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• W2036552662 abstract "Human embryonic stem cells will remain undifferentiated or undergo differentiation when grown in conditioned or non-conditioned medium, respectively. The factors and signaling events that control the maintenance of the undifferentiated state are not well characterized and their identification is of major importance. Based on the data from global expression analyses, we set out to identify genes and the signaling pathways controlling them that are regulated in the early phase of the differentiation process. This study shows that nodal and the inhibitors of Nodal signaling, lefty-A and lefty-B, are down-regulated very early upon differentiation. High expression of these genes in undifferentiated cells is maintained by activation of the transcription factor Smad2/3, downstream of the activin-linked kinases (ALK) 4/5/7. Treatment of differentiating cells with Activin A leads to activation of Smad2/3 and expression of nodal, lefty-A and lefty-B, while inhibition of ALK4/5/7 by the kinase inhibitor SB-431542 blocks activation of Smad2/3 and expression of these genes in the undifferentiated state. In addition, when cells are maintained undifferentiated by treatment with the GSK3-inhibitor, BIO, high expression of nodal, lefty-A, and lefty-B also requires activation of ALK4/5/7. Conversely, BMP signaling leading to Smad1/5/8 activation via ALK2/3/6 is blocked in undifferentiated cells and becomes activated upon differentiation. Taken together, these observations establish that Smad2/3 is activated in undifferentiated hESCs and required for the expression of genes controlling Nodal signaling. Moreover, there appears to be cross-talk between inhibition of GSK3, a hallmark of Wnt signaling and the Activin/Nodal pathway. Human embryonic stem cells will remain undifferentiated or undergo differentiation when grown in conditioned or non-conditioned medium, respectively. The factors and signaling events that control the maintenance of the undifferentiated state are not well characterized and their identification is of major importance. Based on the data from global expression analyses, we set out to identify genes and the signaling pathways controlling them that are regulated in the early phase of the differentiation process. This study shows that nodal and the inhibitors of Nodal signaling, lefty-A and lefty-B, are down-regulated very early upon differentiation. High expression of these genes in undifferentiated cells is maintained by activation of the transcription factor Smad2/3, downstream of the activin-linked kinases (ALK) 4/5/7. Treatment of differentiating cells with Activin A leads to activation of Smad2/3 and expression of nodal, lefty-A and lefty-B, while inhibition of ALK4/5/7 by the kinase inhibitor SB-431542 blocks activation of Smad2/3 and expression of these genes in the undifferentiated state. In addition, when cells are maintained undifferentiated by treatment with the GSK3-inhibitor, BIO, high expression of nodal, lefty-A, and lefty-B also requires activation of ALK4/5/7. Conversely, BMP signaling leading to Smad1/5/8 activation via ALK2/3/6 is blocked in undifferentiated cells and becomes activated upon differentiation. Taken together, these observations establish that Smad2/3 is activated in undifferentiated hESCs and required for the expression of genes controlling Nodal signaling. Moreover, there appears to be cross-talk between inhibition of GSK3, a hallmark of Wnt signaling and the Activin/Nodal pathway. Embryonic stem cells (ESCs) 1The abbreviations used are: ESCs, embryonic stem cells; TGFβ, transforming growth factor β; ALK, activin-linked kinase; BMP, bone morphogenic protein; GDF, growth differentiation factor; TDGF, teratoma-derived growth factor; LIF, leukemia inhibitory factor; SB, SB-431542; BIO, 6-bromoindirubin-3′oxime; CM, conditioned medium; non-CM, non-conditioned medium; MEFs, mouse embryo fibroblasts; mESCs, mouse ESCs; hESCs, human ESCs; HSP, homology sequence pair; RT, reverse transcriptase; ca, constitutively activated.1The abbreviations used are: ESCs, embryonic stem cells; TGFβ, transforming growth factor β; ALK, activin-linked kinase; BMP, bone morphogenic protein; GDF, growth differentiation factor; TDGF, teratoma-derived growth factor; LIF, leukemia inhibitory factor; SB, SB-431542; BIO, 6-bromoindirubin-3′oxime; CM, conditioned medium; non-CM, non-conditioned medium; MEFs, mouse embryo fibroblasts; mESCs, mouse ESCs; hESCs, human ESCs; HSP, homology sequence pair; RT, reverse transcriptase; ca, constitutively activated. are pluripotent cells, which can proliferate indefinitely and contribute to the formation of basically all cell types in vitro and in vivo. Over the last decade the study of mammalian embryonic stem cells, especially mouse ESCs, has provided valuable insights into early embryogenesis in mammals. More recently, researchers have started to analyze human ESCs (hESCs) allowing a glimpse into early human embryogenesis and the development of tools for regenerative medicine. While our understanding of the basic biology of mouse ESCs (mESCs) and their in vivo properties is quite advanced, we know very little about the biological properties of hESC. Studying human cells is of special importance since recent molecular studies have shown that their properties differ from mouse cells (1Sato N. Meijer L. Skaltsounis L. Greengard P. Brivanlou A.H. Nat. Med. 2004; 10: 55-63Crossref PubMed Scopus (1714) Google Scholar, 2Reubinoff B.E. Pera M.F. Fong C.Y. Trounson A. Bongso A. Nat. Biotechnol. 2000; 18: 399-404Crossref PubMed Scopus (2284) Google Scholar, 3Smith A.G. Annu. Rev. Cell Dev. Biol. 2001; 17: 435-462Crossref PubMed Scopus (1027) Google Scholar). These findings are not unexpected given the differences in early embryonic development between mice and humans (4Ko M.S. Trends Biotechnol. 2001; 19: 511-518Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar).One of the foremost questions in the study of hESCs is the identification of factors required for maintenance of the undifferentiated state. hESCs, unlike mESCs (reviewed in Ref. 3Smith A.G. Annu. Rev. Cell Dev. Biol. 2001; 17: 435-462Crossref PubMed Scopus (1027) Google Scholar), cannot be maintained undifferentiated with leukemia inhibitory factor (LIF, Refs. 1Sato N. Meijer L. Skaltsounis L. Greengard P. Brivanlou A.H. Nat. Med. 2004; 10: 55-63Crossref PubMed Scopus (1714) Google Scholar and 2Reubinoff B.E. Pera M.F. Fong C.Y. Trounson A. Bongso A. Nat. Biotechnol. 2000; 18: 399-404Crossref PubMed Scopus (2284) Google Scholar). However, both human and mouse ESCs stay undifferentiated on feeder layers of mouse embryo fibroblasts (MEFs) or in the presence of MEF-conditioned medium (CM), suggesting that MEFs produce soluble factors that are sufficient to promote the undifferentiated state. Embryonic stem cells have the potential to differentiate into cell types of all different lineages, i.e. ecto-, endo-, and mesodermal and extra-embryonic and germ cell lineages (reviewed in Ref. 3Smith A.G. Annu. Rev. Cell Dev. Biol. 2001; 17: 435-462Crossref PubMed Scopus (1027) Google Scholar). Complex signaling networks control these differentiation processes in vivo (reviewed in Ref. 5Loebel D.A. Watson C.M. De Young R.A. Tam P.P. Dev. Biol. 2003; 264: 1-14Crossref PubMed Scopus (211) Google Scholar).A recent study reported global transcriptional profiling in which the entire repertoire of transcripts in mouse (6Ramalho-Santos M. Yoon S. Matsuzaki Y. Mulligan R.C. Melton D.A. Science. 2002; 298: 597-600Crossref PubMed Scopus (1419) Google Scholar) and hESCs, grown both under pluripotent and differentiating conditions were compared (7Sato N. Sanjuan I.M. Heke M. Uchida M. Naef F. Brivanlou A.H. Dev. Biol. 2003; 260: 404-413Crossref PubMed Scopus (397) Google Scholar). Interestingly, 24% overlap was observed between the two sets. The genes that are down-regulated in both mouse and human ESCs, include members of the transforming growth factor β (TGFβ) signaling pathway, such as the teratoma-derived growth factor-1 (TDGF-1/Cripto), which is an EGF-CFC co-receptor for Nodal signaling, as well as lefty-A (EBAF, lefty2 in the mouse) and lefty-B (lefty1 in the mouse), which are inhibitors of Nodal signaling. It has also been shown that nodal itself is highly expressed in undifferentiated hESCs and mESCs (6Ramalho-Santos M. Yoon S. Matsuzaki Y. Mulligan R.C. Melton D.A. Science. 2002; 298: 597-600Crossref PubMed Scopus (1419) Google Scholar, 8Rosler E.S. Fisk G.J. Ares X. Irving J. Miura T. Rao M.S. Carpenter M.K. Dev. Dyn. 2004; 229: 259-274Crossref PubMed Scopus (322) Google Scholar, 9Zeng X. Miura T. Luo Y. Bhattacharya B. Condie B. Chen J. Ginis I. Lyons I. Mejido J. Puri R.K. Rao M.S. Freed W.J. Stem. Cells. 2004; 22: 292-312Crossref PubMed Scopus (140) Google Scholar).TGFβ signaling is involved in a variety of cell fate decisions in early embryogenesis (reviewed in Ref. 10Harland R. Gerhart J. Annu. Rev. Cell Dev. Biol. 1997; 13: 611-667Crossref PubMed Scopus (677) Google Scholar). The TGFβ superfamily of ligands signals through two main branches: the BMP branch activates Activin-linked kinases (ALK) 2/3/6 (TGFβ type I receptors), leading to the phosphorylation and activation of the transcription factors Smad1/5/8. The Activin/Nodal branch involves the activation of the type I receptors ALK4/5/7 and subsequent phosphorylation and activation of the effectors Smad2/3. Smad7 inhibits both branches of TGFβ signaling and provides a repressive input on these pathways (reviewed in Ref. 11Shi Y. Massague J. Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4737) Google Scholar).Activation of Smad2 downstream of Nodal signaling is involved in several processes in the development of the embryo. Nodal induces mesoderm and endoderm, patterns the nervous system, and determines left-right asymmetry in vertebrates (reviewed in Ref. 12Schier A.F. Annu. Rev. Cell Dev. Biol. 2003; 19: 589-621Crossref PubMed Scopus (512) Google Scholar). Lefty1 blocks Nodal signaling by a dual mechanism, it binds Nodal directly and also binds EGF-CFC co-receptors, such as TDGF-1/Cripto, thus preventing the assembly of an active Nodal/Activin receptor complex (13Chen C. Shen M.M. Curr. Biol. 2004; 14: 618-624Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). A similar mechanism has been proposed for the highly homologous factor Lefty2. Loss of function analysis of Nodal leads to a phenotype where no mesoderm is formed (14Conlon F.L. Lyons K.M. Takaesu N. Barth K.S. Kispert A. Herrmann B. Robertson E.J. Development. 1994; 120: 1919-1928Crossref PubMed Google Scholar) while Lefty2-deficient mice show excessive mesoderm formation and expansion of the primitive streak as a consequence of overactivation of Nodal signaling (15Meno C. Gritsman K. Ohishi S. Ohfuji Y. Heckscher E. Mochida K. Shimono A. Kondoh H. Talbot W.S. Robertson E.J. Schier A.F. Hamada H. Mol. Cell. 1999; 4: 287-298Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). No obvious defects in the stem cell compartment, the inner cell mass, were observed in Lefty2-deficient animals. However, redundancy between Lefty1 and Lefty2 might explain the lack of phenotypic consequences. The Lefty1-deficient mouse shows a phenotype in which the left-right axis is disturbed (16Meno C. Shimono A. Saijoh Y. Yashiro K. Mochida K. Ohishi S. Noji S. Kondoh H. Hamada H. Cell. 1998; 94: 287-297Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). There are currently no double knockouts reported. Nodal and both Lefties are under the control of Activin/Nodal and Smad2/3 signaling in the embryonic node (reviewed in Ref. 17Hamada H. Meno C. Watanabe D. Saijoh Y. Nat. Rev. Genet. 2002; 3: 103-113Crossref PubMed Scopus (441) Google Scholar).This study demonstrates that nodal, lefty-A, and lefty-B, are the earliest genes showing decreased expression upon differentiation in non-conditioned medium (non-CM). It is shown that Activin/Nodal signaling in hESCs regulates all three genes. Activin A treatment induces expression of these genes, whereas treatment with the ALK4/5/7 kinase inhibitor SB-431542 (18Inman G.J. Nicolas F.J. Callahan J.F. Harling J.D. Gaster L.M. Reith A.D. Laping N.J. Hill C.S. Mol. Pharmacol. 2002; 62: 65-74Crossref PubMed Scopus (1315) Google Scholar) inhibits their expression in undifferentiated cells. In hESCs grown under undifferentiated conditions in CM, Smad2/3 is phosphorylated, reflecting its activation, but its phosphorylation decreases when hESCs undergo differentiation in non-CM. When the regulatory sequence of lefty-A, which has multiple binding sites for the forkhead transcription factor FoxH1 (Fast1), a coactivator for Smad2/3, and sites for Smad2/3 itself, was fused to a luciferase reporter, comparable regulation during both pluripotency and differentiation was observed compared with the endogenous lefty-A expression. Luciferase expression increased when constitutively active ALK4 was co-expressed while co-expression of the repressive factor Smad7 inhibited luciferase expression. In addition, it has recently been reported that inhibition of GSK3, a hallmark of Wnt-signaling, using the kinase inhibitor 6-bromoindirubin-3′oxime (BIO, 1Sato N. Meijer L. Skaltsounis L. Greengard P. Brivanlou A.H. Nat. Med. 2004; 10: 55-63Crossref PubMed Scopus (1714) Google Scholar,19Meijer L. Skaltsounis A.L. Magiatis P. Polychronopoulos P. Knockaert M. Leost M. Ryan X.P. Vonica C.A. Brivanlou A. Dajani R. Crovace C. Tarricone C. Musacchio A. Roe S.M. Pearl L. Greengard P. Chem. Biol. 2003; 10: 1255-1266Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar), is sufficient to block the decision to undergo differentiation in both mouse and human ESCs (1Sato N. Meijer L. Skaltsounis L. Greengard P. Brivanlou A.H. Nat. Med. 2004; 10: 55-63Crossref PubMed Scopus (1714) Google Scholar). Here it is shown that undifferentiated hESCs grown in BIO without CM, maintain high mRNA levels for nodal, lefty-A, and lefty-B as well as phosphorylation of Smad2/3 utilizing an active ALK4/5/7 receptor complex. These results suggest an interaction between inhibition of GSK-3 and Activin/Nodal signaling.EXPERIMENTAL PROCEDURESReagents and Antibodies—Human Activin A, and BMP4 were purchased from R&D. The TGFβ inhibitor SB-431542 (18Inman G.J. Nicolas F.J. Callahan J.F. Harling J.D. Gaster L.M. Reith A.D. Laping N.J. Hill C.S. Mol. Pharmacol. 2002; 62: 65-74Crossref PubMed Scopus (1315) Google Scholar) was purchased from Tocris. The GSK-3 inhibitor, 6-bromoindirubin-3′oxime (BIO, Refs. 1Sato N. Meijer L. Skaltsounis L. Greengard P. Brivanlou A.H. Nat. Med. 2004; 10: 55-63Crossref PubMed Scopus (1714) Google Scholar and 19Meijer L. Skaltsounis A.L. Magiatis P. Polychronopoulos P. Knockaert M. Leost M. Ryan X.P. Vonica C.A. Brivanlou A. Dajani R. Crovace C. Tarricone C. Musacchio A. Roe S.M. Pearl L. Greengard P. Chem. Biol. 2003; 10: 1255-1266Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar) was generously provided by A. H. Brivanlou (The Rockefeller University, New York). All other reagents were purchased from Sigma.Antibodies used were rabbit polyclonal anti-phospho-Smad1/5/8 and Smad2/3 (Cell Signaling); rabbit polyclonal Smad1/5/8 and Smad2/3 (Upstate Biotechnology) and mouse monoclonal α-tubulin (Santa Cruz Biotechnology), and secondary anti-mouse and anti-rabbit antibodies linked to horseradish peroxidase (Jackson Immunoresearch Laboratories).Cell Culture—HESCs lines H1 (WiCell Research Institute) and BGN2 (BresaGen) were cultured on matrigel (BD Biosciences)-coated tissue culture plates in MEF-conditioned F12 medium supplemented with 20% knock-out serum replacement (KSR), non-essential minimal amino acids, penicillin/streptomycin, l-glutamine, β-mercaptoethanol, and 4 ng/ml FGF-2. The medium was conditioned by incubating 12 ml of medium for 24 h on hygromycin-resistent MEFs culture (5 × 106 cells/100 mm-diameter dish). MEFs (Specialty Media) were rendered growth-incompetent by treatment with mitomycin C by the manufacturer. MEFs were plated in Dulbecco's modified Eagle's medium supplemented with 10% calf serum and penicillin/streptomycin for the first 24 h until used for conditioning stem cell medium. All tissue-culture reagents were purchased from Invitrogen and used in the recommended concentration unless noted otherwise.Plasmid and Probes—The leftyA-luc construct was generated by PCR from bacterial artificial chromosome CITBI-E1–2519G10 (Research Genetics). The lefty-A promoter region (+42 bp to -4805 bp in regard to the transcription start site) was amplified using a primer pair from Integrated DNA Technologies: forward primer: 5′-ttccctgttcttcaaacaccgtcc-3′, reverse primer: 5′-tcctctagggaggttgaaggagg-3′. The amplified PCR fragment was purified with the PCR purification kit (Qiagen) and subcloned into pGL2-basic (Promega). pRL-null (Renilla) luciferase (Promega) was used as a control for transfection efficiency. Cells were co-transfected with components of the TGFβ signaling pathway: constitutive active ALK3 and ALK4 (kindly provided by R. Harland, University of California, Berkeley) and Smad7 in pcDNA3, (kindly provided by P. Ten Dijke, Ludwig Institute, Amsterdam, Netherlands, Ref. 20Nakao A. Afrakhte M. Moren A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.E. Heldin C.H. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1546) Google Scholar).RT-PCR Assays—Total RNA was isolated from BGN2 stem cells using TRIzol (Invitrogen) and reverse-transcribed using MMLV reverse transcriptase (Invitrogen). PCR amplification was performed on a GeneAmp PCR system 9600 (PerkinElmer Life Sciences) using TaqDNA polymerase (Promega). The PCR reaction for all primer pairs consisted of 25 cycles, apart from β-actin (21 cycles) and lck (28 cycles). Oligonucleotides used for RT-PCR were custom-made by Integrated DNA Technologies (for primer sequences see Supplemental Data).Cell Transfection, Western Blotting, and Luciferase Assays—H1 and BGN2 cells were transfected using LipofectAMINE plus reagent (Invitrogen) as described by the manufacturer. Cells were either harvested for preparation of whole cell extracts (extraction buffer: 50 mm HEPES-NaOH, pH 7.4, 150 mm NaCl, 1% Triton X-100, 10% glycerol, 10 mm EDTA, 1 mm Na3VO4, and complete miniprotease inhibitor mixture (Roche Applied Science)) or luciferase extracts following the protocol of the manufacturer of the dual-luciferase assay (Promega). Cells for luciferase experiments were harvested 36 h after transfection. Firefly and Renilla luciferase activity was determined as recommended by Promega in a dual-luciferase luminometer (Lumat LB-9507, Berthold Technologies) and Western blotting of the respective whole cell extract (25 μg) was carried out following standard methods (21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and instructions by the manufacturer of the polyvinylidene difluoride blotting membranes (Millipore) and the immunodetection system (Amersham Biosciences).RESULTSDecrease of nodal, lefty-A, and lefty-B Expression Is the Earliest Indication of Differentiation in hESCs—A previous report has identified 918 genes in H1 hESCs as the signature of the undifferentiated pluripotent state (7Sato N. Sanjuan I.M. Heke M. Uchida M. Naef F. Brivanlou A.H. Dev. Biol. 2003; 260: 404-413Crossref PubMed Scopus (397) Google Scholar). 24% of this set, 204 genes, was also present in mESCs analyzed by similar means (6Ramalho-Santos M. Yoon S. Matsuzaki Y. Mulligan R.C. Melton D.A. Science. 2002; 298: 597-600Crossref PubMed Scopus (1419) Google Scholar). In order to identify genes and/or signaling pathways that are involved in maintaining the undifferentiated state within this pool, 64 genes were selected based on their potential involvement in signal transduction and/or transcriptional regulation. The expression of these genes was assessed by comparative RT-PCR in H1 cells in a time course after withdrawal of MEF-conditioned medium (CM) (see Supplemental Data). The time-dependent regulation of 6 of the 64 genes is shown in Fig. 1A. Among the different genes tested in hESCs, nodal, lefty-A and lefty-B showed the earliest down-regulation after withdrawal of CM (Supplemental Data and Fig. 1). The level of nodal, lefty-A, and lefty-B transcripts drop to about 3% of their level in undifferentiated H1 cells within 24 h of CM withdrawal while the levels for all other 64 genes was still at 20% or higher of their initial value (Fig. 1B and Supplemental Data). These genes are also regulated in a similar way in another hESC line, BGN2 (Fig. 1C). Nodal, lefty-A, and lefty-B mRNA levels decreased the most rapidly upon differentiation compared with all other genes tested. For this reason and in consideration of the fact that Nodal and Lefty-A are involved in early cell fate decisions in the induction of endo- and mesoderm, we decided to analyze their regulation in further detail.Activin/Nodal Signaling Is Required for the Regulation of nodal, lefty-A, and lefty-B—As mentioned above, nodal, lefty-A, and lefty-B are regulated in response to Smad2 activation in the embryonic node (reviewed in Ref. 17Hamada H. Meno C. Watanabe D. Saijoh Y. Nat. Rev. Genet. 2002; 3: 103-113Crossref PubMed Scopus (441) Google Scholar). Therefore, we analyzed their regulation by modulation of the Activin/Nodal signaling pathway, which regulates the activation of Smad2 (Fig. 2A). Activation of the pathway was achieved by treatment of BGN2 cells with Activin A whereas inhibition was carried out by treatment of the cells with the ALK4/5/7 kinase inhibitor SB-431542 (18Inman G.J. Nicolas F.J. Callahan J.F. Harling J.D. Gaster L.M. Reith A.D. Laping N.J. Hill C.S. Mol. Pharmacol. 2002; 62: 65-74Crossref PubMed Scopus (1315) Google Scholar). Expression of all three genes decreased in cells grown in non-CM compared with cells grown in CM. Activin A led to increased expression of lefty-A in cells grown in non-CM while SB-431542 decreased its expression in cells grown in CM, both in a dose-dependent manner. When the cells were treated with Activin A and SB-431542 together, the levels of the genes were low, consistent with the fact that Activin A binds to ALK4 to activate Smad2/3. These results, taken together, demonstrate that nodal, lefty-A, and lefty-B are selectively responsive to Activin/Nodal signaling in hESCs.Fig. 2Activin/Nodal signaling regulates nodal, lefty-A, and lefty-B expression and expression of components of the TGFβ signaling pathway in hESCs and MEFs. A, RT-PCR analysis for the expression levels for nodal, lefty-A, and lefty-B mRNA (as indicated) in BGN2 cells. Cells were grown in CM (lane 1), differentiated in non-CM over time (24 and 72 h, lanes 2 and 3), in non-CM with increasing amounts (10, 30, and 100 ng/ml) of recombinant Activin A (lanes 4–6), in CM with increasing amounts of SB-431542 (1, 3, 10 μm) (lanes 7–9), in non-CM with BIO (2 μm) alone (lane 11) or in non-CM in the presence of SB-431542 (10 μm) with Activin A (30 ng/ml, lane 10) or BIO (2 μm, lane 12). The treatment with Activin-A, SB-431542, BIO, or combination of these agents was done for 72 h in CM or non-CM as indicated. β-Actin expression was used as loading control and as negative control for omission of reverse transcriptase (β-actin -RT). B, RT-PCR analysis for mRNA expression levels of tgfβ-1,2,3, inhibin α, inhibin βA, inhibin βB, and gdf1 in MEFs. hprt expression was used as positive control and as negative control for omission of reverse transcriptase (hprt-RT). Expression of inhibin βC, inhibin βE, and nodal was not detected. C, RT-PCR analysis of activin type I receptors alk1–7, as described in Fig. 1, for undifferentiated (CM) and differentiated for 72 h (72) H1 and BGN2 cells.View Large Image Figure ViewerDownload (PPT)BIO Requires Activation of Smad2/3 for the Regulation of nodal, lefty-A, and lefty-B—It has been shown that inhibition of GSK3 by a small molecular weight compound, called BIO (1Sato N. Meijer L. Skaltsounis L. Greengard P. Brivanlou A.H. Nat. Med. 2004; 10: 55-63Crossref PubMed Scopus (1714) Google Scholar, 19Meijer L. Skaltsounis A.L. Magiatis P. Polychronopoulos P. Knockaert M. Leost M. Ryan X.P. Vonica C.A. Brivanlou A. Dajani R. Crovace C. Tarricone C. Musacchio A. Roe S.M. Pearl L. Greengard P. Chem. Biol. 2003; 10: 1255-1266Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar), is sufficient to maintain the undifferentiated state of hESCs and high expression of marker genes such as oct3/4, nanog, and rex-1 (1Sato N. Meijer L. Skaltsounis L. Greengard P. Brivanlou A.H. Nat. Med. 2004; 10: 55-63Crossref PubMed Scopus (1714) Google Scholar). We tested the effect of BIO on the expression levels of nodal, lefty-A, and lefty-B with or without SB-431542. Fig. 2A shows that BIO in the absence of CM maintained the endogenous expression of all three genes to levels comparable with the expression in CM, while SB-431542 blocked induction of the genes induced by BIO. These results suggest that the induction of the genes whether it derives from conditioned medium or BIO treatment, requires a functional ALK4/5/7 pathway.Expression Status of Components of the TGFβ Signaling Pathway—A list of TGFβ pathway components present in the H1 line is shown in Table I. The values were determined by global gene expression analysis (7Sato N. Sanjuan I.M. Heke M. Uchida M. Naef F. Brivanlou A.H. Dev. Biol. 2003; 260: 404-413Crossref PubMed Scopus (397) Google Scholar) of H1 cells maintained undifferentiated or induced to differentiate. The list comprises 44 different molecules involved in TGFβ signaling either as ligands, receptors, Smads, or modulators of the pathway. The table compares the expression values for the molecules in undifferentiated (CM) and differentiated (non-CM) conditions and gives the relative indices for the expression (Fold CM/n-CM). While most genes are not regulated (fold value between 0.5 and 2) several genes show a significantly higher expression in undifferentiated cells, including lefty-A, lefty-B, and tdgf-1 (cripto), as stated above, as well as cerberus, gdf3, foxH1, and oaz. Other genes, such as TGFβ2, follistatin splice variant 344 (FST sv344), TGFβR-II, and ltbp-1, are up-regulated upon differentiation.Table IComponents of the TGFβ signaling pathway in H1 hESCs Table I shows the expression levels of several TGFβ pathway components as observed in a global expression profile using microarrays on the Affymetrix U133A Chip (7Sato N. Sanjuan I.M. Heke M. Uchida M. Naef F. Brivanlou A.H. Dev. Biol. 2003; 260: 404-413Crossref PubMed Scopus (397) Google Scholar). The second column gives the relative expression value for the respective gene in the presence of CM. The third column gives the relative expression value in the absence of CM (non-CM) for three weeks. The fourth column gives the index for the expression between CM and non-CM, thus a measure whether the gene decreases (>1), increases (<1) or remains unchanged upon differentiation (=1). A value between 0.5 and 2 (not bolded) does not allow a conclusive statement on the gene regulation since this value falls into the range of uncertainty (2-fold).CMnon-CMFold (CM/non-CM)Ligandstgfβ2417740.05inhibin βC4712571.8inhibin βE1201131.1bmp-21501461bmp-71561051.5gdf33721362.7nodalaThere were relevant genes, e.g. nodal, that are not represented on the U133A chip. However, nodal was found to be expressed in hESCs in other studies (8, 9)PresentExtracellular inhibitorsinhibin α671290.5lefty-A10441616.5lefty-B406010041decorin9057811.2tmeff110183532.9cerberus9372194.3follistatin (sv317)1371760.8follistatin (sv344)2968880.3follistatin-like 1240945430.5chordin-like145861.7drm/gremlin59660.9Type I receptorsbNote that genes such as ALK4/5/7, which were detected by RT-PCR (Fig. 2C), were not detected in the microarray analysisbmpRIA/ALK37744031.9actRI/ALK23163920.8Type II receptorstβR-II1043500.3actRII2302700.9actRIIB3833161.2Co-receptorstdgf1/cripto40635267.7Miscellaneous regulatorsltbp-11485230.3fkbp12126217250.7arip-1/magi1131031.1tak13303510.9Smadssmad25466880.8smad3771590.5smad44804681smad51561700.9smad72823740.8Smad-Adaptersara1592070.8Ubiquitin E3-Ligasesmurf-12293660.6smurf-24389640.5Nuclear transportnup2142682311.2nup15310968881.2Co-activatorfoxH1/fast-13431272.7oaz6201773.5Transcription repressorsdrap-12264250.5ecsit1591411.1yy1217316361.3ski120681.8ski-1541360.4a There were relevant genes, e.g. nodal, that are not represented on the U133A chip. However, nodal was found to be expressed in hESCs in other studies (8Rosler E.S. Fisk G.J. Ares X. Irving J. Miura T. Rao M.S. Carpenter M.K. Dev. Dyn. 2004; 229: 259-274Crossref PubMed Scopus (322) Google Scholar, 9Zeng X. Miura T. Luo Y. Bhattacharya B. Condie B. Chen J. Ginis I. Lyons I. Mejido J. Puri R.K. Rao M.S. Freed W.J. Stem. Cells. 2004; 22: 292-312Crossref PubMed Scopus (140) Google Scholar)b Note that genes such as ALK4/5/7, which were detected by RT-PCR (Fig. 2C), were not detected in the microarray analysis Open table in a new tab Next, the expression of TGFβ ligands and receptors in MEFs and hESCs, respectively, was examined. MEFs secrete factor/s into the conditioned medium, which are able to maintain the hESCs undifferentiated. Transcripts for several TGFβ ligands were present in MEFs (Fig. 2B) including tgfβ-1,2,3, activin A,B and A/B (homo-(A or B) or heterodimer (A/B) of two inhibin β chains) and gdf1.RT-PCR analysis showed that all TGFβ type I receptors except ALK1 are present in H1 and BGN2 hESCs, making them competent to respond to Activin/Nodal/TGFβ signaling leading to Smad2/3 activation downstream of ALK4/5/7 (Fig. 2" @default.
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