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First published online December 20, 2006
doi: 10.1242/10.1242/jcs.03296

Journal of Cell Science 120, 55-65 (2007)
Published by The Company of Biologists 2007
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Activin-Nodal signaling is involved in propagation of mouse embryonic stem cells

Kazuya Ogawa1,*, Akira Saito2,*, Hisanori Matsui1, Hiroshi Suzuki2, Satoshi Ohtsuka1, Daisuke Shimosato1,3, Yasuyuki Morishita2, Tetsuro Watabe2, Hitoshi Niwa1,3,{ddagger} and Kohei Miyazono2,4,{ddagger}

1 Laboratory for Pluripotent Cell Studies, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan
2 Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
3 Department of Developmental and Regenerative Medicine, Graduate School of Medicine, Kobe University, 7-5-1, Kusunokicho, Chuo-ku, Kobe 650-0017, Japan
4 Department of Biochemistry, The Cancer Institute of the Japanese Foundation for Cancer Research, Toshima-ku, Tokyo 170-8455, Japan


Figure 1
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  		Fig. 1. Forced Smad7 expression inhibited ES cell propagation. (A) Luciferase reporter assay in MGZ5 cells transfected with reporter constructs (ARE-luc reporter or Id-1-luc reporter) and each expression plasmid. Each bar represents the mean ± s.e.m. (n=3). (B) Colony morphologies of supertransfectants. MGZ5 cells were transfected with Smad7-expressing plasmid (middle), Smad6 plasmid (right), or empty vector (left), seeded at 2000 cells/well in six-well plates, and cultured for 1 week in medium supplemented with puromycin. Numbers indicate numbers of colonies appearing (n=3). The lower panel shows AP staining. Bars, 5 mm (upper panels); 200 µm (lower panels). (C) Relative numbers of each transfectant present on Day 7 of culture compared with that at Day 0. Each bar represents the mean ± s.e.m. (n=3). (D) Relative proliferation intensity is shown as total cell number/number of colonies appearing, compared with that of empty vector transfectants (100%). Each bar represents the mean ± s.e.m. (n=3).

 

Figure 2
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  		Fig. 2. SB-431542 inhibited ES cell propagation. (A) Colony morphologies of MGZ5 cells treated with SB-431542. MGZ5 cells were seeded at 2000 cells/well in six-well plates and cultured in FCS-containing medium supplemented with 10 µM SB-431542 (right) or DMSO (as control, left) for 1 week. Numbers indicate numbers of colonies appearing (n=3). Each lower panel shows AP staining. Bars, 5 mm (upper panels); 200 µm (lower panels). (B) Relative numbers of cells treated with SB-431542 or DMSO present on Day 7 of culture compared with that at Day 0. Each bar represents the mean ± s.e.m. (n=3). (C) Relative proliferation intensity is shown as total cell number/number of colonies appearing, compared with that of cells treated with DMSO (100%). Each bar represents the mean ± s.e.m. (n=3). (D) Luciferase reporter assay of MGZ5 cells transfected with reporter constructs (ARE-luc reporter or Id-1-luc reporter) and treated with SB-431542 or DMSO (as a control). Each bar represents mean ± s.e.m. (n=3).

 

Figure 3
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  		Fig. 3. The inhibitory effect of Smad7 is reversible. (A) Strategy for generation of fSmad7-10+ and fSmad7-10- ES cells. fSmad7-10+ was generated by stable integration of floxed-Smad7 cDNA transgene under the CAG promoter into EB3 ES cells. After elctroporation of pCAGGS-Cre into fSmad7-10+, fSmad7-10- was free of Smad7 cDNA and expressed DsRed. (B) Colony morphologies of fSmad7-10+ (left) and Smad7-10- (right) cells. EB3 (control), fSmad7-10+ and fSmad7-10- ES cells were seeded at 2000 cells/well in six-well plates, and cultured for 1 week in the medium supplemented with puromycin. The lower panel shows expression of DsRed. (C) Colony morphologies of fSmad7-10+, fSmad7-10-, and control ES cells. fSmad7-10+ (middle), fSmad7-10- (right) and EB3 (as control, left) ES cells were cultured in FCS-containing medium for 5 days. Numbers indicate numbers of colonies appearing (n=3). The lower panel shows AP staining. Bars, 5 mm (upper panels); 200 µm (lower panels). (D) Relative numbers of each transfectant present on Days 1, 3, and 5 of culture compared with Day 0. Each bar represents the mean ± s.e.m. (n=4). (E) Relative proliferation intensity is shown as total cell number on Day 5/number of colonies appearing, compared with that of EB3 ES cells (100%). Each bar represents the mean ± s.e.m. (n=3). (F) Expression of stem cell marker genes Oct3-4, Sox2, and Rex-1 in fSmad7-10+, fSmad7-10- and EB3 (as control) ES cells examined by northern blot analysis. ES cells were cultured in FCS-containing medium for 5 days. Gapdh was detected as a loading control. (G) Chimeric mice derived from fSmad7-10- cells that had been cultured for at least 1 month. Chimeric mice (upper panel) expressed DsRed, whereas control mice (lower panel) exhibited no DsRed fluorescence.

 

Figure 4
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  		Fig. 4. Inhibitory effect of Smad7 in serum-free conditions. (A) Colony morphologies of fSmad7-10+ and fSmad7-10- ES cells in serum-free medium. fSmad7-10+ (middle), fSmad7-10- (right), and EB3 (as control, left) ES cells were seeded at 2000 cells/well in six-well plates, and cultured in FCS-containing medium for 1 week. Numbers indicate numbers of colonies appearing (n=3). Bars, 5 mm. (B) Relative numbers of each transfectant present on Day 7 of culture compared with Day 0. Each bar represents the mean ± s.e.m. (n=4). (C) Relative proliferation intensity is shown as total cell number/number of colonies appearing, compared with that of EB3 ES cells (100%). Each bar represents the mean ± s.e.m. (n=3).

 

Figure 5
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  		Fig. 5. Effects of TGFß superfamily signaling on ES cell proliferation in serum-free conditions. (A) Effects of various TGFß superfamily molecules on Smad2 phosphorylation in EB5 ES cells. EB5 cells were treated with the indicated factors for 1 hour, and subjected to western blot analysis using anti-phospho-Smad2 antibody (upper panel) and anti-Smad2/3 antibody (lower panel). (B) Colony morphologies of EB5 ES cells in serum-free medium supplemented with various TGFß superfamily molecules. EB5 colonies were grown for 7 days in serum-free medium supplemented with no factor (as control), 30 ng/ml activin, 1 µg/ml Nodal, 2 µg/ml Nodal or 30 ng/ml BMP-4. Numbers indicate numbers of colonies appearing (n=3). Bars, 5 mm. (C,D) Relative numbers of cells treated with TGFß superfamily molecules present on Day 3 (C) and Day 7 (D) of culture compared with that at Day 0. Each bar represents the mean ± s.e.m. (n=4).

 

Figure 6
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  		Fig. 6. Maintenance of undifferentiated state of ES cells cultured in serum-free medium with Nodal or activin. (A) Examination of expression of stem cell marker genes in EB5 ES cells clonally expanded and maintained for at least 1 month in serum-free medium supplemented with either 30 ng/ml activin or 1 µg/ml Nodal by RT-PCR analysis. Gapdh was detected as a loading control. (B) Hematoxylin and eosin staining of teratomas derived from EB5 ES cells cultured with Nodal protein. Tissues derived from each of the three germ layers were formed. Neuroectoderm (ectoderm; top left), cartilage (mesoderm; top right), mucus-producing epithelium (endoderm; bottom left) and ciliated epithelium (endoderm; bottom right) were observed. (C) Chimeric mouse derived from EB3 ES cells cultured with 2 µg/ml Nodal.

 

Figure 7
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  		Fig. 7. ES cells produce activin-Nodal activities for self-propagation. (A) Luciferase reporter assay of MGZ5 cells with reporter constructs (ARE-luc reporter or Id-1-luc reporter). Control, cells transfected with reporter and control vectors only; Smad7, Smad6, empty, cells transfected with each expression plasmid; SB-431542, DMSO, cells cultured in medium supplemented with SB-431542 or DMSO (as control for SB-431542). Each bar represents the mean ± s.e.m. (n=3). (B) Colony morphologies of MGZ5 cells treated with SB-431542. MGZ5 cells were seeded at 2000 cells/well in six-well plates and cultured in serum-free medium supplemented with 3 µM SB-431542 (right) or DMSO (as control, left) for 1 week. Numbers indicate numbers of colonies appearing (n=3). Bars, 5 mm. (C) Relative numbers of cells treated with SB-431542 or DMSO present on Day 7 of culture compared with that at Day 0. Each bar represents the mean ± s.e.m. (n=3). (D) Relative proliferation intensity is shown as total cell number/number of colonies appearing, compared with that in cells treated with DMSO (100%). Each bar represents the mean ± s.e.m. (n=3).

 




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