Smad2 is essential for maintenance of the human and mouse primed pluripotent stem cell state

Abstract

Human embryonic stem cells and mouse epiblast stem cells represent a primed pluripotent stem cell state that requires TGF-β/activin signaling. TGF-β and/or activin are commonly thought to regulate transcription through both Smad2 and Smad3. However, the different contributions of these two Smads to primed pluripotency and the downstream events that they may regulate remain poorly understood. We addressed the individual roles of Smad2 and Smad3 in the maintenance of primed pluripotency. We found that Smad2, but not Smad3, is required to maintain the undifferentiated pluripotent state. We defined a Smad2 regulatory circuit in human embryonic stem cells and mouse epiblast stem cells, in which Smad2 acts through binding to regulatory promoter sequences to activate Nanog expression while in parallel repressing autocrine bone morphogenetic protein signaling. Increased autocrine bone morphogenetic protein signaling caused by Smad2 down-regulation leads to cell differentiation toward the trophectoderm, mesoderm, and germ cell lineages. Additionally, induction of Cdx2 expression, as a result of decreased Smad2 expression, leads to repression of Oct4 expression, which, together with the decreased Nanog expression, accelerates the loss of pluripotency. These findings reveal that Smad2 is a unique integrator of transcription and signaling events and is essential for the maintenance of the mouse and human primed pluripotent stem cell state.

Keywords: Bone Morphogenetic Protein (BMP); Cell Differentiation; Nanog; Pluripotency; Smad Transcription Factor; Stem Cells; Transforming Growth Factor β (TGFβ).

FIGURE 1.

SMAD2, but not SMAD3, is required for pluripotency of hESCs (A–D) and mEpiSCs (E–H). A, down-regulation of SMAD2 (left panel) but not SMAD3 (right panel) expression for 5 days leads to decreased OCT4 and NANOG but not SOX2 expression shown by immunoblotting. B, colony morphology following down-regulation of SMAD2 or SMAD3 expression at day 5 after lentiviral shRNA expression. C, immunofluorescence shows loss of OCT4 (green) or NANOG (red) expression, upon down-regulation of SMAD2 but not SMAD3 expression for 5 days. DAPI serves as nuclear counterstain. D, proliferation of control cells or cells with decreased SMAD2 (red lines) or SMAD3 (blue lines) expression. KD1 and KD2 cells express different shRNAs. E, down-regulation of Smad2 (left panel) but not Smad3 (right panel) expression for 5 days leads to decreased Nanog expression, as shown by immunoblotting. F, proliferation of control cells or cells with decreased Smad2 (red lines) or Smad3 (blue lines) expression. G, immunoblotting shows decreased Smad2 or Smad3 expression using lentiviral shRNA vectors encoding GFP. H, immunofluorescence reveals loss of Nanog and Oct4 expression in cells infected to express Smad2 shRNA and GFP for 10 days. GFP-positive cells lacked Nanog and Oct4 expression. Decreasing Smad3 expression did not affect Nanog or Oct4 expression in GFP-positive cells. DAPI served as nuclear counter-stain.

FIGURE 2.

Decreasing Smad2 expression affects stem cell differentiation. A, SOX17, BRACHYURY, TUJ1, and CDX2 mRNA expression in control hESCs and hESCs with decreased SMAD2 or SMAD3 expression for 5 days, as quantified by qRT-PCR. B, immunoblots of CDX2, SOX17, BRACHYURY, and TUJ1 expression in control hESCs and hESCs with decreased SMAD2 expression at days 7 or 10 after infection with lentiviral shRNA vector. Lane C, control. C and D, SOX17, FOXA2, GATA4, and CER (endodermal markers) mRNA levels (C) and CDX2, DLX3, HAND1, and MSX2 (trophectodermal markers) mRNA levels (D) in control hESCs and hESCs with decreased SMAD2 expression at day 5 after infection with lentiviral shRNA vector. E, Sox17, Brachyury, Tuj1, and Cdx2 mRNA expression in control mEpiSCs and mEpiSCs with decreased Smad2 or Smad3 expression for 5 days, as quantified by qRT-PCR. F, mRNA levels of CER, FOXA2, and SOX17 (endodermal markers), KDR, MHC, and RUNX2 (mesodermal markers), and NESTIN, SOX1, and TUJ1 (neuroectoderm markers) in embryoid bodies from control hESCs or hESCs with decreased SMAD2 expression. G, immunofluorescence of FOXA2, SMA, and TUJ1 expression during spontaneous differentiation of embryoid bodies of control hESCs and hESCs with decreased SMAD2 expression. n = 3 assays. *, p < 0.05; **, p < 0.01, as compared with control cells by Student's t tests.

FIGURE 3.

Smad2, but not Smad3, regulates Nanog expression through direct binding to the Nanog promoter. A, effects of activin, TGF-β, SB431542, or basal TGF-β/activin signaling (No Treatment) on endogenous NANOG mRNA expression in control hESCs and hESCs with decreased SMAD2 or SMAD3 expression at day 7 after lentiviral shRNA vector infection. B, binding of endogenous SMAD2 or SMAD3 to the NANOG promoter, assessed by chromatin immunoprecipitation, in cells with or without ligand stimulation and after SB431542 treatment, at day 7 after lentiviral shRNA vector infection. C, binding of endogenous SMAD2 and SMAD3 to the SMAD7 promoter, assessed by chromatin immunoprecipitation, in cells with or without ligand stimulation and after SB431542 treatment. n = 3 assays. *, p < 0.05; **, p < 0.01, compared with SB431542-treated cells (A–C) by Student's t tests. D, association of endogenous SMAD4 with the SMAD7 promoter but not the NANOG promoter, as assessed by chromatin immunoprecipitation, in cells with or without ligand stimulation after SB431542 (SB) treatment. n = 3 assays. **, p < 0.01, compared with SMAD4 ChIP with SB431542 treatment by Student's t tests. E, immunoblots of SMAD4, SMAD2/3, NANOG, OCT4, and GAPDH in control hESCs and hESCs with down-regulated SMAD4 expression at 96 h after siRNA transfection.

FIGURE 4.

Increased BMP responsiveness in cells with decreased Smad2 expression leads to increased BMP target gene expression. A, decreased SMAD2 expression in hESCs results in higher levels of phosphorylated SMAD1/5 at day 6 after lentiviral shRNA infection. Noggin or BMP were added at 16 or 1 h before analysis. Lane C, control. B, BRACHYURY (Bra) and CDX2 mRNA expression in control hESCs and hESCs with decreased SMAD2 expression in the presence or absence of Noggin from day 3 to day 8 after infection. C, immunofluorescence for CDX2 and OCT4 in control hESCs and hESCs with decreased SMAD2 expression at day 8 after infection. Noggin or BMP were added from day 3 to 8. DAPI served as nuclear counterstain. D, mRNA expression of primodial germ cell markers, BLIMP1 and PRDM14, in control mEpiSCs and mEpiSCs with decreased Smad2 expression at day 6 after infection. E, Blimp1, Oct4, and Nanog mRNA expression in embryoid bodies from control mEpiSCs or mEpiSCs with decreased Smad2 expression. Cells were dissociated at day 6 after infection and subjected to suspension culture for 4 days. Embryoid bodies were transferred to gelatin-coated plastic plates for two-dimensional culture for 2 more days. BMP4 or vehicle were added to the two dimensional culture for 2 days. n = 3 assays. *, p < 0.05; **, p < 0.01, compared with control cells by Student's t tests.

FIGURE 5.

Decreased Oct4, but not Nanog expression, is rescued by silencing Cdx2 expression. A, CDX2 mRNA expression in control hESCs and hESCs with CDX2 shRNAs. B, decreasing CDX2 expression in hESCs using CDX2 shRNA for 4 days restores expression of OCT4, but not NANOG, as assessed by immunoblots. C, immunofluorescence of CDX2 or NANOG expression in control hESCs and hESCs with decreased SMAD2 expression at day 8 after shRNA infection. Noggin or BMP4 was added from day 3 to day 8. DAPI served as a nuclear counter-stain. D, NANOG and CDX2 mRNA expression in control hESCs and hESCs with decreased SMAD2 expression, in the presence or absence of Noggin or BMP4 between day 3 and day 8 after infection. n = 3 assays. *, p < 0.05; **, p < 0.01, compared with control cells by Student's t tests; ***, p <0.005.

FIGURE 6.

NANOG down-regulation does not induce CDX2 expression or increased BMP responsiveness in undifferentiated hESCs. A, CDX2, NANOG, and OCT4 mRNA in control hESCs or cells with down-regulated NANOG or OCT4 expression. Reduction of NANOG expression does not result in enhanced CDX2 expression. B, down-regulation of Nanog expression does not increase BMP responsiveness, as assessed by immunoblotting for phosphorylated SMAD1/5, as high as in cells with down-regulated SMAD2 expression. Noggin (25 ng/ml) or BMP (1 ng/ml) was added at 48 or 1 h before analysis. C, CDX2 and BRACHYURY mRNA in control hESC or hESCs with down-regulated NANOG expression, with or without BMP stimulation. Noggin (25 ng/ml) or BMP (10 ng/ml) was added for 2 days. D, decreased Nanog expression does not induce Cdx2 expression in mEpiSCs. qRT-PCR analysis of Nanog, Oct4, and Cdx2 mRNAs in mEpiSCs at day 5 after lentiviral infection with control, Nanog-expressing, or Nanog shRNA-expressing vectors. n = 3 assays. *, p < 0.05; **, p < 0.01, compared with control cells by Student's t tests.

FIGURE 7.

Schematic model of the dual functions of Smad2 in the maintenance of hESC and mEpiSC pluripotency. Autocrine BMP activity and differentiation upon activation of the BMP pathway (green area) need to be suppressed by Smad2. BMP signaling induces the expression of master regulators of differentiation, including Cdx2, and drives differentiation toward mesoderm, PGC, and trophectoderm. Smad2 directly induces Nanog expression and maintains expression of other pluripotency regulators such as Oct4 through enhanced Nanog expression (pink area). Suppression of Cdx2 expression by antagonizing BMP signaling is essential to maintain Oct4 expression.