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Published as doi: 10.1096/fj.08-111203.
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(The FASEB Journal. 2009;23:114-122.)
© 2009 FASEB

Directed induction of anterior and posterior primitive streak by Wnt from embryonic stem cells cultured in a chemically defined serum-free medium

Mio Nakanishi*, Akira Kurisaki{dagger}, Yohei Hayashi*, Masaki Warashina{ddagger}, Shoichi Ishiura*, Miho Kusuda-Furue§,|| and Makoto Asashima1,*,{dagger},||

* Department of Life Sciences (Biology), Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan;

{dagger} Organ Development Research Laboratory, National Institute of Advanced Industrial Science and Technology, Ibaraki, Japan;

{ddagger} Genome Research Laboratories, Wako Pure Chemical Industries, Ltd., Hyogo, Japan;

§ Division of Bioresources, National Institute of Biomedical Innovation, Osaka, Japan; and

|| International Cooperative Research Project (ICORP), Japan Science and Technology Agency, Tokyo, Japan

1 Correspondence: Department of Life Sciences (Biology), Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1, Komaba, Meguro-ku, Tokyo, 153-8902, Japan. E-mail: asashi{at}bio.c.u-tokyo.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Formation of the primitive streak (PS) is the initial specification step that generates all the mesodermal and endodermal tissue lineages during early differentiation. Thus, a therapeutically compatible and efficient method for differentiation of the PS is crucial for regenerative medicine. In this study, we developed chemically defined serum-free culture conditions for the differentiation of embryonic stem (ES) cells into the PS-like cells. Cultures supplemented with Wnt showed induction of expression of a PS marker, the brachyury gene, followed by induction of the anterior PS markers goosecoid and foxa2, a posterior PS marker, evx1, and the endoderm marker sox17. Similar differentiation of PS by Wnt was also observed in human ES cells. Moreover, we revealed that the activation of the Wnt canonical pathway is essential for PS differentiation in mouse ES cells. These results demonstrated that Wnt is an essential and sufficient factor for the induction of the PS-like cells in vitro. These conditions of induction could constitute the initial step in generating therapeutically useful cells of the definitive endoderm lineage, such as hepatocytes and pancreatic endocrine cells, under chemically defined conditions.—Nakanishi, M., Kurisaki, A., Hayashi, Y., Warashina, M., Ishiura, S., Kusuda-Furue, M., Asashima, M. Directed induction of anterior and posterior primitive streak by Wnt from embryonic stem cells cultured in a chemically defined serum-free medium.


Key Words: mesendoderm • endoderm • regenerative medicine • brachyury • sox17


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DURING GASTRULATION, A SPECIFIC subgroup of epiblast cells ingresses into the primitive streak (PS) and subsequently generates the mesoderm and definitive endoderm (1 , 2) . PS contains the progenitors of these cells at the early- and mid-PS stages of gastrulation (3 4 5) . Therefore, PS formation is an essential specification step in the generation of all the mesodermal and endodermal tissue lineages, including the pancreas, liver, and heart.

Recent studies using knockout mice suggested that Wnt signaling is indispensable for the differentiation of the PS. In Wnt3a-homozygous knockout embryos, epiblast cells that are ingressing into the PS are diverted to a neuroectodermal fate rather than forming paraxial mesodermal cells (6) . Double-homozygous mutants of coreceptors for the transduction of Wnt signals Lrp5;Lrp6 also fail to establish the PS (7) . Moreover, ablation of β-catenin in embryonic endoderm changes cell fate from endoderm to precardiac mesoderm (8) . These reports support the importance of Wnt signaling in the differentiation of the PS, mesoderm, and endoderm in vivo.

In addition to Wnt signaling, transforming growth factor-β (TGF-β) signaling is important for PS differentiation. Mouse embryos deficient for one of the transforming growth factor-β members, Nodal, fail to form both the mesoderm and the definitive endoderm after implantation (9) . Moreover, animals that lack one allele of Smad2 and Smad3 exhibit defects in the definitive endoderm (10) . Loss of Smad3 in the context of one wild-type allele of Smad2 results in impaired production of the anterior axial mesendoderm, while Smad2-Smad3 double-homozygous mutants completely lack mesoderm and fail to gastrulate (11) . Collectively, these results suggest that dose-dependent TGF-β signaling via Smad2/3 mediates cell fate in the early stage of mesoderm and endoderm formation.

In vitro differentiation of PS from both human and mouse embryonic stem (ES) cells using activin A, which is a member of the TGF-β superfamily, has been reported (12 13 14 15) . In contrast, the role of Wnt signaling in the differentiation of ES cells to PS is not well-documented, although Wnt signaling has been shown to maintain pluripotency in human and mouse EScells (16) .

In this study, we developed a simple culturing method using a chemically defined medium (ESF) to induce specifically the PS-like cells from mouse ES cells. Previously, we established that this medium could be used to propagate mouse ES cells without feeder cells (17 , 18) . Here, we demonstrate that treatment with Wnt efficiently induces directed induction of tissue culture analogs of the PS from both mouse and human ES cells. Furthermore, we show that Wnt selectively activates canonical signaling during PS differentiation of ES cells. Our results verified that Wnt signaling is essential and sufficient for PS differentiation from ES cells in vitro. Moreover, our method could be used as a basic protocol for the preparation of endodermal and mesodermal tissues under chemically defined conditions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
The mouse ES cell D3 line (American Type Culture Collection, Manassas, VA, USA) was routinely cultured in a tissue culture dish coated with type I collagen (Nitta Gelatin Inc., Osaka, Japan) in a humidified atmosphere of 5% CO2 at 37°C in a chemically defined medium, designated ESF7, as described previously (17 , 18) . For subculturing, mouse ES cells were dissociated with 0.2 mg/ml EDTA in PBS and seeded at 1.7 x 103 cells/cm2 every 4 days.

For the induction of PS differentiation, mouse ES cells were dissociated with 20 µg/ml EDTA · 4Na in PBS and seeded onto tissue culture dishes coated with laminin (Sigma, St. Louis, MO, USA) at 0.75–2.5 x 104 cells/cm2. The ES cells were cultured in a chemically defined differentiation medium A [ESF basal medium (Cell Science & Technology Institute, Sendai, Japan) that contains 10 µg/ml insulin, 5 µg/ml apo-transferrin, 10 µM 2-mercaptoethanol, 10 µM ethanolamine, 10 µM sodium selenite, and 0.5 mg/ml BSA (Sigma)] supplemented with the indicated concentration of Wnt-3a (R&D Systems, Minneapolis, MN, USA) or activin A. Besides these supplements, recombinant mouse Dkk1 (R&D Systems) was supplemented where indicated. The culture medium was renewed every 2 days.

Human ES cells (KhES-1) were obtained from the Institute for Frontier Medical Science, Kyoto University (Kyoto, Japan), with approval for human ES cell use granted by the Minister of Education, Culture, Sports, Science, and Technology of Japan. The Review Board of the University of Tokyo approved this research. The entire study was conducted in accordance with the Declaration of Helsinki. Human ES cells were maintained on a feeder layer of mouse embryonic fibroblasts pretreated with 10 µg/ml mitomycin C (Sigma) for 3 h. Human ES cells were maintained in Dulbecco modified Eagle medium (DMEM)/F-12 supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM nonessential amino acids, 2 mM L-glutamine, penicillin/streptomycin, 20% knockout serum replacement (KSR; Invitrogen, Carlsbad, CA, USA) and 5 ng/ml bFGF (Upstate Biotechnology, Lake Placid, NY, USA) in a humidified atmosphere of 3% CO2 and 97% air at 37°C. ES cells were partially dissociated with human ES cell dissociation solution [0.25% trypsin and 0.1 mg/ml collagenase IV (Gibco, Carlsbad, CA, USA) in PBS that contained 1 mM CaCl2 and 20% KSR] and subcultured every 3 or 4 days.

For the induction of PS-like cells, human ES colonies were partially dissociated into clumps with the above-mentioned human ES cell dissociation solution; this was followed by gentle trituration. The human ES clumps (300 cells/clump) were then cultured for 3 days in a laminin-coated tissue culture test plate (2–2.5 clumps/cm2) in hESF-dif medium (Nipro, Osaka, Japan) that was supplemented with 10 µg/ml insulin, 5 µg/ml apo-transferrin, 10 µM 2-mercaptoethanol, 10 µM ethanolamine, 10 µM sodium selenite, 0.5 mg/ml BSA (Sigma), and 50 ng/ml of Wnt-3a (R&D Systems) or activin A. The chemical components of hESF-dif are same as those of mouse ESF except HEPES. HEPES is excluded from hESF-dif medium.

Embryoid body assay for mouse ES cells
We performed two kinds of embryoid body (EB) assays in this study. For the differentiation of mouse ES cells into an ectodermal lineage (see Fig. 3 ), mouse ES cells treated with Wnt-3a for 3 days as described above or with undifferentiated ES cells were dissociated with 10 µg/ml trypsin and 20 µg/ml EDTA · 4Na in PBS and cultured in round-bottom low-cell-binding plates (Nunc, Roskilde, Denmark) (2000 cells/50 µl) using a fetal bovine serum (FBS) -containing differentiation medium B [DMEM supplemented with 15% FBS (Gibco) and penicillin-streptomycin] for 24 h to prepare EBs. The EBs were then transferred to gelatin-coated 24-well test plates and cultured for 12 days in differentiation medium B, which was replenished every 4 days.


Figure 1
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  		Figure 1. Specific induction of the PS and early endoderm marker genes by Wnt-3a in mouse ES cells. A) Undifferentiated mouse ES cells cultured in ESF medium with LIF. B) ES cells differentiated into PS cells by Wnt-3a for 3 days. Scale bars = 100 µm. C) Summary of specific markers for PS, mesoderm, and endoderm. D–I) Quantitative analysis of marker gene expression. Mouse ES cells were cultured with 50 ng/ml Wnt-3a or activin A from day 0 to day 6. Duplicates of the cultures were harvested at the indicated times, and the expression levels of the indicated genes were analyzed by quantitative RT-PCR. Values were normalized to gapdh mRNA, and the control values were arbitrarily set to day 0 (undifferentiated ES cells). J) Titration of Wnt-3a and activin concentration for the differentiation to PS-like cells. Duplicate cultures were harvested on day 3, and the expression levels of the indicated genes were analyzed by quantitative RT-PCR as described above. EB indicates the spontaneously differentiated cells by forming EB in a chemically defined serum-free differentiation medium for 3 days as described in Materials and Methods.


Figure 2
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  		Figure 2. Proportions of PS marker-positive cells in the Wnt-3a treated cell population. A–I) Immunofluorescence staining of mouse ES cells treated with 5 ng/ml (left panels) or 50 ng/ml (middle panels) Wnt-3a or 50 ng/ml activin A (right panels) from day 0 to day 3. Cells were immunostained (red) with antibodies against Brachyury (A–C), Goosecoid (D–F), or Foxa2 (G–I). Nuclei were stained with DAPI (blue). J, K) Bar graphs show ratios of Brachyury-positive (J) and Goosecoid-positive (K) cells to the total number of cells. L–O) Immunofluorescence staining (red) with anti-E-cadherin (L, M) or anti-Sox17 (N, O) antibody of ES cells treated with 50 ng/ml Wnt-3a from day 0 to day 3 (L, N) or day 5 (M, O). Nuclei are stained with DAPI (blue). Scale bars = 10 µm.


Figure 3
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  		Figure 3. Reduced potential of Wnt-3a-treated mouse ES cells to differentiate into non-PS ectodermal lineages. EBs generated from undifferentiated mouse ES cells or ES cells cultured with 50 ng/ml Wnt-3a for 3 days were spontaneously differentiated. After 12 days of culture, EBs were immunostained with an antibody against β3-tubulin (red). Nuclei were stained with DAPI (blue). A) β3-Tubulin-positive neurons in the EBs formed from the undifferentiated ES cells. B) Differentiated cells from Wnt-3a-treated ES cells. C) Ratio of β3-tubulin-positive cells. The ratios are expressed as the percentage of β3-tubulin-positive EBs to the total number of EBs. Scale bars = 10 µm.

For the experiment shown in Fig. 1J , mouse ES cells were dissociated with 10 µg/ml trypsin and 0.2 µg/ml EDTA in PBS and cultured in round-bottom low-cell-binding plates (2000 cells/50 µl) using the chemically defined differentiation medium A without Wnt-3a or activin for 24 h. The next day, 150 µl of differentiation medium A was added to each well, and the EBs were further cultured for 2 days.

Real-time quantitative polymerase chain reaction (PCR)
Total RNA was isolated from duplicated samples using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany), and 500–2000 ng of RNA was used for reverse-transcription with the SuperScript First-Strand System (Invitrogen). PCRs were carried out using 1/50–1/100 dilutions of the cDNA per reaction, 500 nM of the forward and reverse primers, and the Quantitect SYBR Green master mix (Qiagen). The following PCR conditions were used: 95°C for 15 min, followed by 45 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. Alternatively, Taqman gene expression assays (Applied Biosystems, Foster City, CA, USA) were used according to the manufacturer’s instructions. Real-time PCR was performed using the ABI PRISM 7700 Sequence Detector (Applied Biosystems). Relative quantification was performed against a standard curve and the values were normalized against the input determined for the housekeeping gene, gapdh. The primer sequences used in this study are described in Table 1 .


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  		Table 1. Oligodeoxynucleotide primers used for real-time PCR

Immunostaining
ES cells were seeded onto laminin-coated Lab-Tek chamber slides (Nunc). The cells were washed with PBS and fixed in cold acetone for 5 min. After washing with PBS, the cells were blocked with 3% BSA in PBS for 30 min at room temperature and incubated with the primary antibody for 16 h at 4°C. The cells were washed twice with PBS, incubated with secondary antibody that was labeled with Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen) for 1 h at room temperature, and mounted in VectaShield Hardset Mounting Medium with 4',6'-diamino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). The cells were observed under a fluorescence microscope and photographed with the ORCA-3 CCD camera controlled by the Luminavision software (Mitani, Fukui, Japan). The primary antibodies used were goat anti-brachyury (4 µg/ml; R&D Systems), mouse anti-β-catenin (1.25 µg/ml; BD Biosciences, Franklin Lakes, NJ, USA), mouse anti-E-cadherin (2.5 µg/ml; BD Biosciences), mouse anti-GSC (10 µg/ml; Abnova, Taipei, Taiwan), goat anti-HNF-3β/FOXA2 (2 µg/ml; R&D Systems), and goat anti-SOX17 (5 µg/ml; Neuromics, Edina, MN, USA).

For immunofluorescent staining of the differentiated EBs, they were fixed in 4% paraformaldehyde in PBS for 25 min. After washing with PBS, the fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 30 min, blocked with 3% BSA in PBS for 30 min, and incubated with the mouse anti-β3-tubulin monoclonal antibody (Chemicon, Temecula, CA, USA) diluted in 1% BSA, 0.1% Tween-20 in PBS for 16 h at 4°C, followed by 2 washes with PBS. Then, the samples were incubated with the secondary antibody labeled with Alexa Fluor 594 (Invitrogen) diluted in 1% BSA, 0.1% Tween- 20 in PBS for 1 h at room temperature. After washing with PBS, cell nuclei were stained with DAPI for 7 min. The EBs were observed under a fluorescence microscope and photographed with AquaCosmos (Hamamatsu Photonics, Hamamatsu, Japan) connected to the ORCA-3 CCD camera.

Immunoblotting
Cells were lysed in a minimal volume of ice-cold lysis buffer [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, protease inhibitor cocktail (Roche, Basel, Switzerland), PhosSTOP (Roche), 1 mM Na3VO4, 25 mM NaF, and 25 mM β-glycerophosphate]. After rotating at 4°C for 1 h, the supernatant was collected by centrifugation at maximum speed for 15 min at 4°C. The proteins were separated by SDS-PAGE and detected by Western blotting with horseradish peroxidase (HRP) -conjugated anti-mouse or anti-goat immunoglobulin G (IgG) as the secondary antibody. For signal detection, SuperSignal West Femto substrate (Pierce, Rockford, IL, USA) was applied, and the membranes were visualized with the LAS-1000plus lumino-image analyzer (Fuji Film, Tokyo, Japan).

The following primary antibodies were used in this study. Mouse anti-actin was purchased from Sigma, and mouse anti-β-catenin was from BD Biosciences. Mouse anti-GSK3 was obtained from Upstate Biotechnology. Rabbit anti-phospho-GSK-3 S9/S21 and rabbit anti-phospho-PKC (pan) were from Cell Signaling (Danvers, MA, USA). Rabbit anti-c-Jun, rabbit anti-phospho-c-Jun, and rabbit anti-PKC were all from Santa Cruz Biotechnology (Santa Cruz, CA, USA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mouse ES cells were cultured in the chemically defined serum-free medium, ESF7. Under these conditions, ES colonies formed without feeder cells (Fig. 1A ). Using this culture medium, we examined the effects of Wnt-3a and activin on the differentiation of ES cells to PS in vitro. Treatment of mouse ES cells with Wnt-3a induced efficient differentiation into flattened monolayer cells (Fig. 1B ). The expression levels of marker genes for the PS, mesoderm, and endoderm in these cells were analyzed by quantitative reverse transcriptase-PCR (RT-PCR). The specific markers used in this study are summarized in Fig. 1C . In the ES cells, Wnt-3a induced up to 370-fold the expression of the PS- and mesoderm-specific transcription factor, brachyury (19 , 20) , with induction starting on day 3 (Fig. 1D ). Activin treatment also induced the expression of brachyury mRNA, albeit at an extremely lower level and more slowly. The expression of the anterior PS-enriched transcription factor goosecoid (21 , 22) was induced by Wnt-3a treatment, and activin A treatment again showed weaker induction of this gene (Fig. 1E ). Another anterior PS marker foxa2 (23 24 25) was dramatically induced up to 1200-fold by Wnt-3a, and an endoderm marker sox17 was also induced (Fig. 1F, G ). The expression levels of foxa2 and sox17 peaked on Day 6. However, activin A did not show significant effect on the expression of foxa2 and sox17. As shown in Fig. 1H , Wnt-3a transiently induced early posterior PS marker, evx1 (26) . In contrast to the PS markers, the expression levels of the ectodermal marker sox1 and the primitive endoderm marker sox7 were down-regulated by Wnt-3a or activin A before the onset of brachyury and foxa2 induction (Fig. 1I , data not shown). These results indicate that Wnt, but not activin, specifically induces the PS-like cells under chemically defined culture conditions.

To eliminate the possibility that these effects were compared under nonoptimal conditions, titration of these ligands was verified by quantitative RT-PCR (Fig. 1J ). The expression of brachyury mRNA was highest when the ES cells were treated with 100 ng/ml of Wnt-3a. In contrast, 25–50 ng/ml of activin induced the highest expression of the brachyury gene. The expression induced by activin, however, was much weaker than that by Wnt-3A. Higher concentrations of Wnt3a and activin than these resulted in decreased expression of this gene. When the titration was performed with the foxa2 or sox1 genes, the optimal concentration was similar to those with brachyury gene, and 50–100 ng/ml of Wnt-3a produced the highest effect. The optimal concentration of activin was also 50–100 ng/ml for these genes. These results indicated that the concentration of Wnt-3a and activin (50 ng/ml) used for the analyses, as shown in Fig. 1D –I, was appropriate, and the effects of these factors on the differentiation into PS were properly analyzed. In addition, we compared the efficiency of differentiation by Wnt-3a treatment and random differentiation by EB formation in a chemically defined medium. Treatment with Wnt-3a increased the expression of the PS marker genes to greater extent than random EB differentiation did (Fig. 1J , right edge); this further supported the fact that Wnt-3a is indeed a specific inducer of PS formation.

To examine further the differentiated PS-like cells, Wnt-3a- or activin A-treated ES cells were immunostained with specific antibodies for Brachyury, Goosecoid, and Foxa2 at day 3 of differentiation. Intense immunostaining for Brachyury was detected in both the nuclei and cytoplasm of the cells (Fig. 2B ). When the strongly immunopositive cells (Fig. 2B ) were counted, 80% of the Wnt-3a (50 ng/ml) -treated ES cells were found to be Brachyury-positive (Fig. 2J ). The anterior PS marker Goosecoid was detected mainly in the cell nuclei after treatment with 50 ng/ml Wnt-3a (Fig. 2E ), and 61% of the cells were Goosecoid-positive (Fig. 2K ). However, treatment with either activin A or a low concentration of Wnt-3a (5 ng/ml) resulted in very weak expression of Brachyury (Fig. 2A, C, J ). Similarly, the number of Goosecoid-positive cells was small in ES cells treated with a low concentration of Wnt-3a (5 ng/ml) or activin A (50 ng/ml) (Fig. 2D, F, K ). In the absence of Wnt or activin A, the ES cells did not attach to the laminin-coated dish and did not grow at all (data not shown). These data further confirmed that Wnt efficiently induces PS differentiation, including the anterior PS, in mouse ES cells.

In addition to the strong expression of Brachyury and Goosecoid in the PS and the axial mesoderm in vivo (19 20 21 22) , E-cadherin also expresses in the PS during development, although the expression level decreases during the mesoderm differentiation (27) . In our system, E-cadherin was detected in all the cells treated with 50 ng/ml Wnt-3a on day 3 (Fig. 2L ), and 30–40% of cells became E-cadherin-negative on day 5 (Fig. 2M ). Concurrently with this change, an early differentiation marker of definitive endoderm, Sox17, started to express on day 5 (Fig. 2N, O ). These data further support our finding that Wnt-3a induces within 3 days a significant number of cells that correspond to the tissue culture analogs of the PS in developing mouse embryos, which subsequently differentiate into the mesoderm and endoderm.

To evaluate the developmental potential of Wnt-3a-induced PS-like cells, mouse ES cells treated with 50 ng/ml of Wnt-3a for 3 days were allowed to form EBs and to differentiate spontaneously in serum-containing medium for 12 days. Control ES cells differentiated into a significant number of β3-tubulin-positive axon-extending neurons (Fig. 3A ), demonstrating the ability to differentiate into the ectodermal lineages. In contrast, EBs differentiated from Wnt-3a-treated ES cells contained few β3-tubulin-positive cells (Fig. 3B ). In the quantification of this immunofluorescence analysis, β3-tubulin-positive cells were detected in 63% of the control EBs, whereas the number of β3-tubulin-positive EBs was significantly decreased to 27% after Wnt-3a-treatment (Fig. 3C ). These results indicate that Wnt-3a-treated ES cells have a markedly reduced ability to differentiate into non-PS ectodermal lineages.

Next, we analyzed the signal specificity of PS differentiation induced by Wnt-3a. Since the initial 2 days of culture at any concentration of Wnt-3a did not induce the expression of PS markers (Fig. 1 , and data not shown), we analyzed the activation of Wnt signaling pathways after 2 days of preculturing of the ES cells in the presence of 5 ng/ml of Wnt-3a. Treatment of the ES cells with 50 ng/ml Wnt-3a after the preculture period markedly increased the expression levels of the PS marker genes brachyury and foxa2 within 24 h (Fig. 4A ). Under these conditions, phosphorylation of GSK-3{alpha}/β almost disappeared 6–12 h after Wnt-3a stimulation. Concomitantly, there was a significant increase in the level of β-catenin protein (Fig. 4B ), whereas the control cells retained the initial levels of β-catenin and GSK-3{alpha}/β phosphorylation. Immunofluorescence analysis clearly showed the accumulation of β-catenin protein in ES cells 12 h after Wnt-3a stimulation (Fig. 4C ). We also examined the activation of PKC and c-Jun, which are implicated in the signal transduction of noncanonical Wnt signaling (28 , 29) . However, no significant activation of PKC or c-Jun was detected (Fig. 4B , and data not shown). Dkk1 has been used as an inhibitor of the Wnt canonical signaling pathway (30) . Treatment of ES cells with Dkk1 strongly inhibited the expression of the brachyury and foxa2 genes induced by exogenous Wnt-3a (Fig. 4D ), indicating that PS differentiation of ES cells by Wnt-3a is dependent on Wnt canonical signaling.


Figure 4
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  		Figure 4. Activation of Wnt canonical pathway during PS differentiation of mouse ES cells. Mouse ES cells cultured with 5 ng/ml Wnt-3a for 2 days and then left unstimulated or stimulated with 50 ng/ml Wnt-3a. A) Expression levels of PS marker genes were analyzed by quantitative RT-PCR at 0 or 24 h after stimulation. B) Immunoblot of key Wnt-signaling molecules, including β-catenin, phospho-GSK-3{alpha}/β (Ser-9/21), GSK-3{alpha}/β, phospho-PKC, PKC, and the control protein {alpha}-actin, at the indicated time points poststimulation. C) Immunofluorescence analysis of stimulated or unstimulated ES cells stained with anti-β-catenin antibody (red) and DAPI (blue) 12 h poststimulation. Images were taken at the same exposure length. Scale bars = 20 µm. D) Specific inhibition of Wnt-3a-dependent PS differentiation by Dkk1. Expression levels of brachyury and foxa2 were analyzed by quantitative RT-PCR 24 h after stimulation with 50 ng/ml Wnt-3a alone or with the combination of the indicated concentration of Dkk1.

Finally, to determine whether Wnt signaling could also induce PS differentiation in human ES cells in a chemically defined serum-free medium, we cultured human ES cells for 3 days in medium that was supplemented with 50 ng/ml Wnt-3a, and analyzed the specific marker genes for PS by quantitative RT-PCR (Fig. 5A, B ). For the differentiation of human ES cells, a commercially available hESF-dif medium for human ES cells was used. KSR and bFGF were not included in the differentiation medium. Although the effects of activin in human ES cells were superior to those in mouse ES cells, Wnt-3a induced a significantly higher expression of several PS-specific markers. In addition to these quantitative data, immunofluorescence analysis was performed using specific antibodies for PS markers. The result of this analysis showed that significant numbers of immunopositive cells expressing Brachyury, Goosecoid, and Foxa2 were observed in the flattened cells that grew out from the clump of ES cells (Fig. 5C-E ). In most of these cells, Brachyury and Goosecoid were colocalized to the nucleus (data not shown). Semiquantitative analysis by counting immunopositive cells revealed that Wnt-3a-treatment induces quite efficient differentiation of human ES cells into PS-like cells (29–53%) as shown in Fig. 5F-H . On the basis of these data, we conclude that Wnt-3a is also an effective and specific inducer of PS differentiation in human ES cells when cultured in a chemically defined medium.


Figure 5
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  		Figure 5. Directed differentiation of human ES cells into PS by Wnt treatment. A, B) Quantitative analysis of marker gene expression of human ES cells treated with 50 ng/ml of Wnt-3a or activin A for 3 days. Duplicate cultures were analyzed by quantitative RT-PCR. C–E) Immunofluorescent staining of Wnt-3a treated human ES cells. Cells treated withWnt-3a for 3 days were immunostained with antibodies against Brachyury (C), Goosecoid (D), and Foxa2 (E). Nuclei were stained with DAPI (blue). Scale bars = 50 µm. F–H) Quantitative analysis of the immunofluorescent staining in C–E. Immunopositive cells in Wnt-3a-treated cells were counted in 5 independent fields.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrate that mouse ES cells cultured in a serum-free chemically defined medium, ESF7, and treated with Wnt-3a produce a highly enriched culture of anterior and posterior PS. Recently, there have been reports that Wnt-3a can induce the differentiation of the posterior PS and mesoderm from ES cells (15 , 31 , 32) . However, in vitro induction of the anterior streak or definitive endoderm has not been achieved with Wnt (12 13 14 15) . In our unique culture system, robust expression of PS marker genes, including anterior PS markers, was readily observed in Wnt-3a-treated cells. Initially a whole PS and the mesoderm marker brachyury were induced, followed by expression of the anterior PS markers goosecoid and foxa2. The endoderm marker sox17 was induced within 6 days. In contrast, genes not expressed in the PS in vivo, such as the ectoderm marker sox1 and the primitive endoderm marker sox7, were suppressed by Day 3. These results indicate that Wnt-3a induces directed differentiation into PS-like cells, including its anterior subdivision, which could be expected to further differentiate into both the mesoderm and definitive endoderm.

Immunofluorescence analysis revealed that a large proportion of the Wnt-3a-treated cells expressed both Brachyury and Goosecoid. Moreover, E-cadherin, which is expressed in the PS but not in the mesoderm, was detected in all the cells treated with Wnt-3a for 3 days. The expression of these markers in the Wnt-3a-treated Day 3 cells was consistent with the expression patterns in the anterior PS. Furthermore, Wnt-3a treatment significantly reduced the potential of ES cells to differentiate into non-PS lineages, such as neural ectodermal cells. These results support our conclusion that Wnt-3a treatment commits ES cells to the PS lineage in the chemically defined medium.

We also analyzed the Wnt signaling pathways during the anterior and posterior PS differentiation. In this study, we demonstrate that Wnt-3a specifically inhibits the phosphorylation of GSK-3{alpha}/β and induces a significant increase in the level of β-catenin protein in ES cells cultured in our chemically defined medium. Moreover, a Wnt canonical pathway-specific inhibitor, Dkk1, abrogated Wnt-dependent induction of brachyury and foxa2 gene expression. A recent study suggested the importance of canonical signaling in the differentiation of mesoderm from ES cells using complex EB formation, although in that study, Wnt activity alone was not sufficient to induce the mesoderm-specific gene expression (32) . Our data clearly demonstrate for the first time that Wnt-3a is sufficient for the induction of both the anterior and posterior PS, from which the definitive endoderm and mesoderm are produced, and that Wnt canonical signaling is essential for this differentiation.

Previous studies have shown that high doses of activin promote the differentiation of endoderm from ES cells (12 , 15) . However, under our culture conditions, cultivation with activin alone had little effect on the expression of sox17, which suggests that the ability of activin to induce the PS and endoderm is quite limited in our system. This discrepancy may reflect differences in the induction methods used. Some of the previous studies used EB formation as the initial step in the differentiation into PS (13 , 15) . EB formation causes changes in the cellular microenvironment, such as high concentrations of locally produced cytokines and various interactions between different cell types within the EB structures (33 34 35 36) . These complex conditions may enable unexpected crosstalk between different signaling pathways. In addition, serum (14) and serum replacement supplements of undisclosed compositions (12) were used to induce mesendodermal differentiation from ES cells. In contrast to these previous studies, we used a completely defined medium that does not contain any inducing factors, with the exception of minimal supplements, such as insulin and transferrin. Our results suggest that unknown factors derived from serum or serum replacement or complex conditions, such as cell-cell interactions in the EB, are required for endoderm induction by activin.

In this study, we demonstrate that Wnt treatment also induces PS differentiation in human ES cells. These results suggest that Wnt is a common inducing factor for both the anterior and posterior PS in mammalian ES cells. Our method can be an innovative approach to obtain enriched cultures of PS cells in a serum-free chemically defined medium. Our method does not entail procedures that might affect reproducibility, such as EB formation, coculturing with other cells, or gene targeting. Although the potential of these induced cells for the generation of terminally differentiated PS derivatives is currently under investigation, further studies are likely to show that this method is an essential step in generating therapeutically useful cells derived from the PS, such as cardiomyocytes, vascular endothelial cells, hepatocytes, and pancreatic β-cells.


   ACKNOWLEDGMENTS
 
We thank Drs. N. Nakatsuji and H. Suemori (Kyoto University, Kyoto, Japan) for providing human KhES-1 cell line. We thank Drs. T. Michiue and H. Danno for discussion. We also gratefully acknowledge Takeda Pharmaceuticals Company, Inc. for its generous support of this study. This work was supported in part by a grant-in-aid for scientific research from the Ministry of Health, Labor and Welfare of Japan, by a grant from the Naito Foundation, and by Wako Pure Chemical Industries, Ltd.

Received for publication April 9, 2008. Accepted for publication August 21, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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