Stem Cells
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

First published online January 10, 2008
Stem Cells Vol. 26 No. 4 April 2008, pp. 842 -849
doi:10.1634/stemcells.2007-0356; www.StemCells.com
© 2008 AlphaMed Press

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
2007-0356v1
26/4/842    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by He, S.
Right arrow Articles by Keefer, C. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by He, S.
Right arrow Articles by Keefer, C. L.

EMBRYONIC STEM CELLS

Lymphoid Enhancer Factor 1-Mediated Wnt Signaling Promotes the Initiation of Trophoblast Lineage Differentiation in Mouse Embryonic Stem Cells

Shuyang He, Disha Pant, Andrew Schiffmacher, Ashley Meece, Carol L. Keefer

Department of Animal and Avian Sciences, University of Maryland, College Park, Maryland, USA

Key Words. Caudal-related homeobox 2 • Cell differentiation • Trophoblast • Stem cells

Correspondence: Correspondence: Carol L. Keefer, Ph.D., Department of Animal and Avian Sciences, University of Maryland, College Park, Maryland 20742, USA. Telephone: 301-405-3933; Fax: 301-314-9059; e-mail: ckeefer{at}umd.edu

Received on May 9, 2007; accepted for publication on January 2, 2008.

First published online in STEM CELLS EXPRESS  January 10, 2008.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Embryonic stem (ES) cells can differentiate into all three embryonic germ layers but rarely into trophectoderm (TE) lineages that contribute to the placenta, although TE differentiation can be initiated by genetic manipulation of key genes involved in TE development. We demonstrate that Wnt signaling can initiate TE lineage differentiation by triggering an appropriate cue, caudal-related homeobox 2 (Cdx2). Overexpression and RNA interference knockdown studies indicate that Cdx2 induction in response to Wnt3a is mediated by lymphoid enhancer factor 1, whose expression is regulated by leukemia inhibitory factor (LIF) and bone morphogenetic protein. Removal of LIF, along with addition of Wnt3a, stimulated Cdx2 expression and induced formation of trophoblast stem (TS) cells. These TS cells were able to differentiate into cells with characteristics of spongiotrophoblast and trophoblast giant cells. This is, to our knowledge, the first evidence that TE lineage differentiation can be induced by Wnt signaling in mouse ES cells.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Mouse embryonic stem (ES) cells are derived from inner cell mass and have the capacity to differentiate into all three germ layers, mesoderm, endoderm, and ectoderm [1, 2]. However, ES cells do not contribute to the trophectoderm (TE) lineage in the host embryo's placenta following chimera production [3]. Furthermore, TE lineage differentiation from wild-type ES cells occurs only at an extremely low level in culture [4]. Recently, several key players in TE lineage development in peri-implantation embryos have been identified, and their functions have been determined [5–7]. Genetic manipulation of these key genes, including forced repression of Oct4, and overexpression of caudal-related homeobox 2 (Cdx2) or Eomes can induce TE lineage differentiation and permit the derivation of trophoblast stem (TS) cells from ES cells in vitro [8–10]. Moreover, ES cells can be induced into TE differentiation by collagen IV, but only for ES cell lines maintained on embryonic feeder cells that may be providing additional signaling factors [11]. These results indicate that ES cells have the capacity to differentiate into TE lineage when given the right cues.

The transcription factor Cdx2 is a crucial cue for TE formation in embryos. Although Cdx2 mutant embryos can initiate blastocyst formation, these mutant blastocysts have a collapsed blastocoel, lack expression of TE markers, and fail to implant [7]. Moreover, TS cells cannot be derived from Cdx2 mutant embryos [7]. In Cdx2-null ES cells, activation of inducible transgene Cdx2 is required for TS cells self-renewal, thus demonstrating that Cdx2 is essential in TS cell initiation and maintenance [9].

What signal triggers Cdx2 expression is not clear, although it is known that Cdx2 can be induced in ES cells by forced repression of Oct4 (also known as Pou5f1) [8, 12, 13]. In mouse ES cells, expression of Oct4 and other pluripotency-related transcription factors is maintained by leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP), which work in concert to maintain ES cell pluripotency [14]. BMP blocks entry into neural lineages, whereas LIF inhibits mesoderm and endoderm differentiation [15]. However, LIF and BMP do not appear to have a role in repression of TE lineage, since ES cells rarely differentiate into TE lineage in the absence of LIF and/or BMP. This suggests that one or more additional signals are required to trigger Cdx2 expression and TE lineage differentiation. Notably, it has been shown that Cdx1 and Cdx4, two members of the Cdx homeobox gene family in mice, are targets of Wnt signaling pathway [16, 17, 18]. Expression of Cdx1 can be induced by multiple Wnt ligands, including Wnt3a in ES cells, as well as in undifferentiated rat embryonic endoderm [16]. Cdx4 is triggered by Wnt3a in mouse embryos ex vivo at embryonic day (E) 8.5 and in P19 embryocarcinoma cells [17]. Moreover, Wnt2 and Wnt7b mutants have defects in placenta development [19, 20]. These observations suggest that Wnt signaling may play a role in Cdx2 regulation, as well as trophoblast lineage differentiation, in early embryo development.

Here, we provide the first evidence that Wnt signaling can induce Cdx2 expression in ES cells, thus providing an appropriate cue for TE lineage differentiation and derivation of TS cells. These results provide new insights into the signals that control trophoblast lineage differentiation and placental development and may significantly aid in studying the etiology of implantation failure and early pregnancy loss.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
ES Cell Maintenance
R1 (SCRC-1011; American Type Culture Collection, Manassas, VA, http://www.atcc.org) and D3 (CRL-11632; American Type Culture Collection) mouse ES cells were grown on 0.1% gelatin-coated six-well plates in the absence of feeder cells. The ES medium consisted of 1,000 U/ml LIF (ESGRO; Chemicon, Temecula, CA, http://www.chemicon.com), 15% Knockout Serum Replacement (KSR; Invitrogen, Carlsbad, CA, http://www.invitrogen.com), and basic medium that included Knockout Dulbecco's modified Eagle's medium (Invitrogen), 2 mM L-glutamine, 1 x nonessential amino acids, and 0.1 mM β-mercaptoethanol.

Cytokine Stimulation Studies
ES cells were cultured in ES medium for 24 hours after passaging, at which time the LIF was removed, and cytokine components within the ES cell medium were altered as appropriate for each experiment. In the Wnt3a (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) supplementation studies, the effects of concentration (0, 10, 50, and 100 ng/ml) and time dependence (0.5–24 hours after supplementation with 50 ng/ml) on Cdx2 expression in R1 mouse ES cells were tested. The effect of protein synthesis inhibition on Wnt induction of Cdx2 expression was tested by preincubation of R1 cells with 10 µM cycloheximide for 30 minutes followed by addition of Wnt3a (100 ng/ml) for 3 hours. In the LIF and BMP4 (R&D Systems) supplementation studies, KSR was also removed, in addition to removal of LIF. After 5 hours of starvation, LIF (2,000 U/ml) alone, BMP4 (50 ng/ml) alone, or a combination of LIF and BMP4 was added to basic medium for 1 hour. R1 ES cells were trypsinized and processed by quantitative real-time polymerase chain reaction (PCR) to detect gene expression in the treatment groups.

ES Cell Differentiation
R1 and D3 ES cells were trypsinized and cultured on six-well plates (2 x 105 cells per well) in ES medium without LIF for 42 hours. Wnt3a (50 ng/ml) was subsequently added to ES culture. After 6 hours of Wnt3a stimulation, ES cells were trypsinized and replated on six-well plates at a low density (1 x 104 cells per well) in TS medium. The TS medium consisted of Glasgow Minimum Essential Medium (Invitrogen) supplemented with 10% (vol/vol) fetal bovine serum, 1 x sodium pyruvate, 1 x nonessential amino acids, 0.1 mM β-mercaptoethanol, 2 µg/ml sodium heparin (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), and 25 ng/ml recombinant fibroblast growth factor 4 (FGF4; Sigma-Aldrich) in the presence of 70% (vol/vol) mouse embryonic fibroblast (MEF)-conditioned medium (CM). Four days after culture in TS medium, TS-like cells were apparent. Colonies were counted, and TS cells were subsequently passaged every 3–5 days.

Knockdown by Small Interfering RNA

Lymphoid Enhancer Factor 1 Knockdown.   Stealth RNA interference (RNAi) duplexes (Invitrogen) were designed to lymphoid enhancer factor 1 (Lef1) (NM_010703 [GenBank] ) using the BLOCK-iT RNAi Designer from Invitrogen. The sense strands were as follows: Lef1, 5'-AUGACUUGAUGUCGGCUAAGUCGCC-3'; control, 5'-GGCAUUCGCCGUACAGAACUAGCAU-3'. To examine the effect of Lef1 small interfering RNA (siRNA) on knockdown of Lef1 mRNA, R1 ES cells were transiently transfected with control stealth siRNA (missense) and Lef1 siRNA in ES medium, and total RNAs were extracted at 24, 48, or 72 hours after transfection. To examine the effect of Lef1 downregulation on Cdx2 expression, ES cells were transiently transfected with control stealth siRNA and Lef1 siRNA and were cultured in ES medium for 24 hours. LIF was withdrawn for 2 hours, followed by the addition of Wnt3a (0 or 50 ng/ml). After 6 hours, total RNA was extracted.

Cdx2 Knockdown.   Validated Stealth RNAi duplexes against Cdx2 were obtained from Invitrogen. The sense strand was as follows: Cdx2, 5'-GACAAGGACGUGAGCAUGUAUCCUA-3'. To examine the effect of Cdx2 siRNA on knockdown of Cdx2 mRNA following Wnt3a stimulation, R1 ES cells were transiently transfected with control (Universal Negative) Stealth siRNA (Invitrogen) and Cdx2 siRNA in ES medium minus LIF in six-well gelatin-coated plates. A dilution control was also prepared in which only the siRNA dilution medium (Opti-MEM; Invitrogen) was added. Medium were replaced after 16 hours, and 26 hours later, Wnt3a was added to the medium (at 42 hours after initial removal of LIF). After 6 hours of Wnt3a supplementation, samples for total RNA extraction were taken, and cells were passaged to TS medium as described above.

Plasmid Construction
The open reading frame (ORF) of Lef1{Delta}6 was cloned into HindIII/XbaI sites of PcDNA3.1+ (Invitrogen). Primers were as follows: Lef1 forward, 5'-GGGCATaagcttATGCCCCAACTTTCCGGA-3'; Lef1 reverse, 5'-ATCTGCtctagaTCAGATGTAGGCAGCTGTCA-TTC-3'. ES cells were transiently transfected with empty vector (control) and Lef1{Delta}6 construct and were cultured in ES medium for 24 hours. LIF was withdrawn for 2 hours, followed by the addition of Wnt3a (0 or 50 ng/ml). After 6 hours, total RNAs were extracted.

Transfection
Transfections were conducted in triplicate for each treatment immediately after ES cells were trypsinized and plated on gelatin-coated culture plates. For plasmid transfection, ES cells (2 x 105 per well) were transfected with 2 µl of Lipofectamine 2000 (Invitrogen) and 0.8 µg of plasmid in 12-well plates following the manufacturer's protocol. For RNAi transfection, ES cells (1 x 105 per well) were transfected with complexes consisting of 80 nM Stealth RNAi and 2 µl of Lipofectamine 2000 in 24-well plates following the manufacturer's protocol.

Reverse Transcription-PCR and Quantitative Real-Time PCR
Total RNA was extracted from cells using an Absolutely RNA Miniprep Kit (Stratagene, La Jolla, CA, http://www.stratagene.com) and treated with RNase-free DNase. An aliquot of RNA (0.5 µg) was reverse transcribed into cDNAs using SuperScript III (Invitrogen). PCR was performed using PfuUltra hot start PCR master mix (Stratagene). Primer sequences used are shown in Table 1. The PCR thermocycling conditions were as follows: an initial denaturation step at 95°C for 3 minutes followed by 15 (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]), 20 (Oct-4), or 35 (Pl-1, Tpbp, Hnf4) cycles of 95°C for 30 seconds, primer-specific annealing temperature for 30 seconds, 72°C for a 45-second extension, and a final extension at 72°C for 10 minutes.


View this table:
[in this window]
[in a new window]

  		Table 1. Primer sequences for reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR

 
Quantitative real-time PCR was performed using the iCycler apparatus (Bio-Rad, Hercules, CA, http://www.bio-rad.com) and PCR products were detected with SYBR Green fluorochrome (IQ SYBR Green Supermix; Bio-Rad). Primer sequences used for real-time PCRs are shown in Table 1. Equivalent amounts of cDNA generated from reverse transcription reactions were used as templates for PCR. Reactions were performed in triplicate for each sample. Expression of genes was normalized to the expression of GAPDH, and the mRNA level of each gene in untreated ES cells (control) was set to 1 or otherwise stated specifically. Data are expressed as relative levels of mRNA compared with untreated ES cells. To ensure the specificity and integrity of the PCR product, melt-curve analyses were performed for all PCR products. Samples without template for each primer pair were included to identify contamination.

Immunocytochemistry Analysis
Trophoblast stem cells were cultured on gelatin-coated glass coverslips. Trophoblast giant cells were generated by removing MEF-CM and FGF4 for 4 days. Trophoblast giant cells, as well as undifferentiated ES cells (tissue-negative control), were fixed in 4% formaldehyde and permeabilized in 0.2% Triton X-100 and 0.1% Tween 20 for 10 minutes. They were blocked with 10% normal goat serum for 30 minutes and then incubated with trophoblastoma antigen-1 (TROMA-1) monoclonal antibody (Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/~dshbwww) diluted 1:50 overnight at 4°C. Cells were washed and incubated with Alexa Fluor 488 goat anti-rat IgG (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) diluted 1:100 for 30 minutes at room temperature. Cells were then washed and stained with 5 µg/ml Hoechst 33342 (Sigma-Aldrich) for 10 minutes and whole-mounted onto slides. Samples were examined with a laser confocal microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com).

Statistical Analysis
Statistical analysis of quantitative real-time PCR data was performed with SAS, version 9.0 (SAS Institute, Cary, NC, http://www.sas.com). Data are presented as mean ± SEM. Data were analyzed by one-way analysis of variance (ANOVA); values in figures marked with asterisks were significantly different (p < .05). Proportions were analyzed using Fisher's exact test (GraphPad Software, Inc., San Diego, http://www.graphpad.com).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Cdx2 Is Induced by Wnt3a
Previous reports showed that Cdx1 and Cdx4 were both induced by Wnt3a [16, 17]. Therefore, we first examined whether Wnt3a can trigger Cdx2. Indeed, Cdx2 transcript was induced by Wnt3a in a dose-dependent and time course-dependent manner (Fig. 1A, 1B). Upregulation of Cdx2 was detected as early as 30 minutes, and the strongest expression of Cdx2 was observed at 6 hours; however, Cdx2 expression decreased to basal level by 24 hours (Fig. 1B). This suggests that one or more negative feedback loops exist to restrain Cdx2 increase. With preincubation of cycloheximide, Cdx2 transcript induction was repressed, suggesting that Cdx2 is not the direct target of Wnt signaling pathway or that cofactors are required (Fig. 1C).


Figure 1
View larger version (13K):
[in this window]
[in a new window]

  		Figure 1. Wnt3a induces Cdx2 when added 2 h after leukemia inhibitory factor withdrawal. (A): Cdx2 induction by Wnt3a is dose-dependent. (B): Cdx2 induction by Wnt3a is time-dependent. (C): Cdx2 transcript induction by Wnt3a is dependent on new protein synthesis. Asterisks denote significant difference (p < .05) between control (*) and treatments (**). Abbreviations: Cdx2, caudal-related homeobox 2; CHX, cycloheximide; h, hour(s).

 
Cdx2 Induction by Wnt3a Is Mediated by Lef1
Wnt signaling is mediated by the β-catenin and Lef1/T-cell factor (Tcf) family of DNA binding factors in the canonical Wnt pathway [21]. Chromatin immunoprecipitation assays revealed that Lef1 and β-catenin bind to the Cdx4 promoter, thus mediating the Wnt signaling pathway [17]. Therefore, we next tested whether Cdx2 induction by Wnt3a is mediated by Lef1. Lef1 mRNA is expressed in mouse ES cells (Fig. 2B). Interestingly, an alternative splicing variant was also detected in addition to full-length Lef1. DNA sequencing revealed that this Lef1 variant lacked exon 6 (Lef1{Delta}6) (Fig. 2A). This isoform has also been reported in other cell types [22]. Exon 6 contains a binding domain of Groucho/TLE factor that represses Lef1/TCF target genes. Lef1{Delta}6 has an intact N-terminal β-catenin-binding site and C-terminal high mobility group DNA domain and therefore is capable of binding β-catenin and target genes, thus transducing Wnt signaling. Lef1{Delta}6 was the dominant isoform in ES cells and was expressed at a level 10-fold higher than full-length Lef1 (Fig. 2C). This expression pattern was also observed in morula and blastocysts, but not in MEF and adult tissues (Fig. 2B).


Figure 2
View larger version (35K):
[in this window]
[in a new window]

  		Figure 2. Lef1 mediates Cdx2 induction by Wnt3a. (A): Schematic of Lef1 indicating exons and binding domains. (B): mRNA expression of Lef1 isoforms in preimplantation embryos, ESC, MEF, thymus, and muscle. (C): Lef1{Delta}6 is the predominant isoform expressed in embryonic stem cells. (D): Overexpression of Lef1{Delta}6 enhances Wnt3a-mediated induction of caudal-related homeobox 2 (Cdx2). (E): Lef1 siRNA knocks down Lef1 expression for at least 72 h after transfection. (F): siRNA knockdown of Lef1 attenuates Wnt3a-mediated induction of Cdx2. Asterisks denote significant difference (p < .05) between control (*) and treatments (**). Abbreviations: h, hour; Lef1, lymphoid enhancer factor 1; MEF, mouse embryonic fibroblast; siRNA, small interfering RNA.

 
Overexpression and siRNA repression of Lef1 were performed to determine whether Lef1 was involved in Cdx2 upregulation in response to Wnt3a. Because Lef1{Delta}6 was much more abundant than full-length Lef1 in ES cells, it was chosen for overexpression. RNAi was designed to knock down both Lef1 isoforms and reduced total Lef1 more than 80% (Fig. 2E). Without Wnt signaling, alteration of Lef1 has little effect on Cdx2. However, in response to Wnt3a, overexpression of Lef1{Delta}6 increased Cdx2 induction (Fig. 2D), whereas siRNA knockdown of total Lef1 inhibited Cdx2 induction (Fig. 2F). This indicates that Lef1 is involved in regulation of Cdx2 in response to Wnt3a.

To increase the level of endogenous Lef1, we next investigated how Lef1 is regulated in ES cells. LIF and BMP signaling pathways act in concert to maintain pluripotency of mouse ES cells [15, 23, 24]. It has been shown that Lef1 expression is altered by BMP and other TGF-β factors in other biological systems [25, 26]. To determine whether BMP and LIF regulate Lef1 expression in ES cells, Lef1 transcripts were examined by quantitative real-time PCR after short-term stimulation using these cytokines. Total Lef1 was strongly induced by BMP4. However, BMP4 induction of Lef1 was significantly repressed by LIF, despite the observation that addition of LIF alone for 1 hour had no effect on Lef1 (Fig. 3A), suggesting crosstalk between BMP and LIF on Lef1 regulation. Lef1{Delta}6 and full-length Lef1 had the same pattern of response to BMP4 and LIF as total Lef1 (data not shown). Smad7, a known target of BMP, was induced only by BMP4 and was not affected by addition of LIF (Fig. 3B). Furthermore, repression of Lef1 expression was dramatically eliminated after LIF withdrawal, as shown by the more than 18-fold increase of Lef1 observed at 48 hours after removal of LIF (Fig. 3C). Despite the increase of Lef1, withdrawal of LIF alone had no effect on Cdx2 (Fig. 3D), which is consistent with the result that overexpression of Lef1 alone did not alter Cdx2 expression (Fig. 2D). In conjunction with the increased Lef1 triggered by LIF withdrawal for 48 hours, Cdx2 mRNA was increased 12-fold and 14-fold by incubation with Wnt3a (LIF–/Wnt3a+) in R1 and D3 ES cells, respectively, which was approximately three- to fourfold the response in ES cells cultured in the presence of LIF (LIF+/Wnt3a+) (Fig. 3E).


Figure 3
View larger version (21K):
[in this window]
[in a new window]

  		Figure 3. LIF represses left expression and Lef1-mediated responses to Wnt3a. Relative levels of Lef1 (A) and Smad7 (B) were affected by 1 h of LIF and/or BMP supplementation in embryonic stem (ES) cells starved of LIF and Knockout Serum Replacement for 5 h. (C): Lef1 was upregulated and continued to increase for 48 h following removal of LIF supplementation. (D): Expression of caudal-related homeobox 2 (Cdx2) was not affected by removal of LIF supplementation. (E): Wnt3a treatment 42 h after LIF withdrawal resulted in stimulation of Cdx2 transcripts in R1 and D3 ES cell lines. (F): Higher efficiencies of TS colony formation were observed 4 days after Wnt3a treatment when LIF withdrawal preceded treatment in both R1 and D3 ES cell lines. Asterisks denote significant difference (p < .05) between control (*) and treatments (**). Abbreviations: BMP, bone morphogenetic protein; h, hour(s); Lef1, lymphoid enhancer factor 1; LIF, leukemia inhibitory factor; TS, trophoblast stem cell.

 
TS Cell Derivation from ES Cells by Manipulation of Wnt3a and LIF
Since overexpression of Cdx2 in ES cells induced TE lineage differentiation [9, 10], we reasoned that TE lineage can be initiated from ES cells with enhanced expression of Cdx2 as triggered by Wnt3a and increased levels of Lef1 (Fig. 3E). To further induce trophoblast cell lineage, ES cells were plated into new six-well plates with TS medium after 6-hour Wnt3a stimulation following 48-hour LIF withdrawal. Cdx2 levels remained high 24 hours (data not shown) after passage to TS medium, which contains FGF4 and MEF-conditioned medium, and with continued maintenance in TS medium (Fig. 4A). High efficiencies (19% and 26% in R1 and D3 cells, respectively) (Fig. 3F) of TS-like colonies were observed from LIF–/Wnt3a+ groups based on morphology (Fig. 4D) after a 4-day culture.


Figure 4
View larger version (41K):
[in this window]
[in a new window]

  		Figure 4. Trophoblast lineage differentiation of ES cells. (A–C): Expression of trophoblast markers in undiff (TS on day 0), diff2, and diff5 cells. (D): TS-like colony. (E, F): Trophoblast giant cells (E) stained for TROMA-1 (F). (G, H): Undiff ESC cells (G), which were negative for TROMA-1 (H). (A, B): Asterisks denote significant difference (p < .05) between control (*) and treatments (**). Abbreviations: Cdx2, caudal-related homeobox 2; Diff2, day 2 differentiating; Diff5, day 5 differentiating; ES, embryonic stem; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Undiff, undifferentiated.

 
These TS-like cells maintained self-renewal for at least 3 weeks for six passages in TS medium. Trophoblast lineage markers, including Cdx2, Eomes, Gcm1, Hand1, and Mash2, were all substantially expressed in these TS-like cells derived from R1 ES cells (Fig. 4A, 4B). After withdrawal of MEF-CM and FGF4, TS markers Cdx2 and Eomes decreased continuously at days 2 and 5. Expression of Gcm1, Hand1, and Mash2 increased in differentiating cells at day 2 but decreased in further differentiated cells at day 5. Placental lactogen/Chorionic somatomammotropin hormone 1 (Pl-1/Csh1) a marker for trophoblast giant cells, was strongly expressed at days 2 and 5, whereas trophoblast-specific protein {alpha} (Tpbp), a marker for spongiotrophoblast, was also detected at day 2 (Fig. 4C). More than 50% of differentiated cells showed characteristics of giant cells with swollen nuclei and extensive cytoplasmic spreading (Fig. 4E). This was further confirmed by positive staining with TROMA-1 antibody, which is specific for trophoblast giant cells (Fig. 4F) and negative for undifferentiated ES cells (Fig. 4G, 4H). With induction of TE lineage differentiation, Oct4 was significantly downregulated, and Hnf4, a marker for endoderm, was not expressed (Fig. 4C). However, brachyury, a marker for mesoderm, was upregulated, which is consistent with other reports [9]. Pl-1 expression and giant cell formation were also observed following withdrawal of MEF-CM and FGF4 from D3-derived TS cells (data not shown).

RNAi designed to knock down Cdx2 mRNA during LIF withdrawal and Wnt3a supplementation resulted in a significant decrease in Cdx2 mRNA to 64% of dilution control (0.64 ± 0.11; p < .05; ANOVA). Cdx2 levels in the dilution and missense controls were not significantly different from each other (1 ± 0.01 and 1.10 ± 0.09, respectively). Since LIF withdrawal and Wnt3a supplementation resulted in a 12-fold increase in Cdx2 mRNA, this reduction in Cdx2 by siRNA still represented approximately 8-fold and 2-fold increases in Cdx2 mRNA compared with Cdx2 levels in LIF+ and LIF+/Wnt3a+ treatments, respectively. The efficiency of TS colony formation was 15% following Cdx2 siRNA treatment, 25% in the missense control, and 30% in the dilution control. Knockdown of Cdx2 did reduce TS colony formation significantly compared with the dilution control (p < .05; Fisher's exact test), but not compared with the missense control. To our knowledge, this is the first evidence that TS cells can be derived at a high efficiency from ES cells by manipulation of cytokines, in this case, Wnt3a and LIF.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
TS cells can be derived from either blastocysts or early postimplantation extraembryonic ectoderm, in the presence of FGF4 and fibroblast-conditioned medium [27]. TS cells can also be derived from ES cells by overexpression of Cdx2 or Eomes [9], a downstream target of Cdx2, or by triggering Cdx2 expression via Oct4 knockdown [8]. More recently, trophectoderm differentiation was obtained following culture of ES cells on collagen IV, but only for cell lines that had been maintained on feeder layers, indicating the requirement for additional inductive cues [11]. Furthermore, this TE differentiation was Cdx2-dependent [11]. Clearly, the evidence indicates that Cdx2 induction is the cue forcing ES cells to differentiate into TE lineage; however, what external signal triggers Cdx2 is largely unknown. Here, we show that Cdx2 expression and TE lineage differentiation can be triggered by Wnt signaling. This new knowledge provides a novel means to derive TS cells from ES cells by controlling exposure to Wnt3a and LIF, but without any forced genetic manipulation.

At first glance, Wnt is not an obvious candidate for induction of TE lineage differentiation. Previous studies have emphasized the role of Wnt and β-catenin in regulating self-renewal in ES cells. It was shown that pluripotency was sustained in human and mouse ES cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor [28]. This positive role of Wnt/β-catenin in mouse ES cell self-renewal is likely accomplished by enhancement of the LIF-STAT3 pathway [29, 30]. However, placenta defects were observed in Wnt2 and Wnt7b mutants, as well as mice with Lef1/Tcf1 double knockout, suggesting a role for Wnt in TE lineage development. More interestingly, two Cdx2 homologs, Cdx1 and Cdx4, were target genes of Wnt3a in ES cells [16, 17]. Accordingly, we investigated whether Cdx2 and further TE lineage differentiation can be triggered by Wnt3a in ES cells.

Our results show that Cdx2 is indeed induced by Wnt3a; however, this induction was only a short-term response. It is not surprising that Cdx2 expression quickly decreased back to the basal level within 24 hours, since Wnt signaling also has a positive role in self-renewal and may send a negative feedback signal to inhibit Cdx2 transcription. This Cdx2 response pattern to Wnt signaling may explain why ES cells maintain compact colonies and show no increase of TE markers after 4–6 days when they were cultured in standard ES medium containing Wnt3a, but without LIF. This suggests that sustained high expression of Cdx2 is required to trigger TE lineage differentiation in ES cells. Sustained levels of Cdx2 are maintained with exposure to the appropriate culture milieu as provided by the TS medium (Fig. 4A).

In embryogenesis, Wnt signaling also has distinct roles in mesoderm development, which is regulated by different members of Lef1/Tcf family [31]. Here, in ES cells, different members of Lef1/Tcf family may also have diverse roles in response to Wnt signaling. A recent report showed that Tcf3 was able to repress Nanog, a key transcription factor for maintenance of pluripotency [32]. However, the predominant isoform of Lef1 in ES cells lacks the critical Groucho domain that is involved in Tcf3 repression of Nanog [32], and neither overexpression of Lef{Delta}6 nor knockdown of total Lef1 by siRNA affected Nanog or Oct4 expression (data not shown). Our results showed that Lef1 mediated Cdx2 induction; however, Lef1 appeared dispensable for upregulation of another Wnt3a-regulated gene, brachyury, since brachyury induction by Wnt3a was not affected by either overexpression or knockdown of Lef1 (data not shown). Others have also reported that Lef1 is dispensable for induction, but not maintenance, of brachyury [33]. Interestingly, Lef1 was coregulated by LIF and BMP, which indicates that LIF and BMP can crosstalk with the Wnt signaling pathway through regulation of Lef1 expression (Fig. 5).


Figure 5
View larger version (18K):
[in this window]
[in a new window]

  		Figure 5. Proposed model for signaling pathways involved in the initiation of trophoblast lineage differentiation. Details are given in the text. Abbreviations: BMP, bone morphogenetic protein; Cdx2, caudal-related homeobox 2; FGF4, fibroblast growth factor 4; Lef1, lymphoid enhancer factor 1; LIF, leukemia inhibitory factor; MEF-CM, mouse embryonic fibroblast-conditioned medium; TS, trophoblast stem cell.

 
After revealing that Cdx2 induction by Wnt3a was mediated by Lef1, whose expression was largely inhibited by LIF, we were able to induce much higher expression of Cdx2 by applying short-term Wnt3a stimulation after removal of LIF for 48 hours. Nevertheless, Cdx2 expression was not sustained, and TE lineage differentiation was not observed when ES cells continued to be cultured in ES medium regardless of the presence or absence of LIF. On the other hand, when favorable conditions for TS differentiation were provided by replating ES cells in TS medium after 6 hours of Wnt3a stimulation, Cdx2 expression was maintained, and TE differentiation was apparent after 4 days. This suggests that some factor(s) in TS medium help to maintain Cdx2 level, thus facilitating TE lineage differentiation. It has been shown that FGF and Wnt signaling can interact in the regulation of Cdx genes during gastrula stages in Xenopus laevis [34]. Furthermore, FGF4 can stimulate ES cells to exit the self-renewal program and respond to inductive cues for lineage differentiation [35]. Therefore, the supplemented FGF4 and other FGFs in MEF-CM may have a role in this maintenance of elevated Cdx2 expression. Although Wnt3a stimulation induced TS lineage differentiation, this should be considered as a context-dependent trigger; that is, TS cells were obtained when Wnt3a-stimulated cells were placed under favorable conditions (TS medium). As the Wnt/β-catenin pathway plays a role in the differentiation of various lineages [21, 36], it should be possible to establish other lineages under the proper conditions. In fact, Bakre et al. have reported that sustained Wnt activation in ES cells can lead to the generation of multipotential mesendodermal progenitors [37].

Our finding that Cdx2 expression is induced by Wnt3a in ES cells suggests a possible role of Wnt signaling in Cdx2 initiation in preimplantation embryos. Other evidence also supports this hypothesis. First, several Wnt ligands, including Wnt3a, are expressed from two-cell to blastocyst stage [38]. Second, nuclear-localized active β-catenin is observed in outer cells of morula and TE of unhatched blastocysts but then disappears in the hatched blastocyst and does not reappear until postimplantation [39]. These studies suggest that Wnt signaling may be active during TE cell lineage specification. However, other studies have indicated that, contrary to ES cells, Wnt/β-catenin signaling is not functional in preimplantation embryos and does not become active until late blastocyst stage (E4.5) [36, 40, 41]. Furthermore, no defects of TE formation and implantation have been observed in Wnt mutant embryos tested so far, although this could be due to maternal expression of Wnt or redundant functions among Wnt proteins known to be expressed in blastocysts, such as Wnt1, Wnt3a, Wnt6, Wnt7b, Wnt9a, and Wnt10b [42]. Moreover, overexpression of Dishevelled proteins, transducers of divergent Wnt pathways, disrupts cell-cell adhesion at early preimplantation stages, suggesting that noncanonical pathways may be active [43]. Clearly, additional studies are needed to answer the many questions concerning the role of Wnt pathways during early preimplantation development.

In this study, the interplay between cytokines and culture milieu was critical for the appropriate derivation of TS cells. Thus, although removal of LIF resulted in upregulation of Lef1, the addition of Wnt3a was required to induce Cdx2 expression. Subsequent culture of the stimulated cells in TS medium, which contains FGF and conditioned medium, was necessary for continued Cdx2 expression and thus provided the appropriate conditions for TS cells derivation. This protocol provides a new system for studying TE lineage without the need for genetically altering ESC.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
S.H. is currently affiliated with the Cellular Therapeutics Division, Celgene Corp., Warren, NJ.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

  1. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981;292:154–156.[CrossRef][Medline]

  2. Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 1981;78:7634–7638.[Abstract/Free Full Text]

  3. Beddington RS, Robertson EJ. An assessment of the developmental potential of embryonic stem cells in the midgestation mouse embryo. Development 1989;105:733–737.[Abstract/Free Full Text]

  4. Hemberger M, Nozaki T, Winterhager E et al. Parp1-deficiency induces differentiation of ES cells into trophoblast derivatives. Dev Biol 2003;257:371–381.[CrossRef][Medline]

  5. Chawengsaksophak K, James R, Hammond VE et al. Homeosis and intestinal tumours in Cdx2 mutant mice. Nature 1997;386:84–87.[CrossRef][Medline]

  6. Russ AP, Wattler S, Colledge WH et al. Eomesodermin is required for mouse trophoblast development and mesoderm formation. Nature 2000;404:95–99.[CrossRef][Medline]

  7. Strumpf D, Mao CA, Yamanaka Y et al. Cdx2 is required for correct cell fate specification and differentiation of trophectoderm in the mouse blastocyst. Development 2005;132:2093–2102.[Abstract/Free Full Text]

  8. Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000;24:372–376.[CrossRef][Medline]

  9. Niwa H, Toyooka Y, Shimosato D et al. Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation. Cell 2005;123:917–929.[CrossRef][Medline]

  10. Tolkunova E, Cavaleri F, Eckardt S et al. The caudal-related protein cdx2 promotes trophoblast differentiation of mouse embryonic stem cells. STEM CELLS 2006;24:139–144.[Abstract/Free Full Text]

  11. Schenke-Layland K, Angelis E, Rhodes KE et al. Collagen IV induces trophoectoderm differentiation of mouse embryonic stem cells. STEM CELLS 2007;25:1529–1538.[Abstract/Free Full Text]

  12. Hay DC, Sutherland L, Clark J et al. Oct-4 knockdown induces similar patterns of endoderm and trophoblast differentiation markers in human and mouse embryonic stem cells. STEM CELLS 2004;22:225–235.[Abstract/Free Full Text]

  13. Hough SR, Clements I, Welch PJ et al. Differentiation of mouse embryonic stem cells after RNA interference-mediated silencing of OCT4 and Nanog. STEM CELLS 2006;24:1467–1475.[Abstract/Free Full Text]

  14. Cavaleri F, Scholer HR. Nanog: A new recruit to the embryonic stem cell orchestra. Cell 2003;113:551–552.[CrossRef][Medline]

  15. Ying QL, Nichols J, Chambers I et al. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003;115:281–292.[CrossRef][Medline]

  16. Lickert H, Domon C, Huls G et al. Wnt/(beta)-catenin signaling regulates the expression of the homeobox gene Cdx1 in embryonic intestine. Development 2000;127:3805–3813.[Abstract]

  17. Pilon N, Oh K, Sylvestre JR et al. Cdx4 is a direct target of the canonical Wnt pathway. Dev Biol 2006;289:55–63.[CrossRef][Medline]

  18. Moon RT, Bowerman B, Boutros M et al. The promise and perils of Wnt signaling through beta-catenin. Science 2002;296:1644–1646.[Abstract/Free Full Text]

  19. Monkley SJ, Delaney SJ, Pennisi DJ et al. Targeted disruption of the Wnt2 gene results in placentation defects. Development 1996;122:3343–3353.[Abstract]

  20. Parr BA, Cornish VA, Cybulsky MI et al. Wnt7b regulates placental development in mice. Dev Biol 2001;237:324–332.[CrossRef][Medline]

  21. Willert K, Jones KA. Wnt signaling: Is the party in the nucleus? Genes Dev 2006;20:1394–1404.[Abstract/Free Full Text]

  22. Cordray P, Satterwhite DJ. TGF-beta induces novel Lef-1 splice variants through a Smad-independent signaling pathway. Dev Dyn 2005;232:969–978.[CrossRef][Medline]

  23. Smith AG, Heath JK, Donaldson DD et al. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 1988;336:688–690.[CrossRef][Medline]

  24. Williams RL, Hilton DJ, Pease S et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 1988;336:684–687.[CrossRef][Medline]

  25. Jamora C, DasGupta R, Kocieniewski P et al. Links between signal transduction, transcription and adhesion in epithelial bud development. Nature 2003;422:317–322.[CrossRef][Medline]

  26. Nawshad A, LaGamba D, Hay ED. Transforming growth factor beta (TGFbeta) signalling in palatal growth, apoptosis and epithelial mesenchymal transformation (EMT). Arch Oral Biol 2004;49:675–689.[CrossRef][Medline]

  27. Tanaka S, Kunath T, Hadjantonakis AK et al. Promotion of trophoblast stem cell proliferation by FGF4. Science 1998;282:2072–2075.[Abstract/Free Full Text]

  28. Sato N, Meijer L, Skaltsounis L et al. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 2004;10:55–63.[CrossRef][Medline]

  29. Hao J, Li TG, Qi X et al. WNT/beta-catenin pathway up-regulates Stat3 and converges on LIF to prevent differentiation of mouse embryonic stem cells. Dev Biol 2006;290:81–91.[CrossRef][Medline]

  30. Ogawa K, Nishinakamura R, Iwamatsu Y et al. Synergistic action of Wnt and LIF in maintaining pluripotency of mouse ES cells. Biochem Biophys Res Commun 2006;343:159–166.[CrossRef][Medline]

  31. Liu F, van den Broek O, Destree O et al. Distinct roles for Xenopus Tcf/Lef genes in mediating specific responses to Wnt/beta-catenin signalling in mesoderm development. Development 2005;132:5375–5385.[Abstract/Free Full Text]

  32. Pereira L, Yi F, Merrill BJ. Repression of Nanog gene transcription by Tcf3 limits embryonic stem cell self-renewal. Mol Cell Biol 2006;26:7479–7491.[Abstract/Free Full Text]

  33. Galceran J, Hsu SC, Grosschedl R. Rescue of a Wnt mutation by an activated form of LEF-1: Regulation of maintenance but not initiation of Brachyury expression. Proc Natl Acad Sci U S A 2001;98:8668–8673.[Abstract/Free Full Text]

  34. Keenan ID, Sharrard RM, Isaacs HV. FGF signal transduction and the regulation of Cdx gene expression. Dev Biol 2006;299:478–488.[CrossRef][Medline]

  35. Kunath T, Saba-El-Leil MK, Almousailleakh M et al. FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment. Development 2007;134:2895–2902.[Abstract/Free Full Text]

  36. Marikawa Y. Wnt/beta-catenin signaling and body plan formation in mouse embryos. Semin Cell Dev Biol 2006;17:175–184.[CrossRef][Medline]

  37. Bakre MM, Hoi A, Mong JC et al. Generation of multipotential mesendodermal progenitors from mouse embryonic stem cells via sustained Wnt pathway activation. J Biol Chem 2007;282:31703–31712.[Abstract/Free Full Text]

  38. Lloyd S, Fleming TP, Collins JE. Expression of Wnt genes during mouse preimplantation development. Gene Expr Patterns 2003;3:309–312.[CrossRef][Medline]

  39. Li J, Zhang JV, Cao YJ et al. Inhibition of the beta-catenin signaling pathway in blastocyst and uterus during the window of implantation in mice. Biol Reprod 2005;72:700–706.[Abstract/Free Full Text]

  40. De Vries WN, Evsikov AV, Haac BE et al. Maternal beta-catenin and E-cadherin in mouse development. Development 2004;131:4435–4445.[Abstract/Free Full Text]

  41. Kemler R, Hierholzer A, Kanzler B et al. Stabilization of beta-catenin in the mouse zygote leads to premature epithelial-mesenchymal transition in the epiblast. Development 2004;131:5817–5824.[Abstract/Free Full Text]

  42. Kemp C, Willems E, Abdo S et al. Expression of all Wnt genes and their secreted antagonists during mouse blastocyst and postimplantation development. Dev Dyn 2005;233:1064–1075.[CrossRef][Medline]

  43. Na J, Lykke-Andersen K, Torres Padilla ME et al. Dishevelled proteins regulate cell adhesion in mouse blastocyst and serve to monitor changes in Wnt signaling. Dev Biol 2007;302:40–49.[CrossRef][Medline]





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
2007-0356v1
26/4/842    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by He, S.
Right arrow Articles by Keefer, C. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by He, S.
Right arrow Articles by Keefer, C. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS