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First published online 9 February 2005
doi: 10.1242/dev.01706

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TGFß/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells

Laboratory of Molecular Vertebrate Embryology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA

View larger version (64K): [in a new window]   Fig. 1. The undifferentiated state of hESCs is characterized by activation of SMAD2/3-mediated signal transduction and inhibition of SMAD1/5-mediated signal transduction. (A) Western blot analysis of H1 hESCs cultured under various conditions. Cells were cultured for 4 days in the presence of: nCM supplemented with 2 µM BIO, non-conditioned medium (nCM), MEF-conditioned medium (CM), CM supplemented with 25 ng/ml BMP4, and nCM supplemented with 25 ng/ml activin A. Membranes were probed with antibodies specific for phosphorylated (P) SMAD2, SMAD2/3, phosphorylated SMAD1/5, SMAD1/5, OCT3/4 and -tubulin (as a control for protein loading). (B) Immunofluorescence microscopy of BGN2 hESCs in the undifferentiated and differentiated state. BGN2 cells were cultured for 5 days in CM, in nCM, in nCM supplemented with 25 ng/ml activin A, and in nCM supplemented with 2 µM BIO. Cells were decorated with an antibody specific for SMAD2/3, as well as SytoxGreen nuclear counterstain. (C) Bright-field images of H1 hESCs cultured in conditions described in A. Scale bars: 50 µm.

View larger version (80K): [in a new window]   Fig. 2. Global SMAD1/5 phosphorylation is increased under differentiation conditions and is evident in mitotic cells in the undifferentiated state. (A) Immunofluorescence microscopy of BGN2 hESCs in the undifferentiated and differentiated state. BGN2 cells were cultured for 5 days in CM, in nCM, in CM supplemented with 25 ng/ml BMP4 (as a control for SMAD1/5 activation) and in nCM supplemented with 2 µM BIO. Cells were decorated with an antibody specific for phosphorylated SMAD1/5, as well as SytoxGreen nuclear counterstain. Arrows indicate mitotic cells. Scale bars: 50 µm. (B) Western blot analysis of colcemide synchronized BGN1 hESCs. Cells were blocked at metaphase by incubation with 100 ng/ml demecolcine solution and harvested at 15 minutes and 4 hours post-release. Cells grown in CM alone and CM supplemented with 25 ng/ml BMP4 were used as controls for asynchronous and SMAD1/5-activated cells, respectively. Membranes were probed with antibodies specific for phosphorylated (P) SMAD1/5, SMAD1/5, phosphorylated Ser CDKs substrate and -tubulin (as a control for protein loading). (C) Immunofluorescence microscopy of blastocyst stage embryo containing mitotic cells. Mouse blastocyst embryos were fixed and labeled with anti-phospho SMAD1/5 antibody and SytoxGreen nuclear counterstain, and then imaged by confocal microscopy. Scale bars: 20 µm.

View larger version (38K): [in a new window]   Fig. 3. Intact SMAD2/3 signaling is required for the maintenance of the undifferentiated state in hESCs and the formation of embryoid bodies. (A) Western blot analysis of H1 hESCs cultured in conditions in which SMAD2/3 signaling is intact or inhibited. Cells were cultured for 4 days in CM, in nCM, in nCM supplemented with 2 µM BIO, in CM supplemented with 10 µM SB431542, and in nCM supplemented with 2 µM BIO and 10 µM SB431542. Membranes were probed with antibodies specific for phosphorylated (P) SMAD2, SMAD2/3, phosphorylated SMAD1/5, SMAD1/5, OCT3/4 and -tubulin (as a control for protein loading). (B) Expression analysis of H1 hESCs. Cells were cultured for 4 days in nCM supplemented with 25 ng/ml activin A, in CM supplemented with 25 ng/ml BMP4, in CM, in nCM, in nCM supplemented with 2 µM BIO in CM supplemented with 10 µM SB431542, and in nCM supplemented with 2 µM BIO and 10 µM SB431542. RT-PCR was performed on these cells using primers for human OCT3/4, nanog, ß-actin (as a loading control) and ß-actin RT minus (as a control for contamination with genomic DNA). (C) Western blot analysis of H1 hESCs cultured in the presence of a cocktail of soluble receptors specific to the activin/nodal pathway. Cells were cultured for 5 days in CM, in nCM, in CM supplemented with 10 µM SB431542, and in CM supplemented with hrActRIB (5 µg/ml), hrActRIIB (5 µg/ml) and hrCripto (250 ng/ml). Membranes were probed with antibodies specific for phosphorylated SMAD2, OCT3/4, NANOG and -tubulin (as a control for protein loading). (D) Histogram describing number of embryoid bodies formed from BGN2 hESCs. Cells were cultured for 7 days in CM, in nCM, in nCM with 25 ng/ml activin A, in CM with 10 µM SB431542, in nCM with 2 µM BIO, and in nCM with 2 µM BIO and 10 µM SB431542. Cells were then detached from substrate and cultured in a suspension of nCM for 7 more days. Histograms and error bars (s.e.m.) represent the experiment performed in triplicate and on two separate passages of BGN2 cells.

View larger version (69K): [in a new window]   Fig. 4. Intact SMAD2/3 signaling is not required for the maintenance of the undifferentiated state of mESCs, but is required for the maintenance of the stem cell compartment of blastocyst outgrowths. (A) Western blot analysis of 129/SVJ mESCs. mESCs were cultured for 3 days in mESC medium with (+) and without (-) LIF, in mESC medium with LIF plus 10 µM SB431542, in mESC medium with 25 ng/ml activin A, in mESC medium with 2 µM BIO, in mESC medium with 2 µM BIO plus 10 µM SB431542, in CM, and in CM with 10 µM SB431542. Membranes were probed with antibodies specific for phosphorylated SMAD2, SMAD2/3, OCT3/4 and -tubulin (as a control for protein loading). (B) Whole-mount immunofluorescent confocal microscopy of pre-implantation stage mouse embryos. Mouse embryos were extracted and fixed at two-cell, four-cell, eight-cell, compacted morula and blastocyst stages. Embryos were decorated with an antibody specific for phosphorylated SMAD2 and SytoxGreen nuclear counterstain. Scale bars: 20 µm. (C) Confocal immunofluorescent microscopy of blastocyst outgrowths. Mouse blastocyst stage embryos were extracted and cultured in the presence of mESC medium supplemented with DMSO or 20 µM SB431542 for 4 days. Outgrowths were decorated with an antibody specific for OCT3/4 and SytoxGreen nuclear counterstain. Table gives percentage of embryos exhibiting OCT3/4-positive cells. Scale bars: 100 µm.