cMyc, and Klf4, generatinginduced pluripotent stem cells (iPSCs) (reviewed in references20 and 38). This process of dedifferentiation was termed re-programming. Here, we addressed the question of whether thesame factors known to reprogram somatic cells would be suf-ficient to overcome extraembryonic lineage restriction andconvert TSCs to the embryonic lineage. We derived extraem-bryonic TSC lines from Oct4-GFP transgenic mice that condi-tionally expressed the four reprogramming factors Oct4, Sox2,Klf4, and cMyc. After transgene induction in TSCs, we ob-served colonies showing typical ESC morphology that had ac-quired full pluripotency. Overall, this is the first report, to ourknowledge, demonstrating successful extraembryonic-to-em-bryonic lineage conversion.

MATERIALS AND METHODS

Derivation of TSC and TSC-iPSC lines. Oct4-GFP (C57BL/6) reporter mice(49) were mated with R26-M2rtTA/Col1a1-tetO-Oct4 (C57BL/6 � 129sv) mice(21). On embryonic day 3.5 (E3.5), uteri were dissected and flushed with medium(Dulbecco’s modified essential medium [DMEM], 10% fetal calf serum [FCS]).Oct4-GFP, tetO-Oct4/rtTA blastocysts were either used directly for TSC gener-ation or transduced with tetO-Sox2, tetO-cMyc, and tetO-Klf4 lentiviruses. Forviral transduction, the zona pellucida was removed, and blastocysts were incu-bated with lentiviral particles overnight as previously described (12). TSCs weregenerated and maintained as previously described (26, 50). After five passages,TSCs were genotyped for carrying the reverse tetracycline transcriptional acti-vator (rtTA) transgene, which is integrated into the ROSA26 locus, and for theinducible Col1a1-tetO-Oct4 transgene. TSC lines from virus-incubated blasto-cysts were genotyped for viral integration. The PCR primer sequences are listedin Table S1 in the supplemental material. ESCs and TSC-iPSCs were cultured onirradiated murine embryonic fibroblasts (MEFs; feeders) in standard ESC me-dium (DMEM supplemented with 15% FCS, essential and nonessential aminoacids, L-glutamine, penicillin-streptomycin, ß-mercaptoethanol, and 1,000 U/mlleukemia inhibitory factor [LIF]). For TSC-iPSC conversion, 1 � 105 TSCs wereplated onto 10-cm dishes containing feeder cells and cultured with fresh ESCmedium supplemented with 1 �g/ml doxycycline (Dox; Sigma). Medium waschanged every 2 days until Oct4-GFP-expressing colonies appeared. We calcu-lated the conversion efficiency by counting Oct4-GFP-positive colonies and cor-relating them to the starting cell number.

Virus production. The pLV-tetO-Sox2, pLV-tetO-Klf4, and pLV-tetO-cMycvectors were a generous gift of Konrad Hochedlinger (Harvard University) (47).Lentiviral production was performed in 293T cells according to the protocol usedby Mitta et al. (29).

Immunohistochemistry. Prior to immunohistochemical staining, all cells werewashed with 1� phosphate-buffered saline (PBS) (Invitrogen), fixed for 10 minin 4% paraformaldehyde (Sigma), and rinsed twice in PBS. Cells then werepermeabilized using 1% Triton X-100 (Sigma) for 10 min and subsequentlyincubated for 30 min in serum blocking buffer (2% bovine serum albumin[Sigma], 0.05% Triton X-100 [Sigma] in PBS). After being blocked, cells wereincubated overnight with a primary antibody diluted in serum blocking buffer at4°C and rinsed with PBS (three times for 5 min each) afterwards. For detection,the appropriate Alexa Fluor-conjugated antibodies were incubated at a 1:500

dilution in PBS for 1 h at room temperature in the dark. After secondaryantibody incubation, the cells were washed three times in PBS and incubated inHoechst-PBS solution, followed by three washes with PBS. Cells stained withoutprimary antibodies served as controls. The following primary antibodies anddilutions were used for immunofluorescence: mouse anti-Oct3/4 (1:200; C-10;Santa Cruz), rabbit anti-Tuj1 (1:1,000; Covance), mouse anti-SSEA1 (1:400;R&D Systems), mouse anti-E-cadherin (1:400; B&D), mouse anti-Cdx2 (1:200;Cdx2-88; BioGenex Inc.), rabbit anti-GFAP (1:500; Dako), rabbit anti-alpha-fetopoprotein (AFP) (1:100; Dako), mouse anti-SMA (1:500; Dako), and mouseanti-Desmin (1:500; Dako). Secondary antibodies included Alexa Fluor 488- andAlexa Fluor 594-conjugated goat-anti-mouse IgG (1:500; Invitrogen) and AlexaFluor 488-conjugated goat-anti-rabbit IgG (1:500; Invitrogen).

RNA extraction, cDNA synthesis, and semiquantitative reverse transcription-PCR (RT-PCR). Total RNA was isolated from cells and tissues by using theRNeasy minikit (Qiagen) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 500 ng total RNA using SuperScript IIIreverse transcriptase (Invitrogen) according to the manufacturer’s instructions.The PCR primer sequences are listed in Table S1 in the supplemental material.

Microarray procedures. Total RNA was extracted from cells using the RNeasyminikit, including DNase digestion (Qiagen). RNA quantity was determined witha NanoDrop 1000 spectrometer (Thermo Fisher Scientific), and quality wasmeasured on a 2100 Bioanalyzer (Agilent Technologies). Affymetrix GeneChipmouse genome 430 2.0 arrays (Affymetrix) were hybridized with 15 �g biotin-labeled cRNA, obtained from 5 �g total RNA, using the GeneChip one-cyclelabeling kit (Affymetrix). After hybridization, arrays were washed and stainedautomatically on a GeneChip Fluidics station 450 (Affymetrix) and scanned witha GeneChip scanner 3000 7G (Affymetrix). Normalization and statistical dataanalyses were determined by a robust multichip average (RMA) algorithm per-formed with GeneSpring GX 10 software (Agilent) and are available under GeneExpression Omnibus accession number GSE25872.

Teratoma formation and histological analyses. We diluted 5 � 106 four-factorTSCs (4FTSCs), 2 � 106 ESCs, or 2 � 106 TSC-iPSCs in DMEM containing 10%FCS and injected them subcutaneously into the dorsal flank of nude mice.Twenty-eight days after injection, TSC-iPSC tumors were surgically dissectedfrom the mice. 4FTSC tumors were dissected at 14, 21, and 28 days. Sampleswere fixed in formaldehyde and embedded in paraffin. For immunohistochem-istry, 1- to 3-�m sections were either stained with hematoxylin-eosin or incubatedwith primary antibodies for 2 h at 37°C. After being washed, sections wereincubated for 30 min at room temperature with the appropriate anti-mouse/rabbit/goat secondary antibodies (1:400; Dako). Signals were visualized using aVectastain ABC kit (Vector Laboratories).

Bisulfite sequencing. The bisulfite treatment of DNA was performed using theEZ-DNA methylation direct kit (Zymo Research) according to the manufactur-er’s instructions. Primer sequences for Oct4 (15) and Nanog (14) were used aspreviously described. Elf5 primers are listed in Table S1 in the supplementalmaterial. Amplified products were purified using gel filtration columns, clonedinto the pCR2.1-TOPO vector (Invitrogen), and sequenced using M13 reverseprimers. CpG methylation was analyzed using Bisma software. For each pro-moter sequence, six randomly selected clones were sequenced.

In vitro differentiation. After removing feeder cells, 2 � 105 ESCs and TSC-iPSCs were resuspended in 10 ml of medium and aggregated in hanging-dropculture (30 �l). After 3 days in hanging-drop culture, the resulting embryoidbodies (EBs) were transferred to uncoated dishes (25 EBs per dish) to preventattachment. Every other day, one-third of the medium was replaced. After 4days, EBs were transferred to culture dishes, allowing attachment.

For spontaneous differentiation toward trophoblast derivatives, 5 � 104 ESCsand TSC-iPSCs were seeded on gelatinized six-well plates in trophoblast stemcell medium supplemented with FGF4 and MEF conditioned medium and cul-tured for 14 days as previously described (50). The culturing of trophoblast stemcells without FGF4 and conditioned medium allowed differentiation into theirvarious derivatives.

Chimera formation. To generate chimeric mice, blastocysts (BALB/c) injectedwith TSC-iPSCs or 4FTSCs were transferred into pseudopregnant foster(CB6F1) mice. For analyzing the germ line transmission of TSC-iPSCs, chimeraswere mated to BALB/c females. F1 embryos were collected at E13.5 and scoredfor Oct4-GFP-driven fluorescence in the gonads. The chimerization of embry-onic and extraembryonic tissue of the 4FTSCs was analyzed by Oct4-GFP geno-typing PCR on E10.5.

Cloning and design of short hairpin RNA (shRNA). The p53-sh-oligonucleo-tide (listed in Table S1 in the supplemental material) was inserted into anHpaI-XhoI-digested pSicoR-mCherry lentiviral vector as previously described(http://web.mit.edu/ccr/labs/jacks/protocols/pSico.html). The p53 target se-quence was GTACTCTCCTCCCCTCAAT (52).

VOL. 31, 2011 CONVERSION OF EXTRAEMBRYONIC TO EMBRYONIC CELLS 1749

Microarray data accession number. Microarray data determined in the courseof this work were deposited in the Gene Expression Omnibus database (GEO)under accession number GSE25872.

RESULTS

Establishment of TSC lines for conversion. TSC lines werederived from mice harboring a doxycycline-inducible Oct4 al-lele and an Oct4-GFP reporter that has been demonstrated tobe activated in cells upon the acquisition of pluripotency (21,45). Oct4-GFP-positive blastocysts were collected at E3.5 and

either used directly for TSC derivation (one-factor TSC, or1FTSC) or transduced with lentiviruses encoding doxycycline-inducible Sox2, Klf4, and cMyc transgenes (4FTSC) (Fig. 1Adepicts the experimental outline). Because the trophectodermlayer of the blastocyst acts as a robust nonpermeable barrier tovirus particles, it effectively shields the cells of the ICM fromviral infection (see Fig. S1A in the supplemental material) (6,12, 28). The integration of the lentiviral vectors and presenceof the transgenes were verified by PCR (Fig. 1C). Six 1FTSCand 13 4FTSC lines were passaged 10 times to establish a

FIG. 1. Generation of extraembryonic trophoblast stem cell-derived iPSCs. (A) Schematic of the protocol for iPSC generation from trophoblaststem cell lines. Blastocysts carrying the Oct4-GFP, tetO-Oct4, and R26rtTA transgenes (E3.5) were used to establish inducible one-factor (Oct4)TSC lines (1FTSC) or inducible four-factor TSC lines (4FTSC) after transduction with doxycycline-inducible Sox2, Klf4, and cMyc lentiviruses(tetO-Sox2, tetO-Klf4, and tetO-cMyc). (B) Oct4-GFP-positive blastocysts (left) used for the experiment to generate 4FTSCs (middle) and theresulting TSC-iPSC colony after 28 days of transgene induction. (C) Genotyping of independently derived 1FTSC, 4FTSC, and Oct4GFP controlTSC lines. (D) RT-PCR of pluripotency- and trophoblast-specific genes indicated in two representative 4FTSC-derived iPSC, ESC, and 4FTSClines. (E) Typical ESC-like morphology of TSC-iPSCs (BF). Shown is the immunohistochemical detection of Oct4, Oct4-GFP, E-cadherin, andSSEA1 in TSC-iPSCs. Cdx2 expression, indicative of trophoblast fate, is no longer detectable. Scale bar, 100 �m. (F) 4FTSCs were infected witheither p53sh (gray bars) or pSicoR-mCherry (empty vector; black bars) lentiviral vectors. After transduction, 10,000 cells were seeded and culturedwith ESC/Lif medium plus doxycycline. GFP-positive colonies were counted 14, 21, and 28 days after transduction. ***, P � 0.005.

1750 KUCKENBERG ET AL. MOL. CELL. BIOL.

population of constantly growing, self-renewing TSCs in thepresence of FGF4 and fibroblast-conditioned media (19, 26).All further analyses were performed with two independentlyderived lines from the 1FTSC and 4FTSC experiments, respec-tively. The comparison of global gene expression profiles re-vealed that the 4FTSC lines show a high similarity to estab-lished TSC lines (37) (see Fig. S1B). In line with publishedresults, the established 4FTSC lines did express low levels ofendogenous cMyc and Sox2 (25) and high levels of the TSCmarkers Cdx2, Eomes, Tpbpa, Pl1, Bmp4, Hand1, and Elf5.TSC lines were negative for Oct4 reporter activity and Oct4expression even when grown in medium supporting pluripo-tency (ESC/Lif without doxycycline; data not shown) (Fig. 1D).These results indicated that the TSC lines generated herein didnot contain ICM-derived persistent, pluripotent cells. In addi-tion, upon the withdrawal of Fgf4, Cdx2 was downregulatedand the cells formed multinucleated, terminally differentiatedgiant cells (see Fig. S1C and D) (18, 50). The differentiationpotential of the TSCs was confirmed by RT-PCR (see Fig.S1E).

Conversion of TSCs to TSC-iPSCs. To induce lineage con-version, 4FTSCs were cultured under ESC/Lif conditions withdoxycycline. After 28 days, several colonies displaying ESCcharacteristic dome-shaped colony morphology and brightOct4-GFP fluorescence could be observed (Fig. 1B; also seeFig. S2A in the supplemental material). Control cells grown forthe same period of time without doxycycline did not showgreen fluorescent protein (GFP) fluorescence. The 4FTSC-derived colonies were isolated mechanically, dissociated bytrypsinization, and plated onto MEFs in ESC medium withoutdoxycycline, demonstrating the independence of exogenousfactors. They will be called TSC-iPSCs (trophoblast stem cell-derived induced pluripotent stem cells). The efficiency of4FTSC conversion was 0.0017 and 0.0055% after 21 and 28days, respectively. For comparison, primary murine embryonicfibroblasts reprogrammed using the same approach showed anefficiency of 0.171 and 0.202% after the same period of time.These data demonstrate that the conversion of 4FTSCs toTSC-iPSCs was approximately 40 to 100 times lower than thatof somatic fibroblast reprogramming (see Fig. S2B) (20). RT-PCR analysis showed the downregulation of trophectodermmarker genes (Cdx2, Eomes, Tpbpa, Pl1, Bmp4, Hand1, andElf5) and the expression of endogenous Oct4, Sox2, Klf4, cMyc,and Nanog in TSC-iPSCs (Fig. 1D). On the protein level,TSC-iPSCs were negative for Cdx2 and positive for E-cadherinand SSEA1, further supporting the fact that they had lost theirTE fate and initiated pluripotency (Fig. 1E).

Because TSCs express low levels of endogenous Sox2 andcMyc (25), and because Klf4 has been demonstrated to bedispensable for somatic reprogramming (30), we next ad-dressed the question of whether Oct4 upregulation in TSCswas sufficient to induce lineage conversion to iPSCs (Fig. 1A).1FTSCs were supplemented with doxycycline and cultured inESC/Lif medium. However, after 28 days of culture, neitherthe ESC-specific morphology nor a GFP signal could be de-tected (not shown). We also tested whether the upregulation ofOct4, in conjunction with the application of small moleculesknown to enhance iPSC generation from somatic cells (23),would enable Oct4-driven TSC-iPSC generation. However, theuse of the MEK inhibitor (PD0325901), ALK4/5/7 inhibitor

(SB431542), and histone deacetylase inhibitor (valproic acid)in conjunction with Oct4 upregulation and culture in ESC/Lifmedium did not result in GFP-positive cells or the appearanceof ESC morphology after 28 days of culture (not shown).Hence, the upregulation of Oct4 in TSCs, combined with basallevels of Sox2 and cMyc, was not sufficient to overcome therestriction between the extraembryonic and embryonic lineageand did not shift the cells toward a pluripotent phenotype.

In somatic cells, the knockdown of p53 improved iPSC gen-eration, demonstrating that the p53-p21 pathway serves as abarrier to iPSC generation (22). To test whether p53 repressesthe formation of TSC-iPSCs, we reduced levels of p53. 4FTSCswere transduced with a lentivirus encoding an shRNA againstp53 and mCherry as a fluorescent marker for transductionefficiency. We observed an earlier appearance and a significantincrease in GFP-positive colonies after p53 knockdown com-pared to those of control cells transduced with empty vector(Fig. 1F). These data demonstrate that the reduction of p53 inTSCs speeds up the process of TSC-iPSC generation and en-hances the efficiency of conversion.

While somatic cells can be reprogrammed quite easily, ter-minally differentiated postmitotic B cells and pancreatic betacells show rather low reprogramming efficiency (9, 46). Here,we tested whether terminally differentiated cells from the ex-traembryonic lineage also could be converted. Differentiationwas induced by culturing 4FTSCs for 14 days without FGF4(18, 26, 50), which resulted in the formation of postmitotictrophoblast giant cells. However, under ESC/Lif plus Dox con-ditions, no proliferating and GFP-positive TSC-iPSCs could bedetected after 28 days (see Fig. S2C to E in the supplementalmaterial).

Analysis of expression profile and epigenetic status. To ex-amine if the extraembryonic lineage-specific mRNA profilewas overcome, the gene expression profiles of TSC-iPSCs andtheir parental 4FTSCs were analyzed by microarray analyses.A scatter plot highlights the differences between the parental4FTSCs and the derived TSC-iPSCs (Fig. 2A). Specifically, theexpression of ESC markers (pluripotency genes) and TSCmarkers differs dramatically between these two cell popula-tions. In contrast, the scatter plot comparing ESCs to TSC-iPSCs showed a high degree of coherence in the expressionprofile, with almost all expression values ranging within thelog2 intervals, indicating high similarity (Fig. 2B).

The successful reprogramming of somatic cells is thought torequire the faithful remodeling of epigenetic modifications,such as DNA methylation. Also, stable lineage conversionshould be accompanied by epigenetic remodeling. Here, weused bisulfite sequencing to assess the methylation status of theElf5, Oct4, and Nanog promoters in the TSC-iPSCs and com-pared them to the respective promoter elements in TSCs andESCs (Fig. 2C to E). Nanog and Oct4 promoter elements werehighly methylated in TSCs (approximately 75%) and demeth-ylated in ESCs (0 to 5.5%) (Fig. 2C and D). In contrast, thepromoter of the trophoblast marker Elf5 was highly meth-ylated in ESCs (90%) and largely demethylated in TSCs(30.8%) (Fig. 2E). The expression of Elf5 leads to a positivefeedback loop with the key trophoblast determinant Cdx2, andit is essential for trophoblast maintenance (31). In TSC-iPSCs,all three promoter elements analyzed displayed a methylationstatus similar to that of ESCs, suggesting that the epigenetic


markers of the extraembryonic lineage had been replaced byESC-specific markers.

Analysis of the in vitro and in vivo differentiation capabilityof TSC-iPSCs. The TSC-iPSC lines generated were highly sim-ilar to ESCs with regard to expression profile and the meth-ylation of key ESC markers. To assess their developmentalpotency in more detail, TSC-iPSCs were aggregated to em-bryoid bodies (EBs) and allowed to adhere to gelatin-coateddishes and to differentiate for 14 days. Immunohistochemicalanalyses showed cells differentiating into mesoderm (Fig. 3A,top, Desmin and SMA), ectoderm (Fig. 3A, middle, GFAP andTuj1), and endoderm (Fig. 3A, bottom, AFP). In addition,contracting cardiomyocytes were observed during differentia-tion (data not shown). The loss of the pluripotency of TSC-iPSCs and the induction of somatic differentiation was con-firmed by RT-PCR. Upon EB formation, the expression of thepluripotency-associated genes Oct4, Nanog, Stella, Fgf4, andFbx15 was downregulated (Fig. 3B, upper), and somatic mark-ers indicative of ectoderm (Fgf5), mesoderm (Brachyury, Flk1),and endoderm (Gata6, Afp) were upregulated (Fig. 3B, mid-dle). The upregulation of the trophoblast-associated markersCdx2, Eomes, Tpbpa, Pl1, Bmp4, Hand1, and Elf5 was compa-rable in ESCs and 4FTSCs (Fig. 3B, lower). Of note, TSCs donot aggregate at all when put into hanging-drop cultures.

We next tested whether TSC-iPSCs retain some kind ofepigenetic memory (20), enabling them to revert spontane-ously into the TE. To address this question, we cultured theTSC-iPSC lines and control ESCs under TSC-specific condi-tions for 14 days and analyzed the TE markers Cdx2 and Cdh3using immunofluorescent staining. In all cultures analyzed, fewCdx2/Cdh3-positive colonies were observed (see Fig. S3A andB in the supplemental material). RT-PCR analyses showed no

increase in the expression of trophoblast markers in TSC-iPSCs compared to that in ESCs (see Fig. S3C).

To examine in vivo differentiation capability, TSC-iPSCswere injected subcutaneously into nude mice, and tumors wereanalyzed after 28 days (Table 1). Histological assessment (Fig.4A) revealed the presence of derivatives of the three embry-onic germ layers, mesoderm (muscle and cartilage), endoderm(pancreas and respiratory epithelium), and ectoderm (squa-mous epithelium and neural tissue), which was confirmed byimmunohistochemical staining for SMA, AFP, and GFAP(Fig. 4B). Areas of Cdx2-positive staining within the teratomawere negative for other trophoblast markers, such as Cdh3,CK8, and PLAP. These cells were identified as glandular cellsof the gastrointestinal tract, which are known to express Cdx2(13) (see Fig. S4A to C in the supplemental material). Thesedata demonstrate that the transplantation of TSC-iPSCs orESCs does not lead to trophoblast derivatives in vivo. Thetransplantation of the parental 4FTSCs did not give rise toteratomas but resulted in transient hemorrhagic tumors(Fig. 4C), as described before (24). Morphological analysis14 days after subcutaneous injection revealed trophoblastgiant cells surrounding blood-filled lacunas (Fig. 4D). Tu-mor size decreased over time (see Fig. S5A and B in thesupplemental material). Apoptosis was detected in the cellsof the tumors, eventually leading to their disappearance 28days after transplantation (Table 1; also see Fig. S5C) (24).This indicates that TSC-iPSCs have acquired embryonic lin-eage restriction because they differentiate into all somaticgerm layers and are not prone to revert into extraembryonicderivatives in vitro and in vivo.

Chimera formation and germ line transmission. Extraem-bryonic and embryonic cell lineages become fate restricted by

FIG. 2. Molecular characterization of TSC-iPSCs. (A and B) Correlation graphs of log2-based intensity values comparing the global geneexpression of TSC-iPSCs to that of 4FTSCs (A) and that of TSC-iPSCs to that of ESCs (B). Specific marker genes are indicated. (C to E)Methylation status of differentially methylated regions (DMR) in the promoters of Pou5f1 (Oct4), Nanog, and Elf5 in ESCs, 4FTSCs, andTSC-iPSCs. Open and filled circles indicate unmethylated and methylated CpGs, respectively.


the time of implantation, with the ICM contributing to theembryo proper and the trophectoderm giving rise to the extra-embryonic cell types of the placenta. If TSC-iPSCs underwentfull lineage conversion, they should be able to chimerize theembryo proper. Therefore, two TSC-iPSC lines derived fromC57/Bl6 mice were selected for injection into blastocysts ofBALB/c mice. Coat color chimerism (see Fig. S6A in the sup-

plemental material) could be detected in 9 of 17 mice pro-duced, indicating that TSC-iPSCs are able to participate in theregular developmental program. Genotyping for the Oct4-GFP transgene demonstrated the contribution of the TSC-iPSCs to somatic tissues in the chimeric mice (see Fig. S6B).To test whether TSC-iPSCs contributed to the germ line,chimeras were crossed with BALB/c females. Black coatcolor in the F1 offspring was indicative of the germ linetransmission of the TSC-iPSCs (Fig. 5A). In addition, wedetected GFP expression in blastocysts and in fetal gonadsof F1 animals on embryonic day 14, indicating the presenceof the Oct4-GFP transgene, which was used initially toscreen for TSC-iPSCs (Fig. 5B to D). Of note, parental4FTSCs did not chimerize the embryo proper (0/13) butrather only the TE compartment (5/13) when injected intoblastocysts (see Fig. S6C and D). These experiments clearlyshow that TSC-iPSCs are capable of contributing to the fullspectrum of somatic differentiation, including the germ line.

FIG. 3. Analysis of in vitro differentiation of TSC-iPSCs. (A) Immunostaining demonstrating in vitro differentiation into all three germ layersof TSC-iPSCs after EB differentiation. Markers detected are indicated. Scale bar, 100 �m. (B) RT-PCR examining the expression of marker genesin TSC-iPSCs, 4FTSCs, and ESCs. Total RNA was isolated from undifferentiated cells and cells differentiated for 14 days.

TABLE 1. Type of cells, number of transplantation experiments,and the number of teratomas obtained from the experiments

Cells injected Days grown No. oftransplantations

No. ofteratomas/tumors

4FTSC 14 8 74FTSC 21 6 34FTSC 28 5 0ESC 28 4 3TSC-IPSC 28 16 9


DISCUSSION

We have demonstrated that extraembryonic TSC lines canbe triggered to undergo lineage conversion into fully inducedpluripotency. Overall, these results demonstrate, for the firsttime, that the overexpression of the reprogramming factorsOct4, Sox2, Klf4, and cMyc in TSCs also is able to overcomethe extraembryonic versus embryonic lineage restriction. Con-verted TSCs give rise to functional somatic cells in vitro and invivo. We demonstrate that transcription factor-induced con-

version across cell lineages can generate epigenetically stablecell fates that closely mirror cell types found in vivo.

In our 4FTSC-derived lines, we observed that the conversionefficiency is much lower than that of reprogramming experi-ments using somatic cells (our data and reference 20). Thismight be due to the fact that the TE lineage is established incompetition to ICM from totipotent blastomeres. The TE lin-eage expresses a set of transcription factors, such as Cdx2,Gata3, Tcfap2c, and Elf5, which are described to repress the

FIG. 4. In vivo pluripotency of TSC-iPSCs. Teratoma formation of TSC-iPSCs. TSC-iPSCs, 4FTSCs, and ESCs were transplanted into nudemice. After 4 weeks, tumors were sectioned and stained with hematoxylin and eosin. (A) Various tissues were present in teratomas derived fromTSC-iPSCs. The mesoderm includes muscle and cartilage, the endoderm includes pancreas and respiratory epithelium, and the ectoderm includessquamous epithelium and neuroectoderm. (B) Immunostaining confirming differentiation into neural tissues (ectoderm), muscles (mesoderm), andendoderm with the markers indicated. (C) 4FTSCs form transient solid tumors with hemorrhagic lesions 14 days after subcutaneous injection.(C and D) Differentiated trophoblast giant cells are arranged around blood-filled lacunas within the tumor. T, trophoblast cells; L, lacuna. Scalebar, 100 �m.


pluripotency network (26, 31, 35, 37). Furthermore, we werenot able to induce lineage conversion in postmitotic tropho-blast giant cells. Therefore, extraembryonic cells represent arepressive context for conversion. Our study also revealed thatlowering the level of p53 leads to increased speed and rate oflineage conversion. This suggests that the p53 pathway restrictsthe rate of lineage conversion in extraembryonic cells as well asin somatic reprogramming (55).

We observed that TSC-iPSCs differentiated into all threegerm layers and gave rise to germ cells. This result indicatesthat the cells derived from TSCs adopted all capabilities toform somatic cells. The issue remains whether the TSC-IPSCsstill are capable of differentiation into the TE derivatives.Upon EB-induced differentiation, the expression of TE-asso-ciated markers was detected. However, we demonstrated thatcontrol ESCs display an identical pattern of marker gene ex-pression. In certain somatic cells during development, thesemarkers also are found upregulated in, e.g., Cdx2 cells of thegastrointestinal tract (13), Eomes in mesoderm and the centralnervous system (1, 27), and Hand1 in cardiomyocytes (2). Thisleads us to conclude that somatic differentiation rather thanreversion to the extraembryonic fate is being observed. In vivo,the TSC-iPSC-derived teratomas did not differentiate into tro-phoblast tissues at all. Hence, it seems unlikely that the TSC-iPSCs retain an epigenetic memory channeling them to differ-entiate preferentially into the cell of origin (20), even whencultured under TSC conditions.

We speculate that conversion from the extraembryonic to

embryonic lineage identity and the acquisition of somatic plu-ripotency requires a complete reset of the extraembryonic epi-genetic program. Again, the reason might be that TE is not aderivative of the ICM but rather represents an alternative andcompetitive lineage. Further experiments should address thequestion of whether the unique epigenetic signature of TE,such as global DNA hypomethylation, histone modifications,specific silencing of the paternal X chromosome, and allele-specific imprinting, are instrumental in orchestrating the mech-anism of the repression of this lineage conversion (16). Theability to convert cells from the extraembryonic lineage opensa door to the molecular dissection of early lineage decisions inmammals.

ACKNOWLEDGMENTS

We thank Sabine Schafer for helping with the embryo dissection,Konrad Hochedlinger for lentiviral constructs, and Mathilde Hau-Liersch and Susanne Steiner for excellent technical assistance.

This study was supported by DFG Grant Scho 503/9 (to H.S.), andP.K. is a scholar of the NRW International Graduate Research School,LIMES–Chemical Biology.

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lineage conversion of murine extraembryonic trophoblast stem cells to pluripotent stem cells

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