Human and mouse embryonic stem cells (HESCs and MESCs, respectively) self-renew indefinitely while maintaining the ability to generate all three germ-layer derivatives. Despite the importance of ESCs in developmental biology and their potential impact on tissue replacement therapy, the molecular mechanism underlying ESC self-renewal is poorly understood. Here we show that activation of the canonical Wnt pathway is sufficient to maintain self-renewal of both HESCs and MESCs. Although Stat-3 signaling is involved in MESC self-renewal, stimulation of this pathway does not support self-renewal of HESCs. Instead we find that Wnt pathway activation by 6-bromoindirubin-3′-oxime (BIO), a specific pharmacological inhibitor of glycogen synthase kinase-3 (GSK-3), maintains the undifferentiated phenotype in both types of ESCs and sustains expression of the pluripotent state-specific transcription factors Oct-3/4, Rex-1 and Nanog. Wnt signaling is endogenously activated in undifferentiated MESCs and is downregulated upon differentiation. In addition, BIO-mediated Wnt activation is functionally reversible, as withdrawal of the compound leads to normal multidifferentiation programs in both HESCs and MESCs. These results suggest that the use of GSK-3-specific inhibitors such as BIO may have practical applications in regenerative medicine.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $18.75 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Smith, A.G. Embryo-derived stem cells: of mice and men. Annu. Rev. Cell Dev. Biol. 17, 435–462 (2001).
Rossant, J. Stem cells from the mammalian blastocyst. Stem Cells 19, 477–482 (2001).
Martin, G.R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. USA 78, 7634–7638 (1981).
Evans, M.J. & Kaufman, M.H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981).
Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).
Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A. & Bongso, A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat. Biotechnol. 18, 399–404 (2000).
Hori, Y. et al. Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells. Proc. Natl. Acad. Sci. USA 99, 16105–16110 (2002).
Kim, J.H. et al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease. Nature 418, 50–56 (2002).
Sato, N. et al. Molecular signature of human embryonic stem cells and its comparison with the mouse. Dev. Biol. 260, 404–413 (2003).
Meijer, L. et al. GSK-3 selective inhibitors derived from Tyrian purple indirubins. Chem. Biol. (in the press).
Xu, C. et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 19, 971–974 (2001).
Okamoto, K. et al. A novel octamer binding transcription factor is differentially expressed in mouse embryonic cells. Cell 60, 461–472 (1990).
Scholer, H.R., Ruppert, S., Suzuki, N., Chowdhury, K. & Gruss, P. New type of POU domain in germ line-specific protein Oct-4. Nature 344, 435–439 (1990).
Rosner, M.H. et al. A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo. Nature 345, 686–692 (1990).
Niwa, H., Burdon, T., Chambers, I. & Smith, A. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev. 12, 2048–2060 (1998).
Ramalho-Santos, M., Yoon, S., Matsuzaki, Y., Mulligan, R.C. & Melton, D.A. “Stemness”: transcriptional profiling of embryonic and adult stem cells. Science 298, 597–600 (2002).
Brivanlou, A.H. & Darnell, J.E., Jr. Signal transduction and the control of gene expression. Science 295, 813–818 (2002).
van Es, J.H., Barker, N. & Clevers, H. You Wnt some, you lose some: oncogenes in the Wnt signaling pathway. Curr. Opin. Genet. Dev. 13, 28–33 (2003).
Moon, R.T., Bowerman, B., Boutros, M. & Perrimon, N. The promise and perils of Wnt signaling through β-catenin. Science 296, 1644–1646 (2002).
Doble, B.W. & Woodgett, J.R. GSK-3: tricks of the trade for a multi-tasking kinase. J. Cell Sci. 116, 1175–1186 (2003).
Korinek, V. et al. Constitutive transcriptional activation by a β-catenin-Tcf complex in APC−/− colon carcinoma. Science 275, 1784–1787 (1997).
Tetsu, O. & McCormick, F. Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 398, 422–426 (1999).
Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).
Hosler, B.A., LaRosa, G.J., Grippo, J.F. & Gudas, L.J. Expression of REX-1, a gene containing zinc finger motifs, is rapidly reduced by retinoic acid in F9 teratocarcinoma cells. Mol. Cell. Biol. 9, 5623–5629 (1989).
Molenaar, M. et al. XTcf-3 transcription factor mediates β-catenin-induced axis formation in Xenopus embryos. Cell 86, 391–399 (1996).
Vonica, A. & Gumbiner, B.M. Zygotic Wnt activity is required for Brachyury expression in the early Xenopus laevis embryo. Dev. Biol. 250, 112–127 (2002).
Chambers, I. et al. Functional expression cloning of nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113, 643–655 (2003).
Mitsui, K. et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113, 631–642 (2003).
Itskovitz-Eldor, J. et al. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol. Med. 6, 88–95 (2000).
Kawasaki, H. et al. Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity. Proc. Natl. Acad. Sci. USA 99, 1580–1585 (2002).
Dani, C. et al. Paracrine induction of stem cell renewal by LIF-deficient cells: a new ES cell regulatory pathway. Dev. Biol. 203, 149–162 (1998).
Niwa, H. Molecular mechanism to maintain stem cell renewal of ES cells. Cell Struct. Funct. 26, 137–148 (2001).
Niwa, H., Miyazaki, J. & Smith, A.G. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet. 24, 372–376 (2000).
Huelsken, J. et al. Requirement for β-catenin in anterior-posterior axis formation in mice. J. Cell Biol. 148, 567–578 (2000).
Conacci-Sorrell, M.E. et al. Nr-CAM is a target gene of the β-catenin/LEF-1 pathway in melanoma and colon cancer and its expression enhances motility and confers tumorigenesis. Genes Dev. 16, 2058–2072 (2002).
Lloyd, S., Fleming, T.P. & Collins, J.E. Expression of Wnt genes during mouse preimplantation development. Gene Expr. Patterns 3, 309–312 (2003).
Korinek, V. et al. Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4. Nat. Genet. 19, 379–383 (1998).
Kubo, F., Takeichi, M. & Nakagawa, S. Wnt2b controls retinal cell differentiation at the ciliary marginal zone. Development 130, 587–598 (2003).
Gat, U., DasGupta, R., Degenstein, L. & Fuchs, E. De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated β-catenin in skin. Cell 95, 605–614 (1998).
Merrill, B.J., Gat, U., DasGupta, R. & Fuchs, E. Tcf3 and Lef1 regulate lineage differentiation of multipotent stem cells in skin. Genes Dev. 15, 1688–1705 (2001).
Chenn, A. & Walsh, C.A. Regulation of cerebral cortical size by control of cell cycle exit in neural precursors. Science 297, 365–369 (2002).
Reya, T. et al. A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 423, 409–414 (2003).
Giles, R.H., van Es, J.H. & Clevers, H. Caught up in a Wnt storm: Wnt signaling in cancer. Biochim. Biophys. Acta 1653, 1–24 (2003).
Kielman, M.F. et al. Apc modulates embryonic stem-cell differentiation by controlling the dosage of β-catenin signaling. Nat. Genet. 32, 594–605 (2002).
Aubert, J., Dunstan, H., Chambers, I. & Smith, A. Functional gene screening in embryonic stem cells implicates Wnt antagonism in neural differentiation. Nat. Biotechnol. 20, 1240–1245 (2002).
Ding, S. et al. Synthetic small molecules that control stem cell fate. Proc. Natl. Acad. Sci. USA 100, 7632–7637 (2003).
Willert, K. et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423, 448–452 (2003).
We thank WiCell Research Institute and Bresagen for providing HESC lines; H. Clevers, L. Gudas, A. Miyawaki and J. Miyazaki for providing plasmid constructs; M. Willey and C. Yang for providing MESC lines; M. Heke and M. Uchida for technical assistance; K. La Perle for histological report of teratoma sections; A. North for confocal microscopic imaging; the Transgenic Core Facility at The Rockefeller University and Memorial Sloan-Kettering Cancer Institute for blastocyst injection; A. Vonica for providing a construct and helpful discussion; and D. Besser and T. Tomoda for helpful advice. A.H.B. is funded by The Rockefeller University. L.M. is supported by the Ministère de la Recherche/INSERM/CNRS 'Molécules et Cibles Thérapeutiques' Programme; his sabbatical leave in the laboratory of P.G. is supported by The Rockefeller University and the CNRS.
The authors declare no competing financial interests.
About this article
Comparative Proteomic Analysis Reveals the Upregulation of Ketogenesis in Cardiomyocytes Differentiated from Induced Pluripotent Stem Cells
Adenoviral delivery of VHL suppresses bone sarcoma cell growth through inhibition of Wnt/β-catenin signaling
Cancer Gene Therapy (2019)
DeepNEU: cellular reprogramming comes of age – a machine learning platform with application to rare diseases research
Orphanet Journal of Rare Diseases (2019)
6-Bromoindirubin-3’-oxime promotes osteogenic differentiation of canine BMSCs through inhibition of GSK3β activity and activation of the Wnt/β-catenin signaling pathway
Anais da Academia Brasileira de Ciências (2019)