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Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor


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.

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Figure 1: LIF-induced Stat-3 activation is not sufficient to maintain the undifferentiated state of HESCs.
Figure 2: MESCs and HESCs can transduce Wnt signaling when treated with BIO.
Figure 3: Activation of Wnt signaling by BIO maintains the undifferentiated state of MESCs.
Figure 4: Activation of Wnt signaling by BIO maintains the undifferentiated state of HESCs.
Figure 5: Wnt activation of HESCs by BIO preserves normal multidifferentiation potential.
Figure 6: MESCs maintain pluripotency through BIO-mediated Wnt activation.


  1. 1

    Smith, A.G. Embryo-derived stem cells: of mice and men. Annu. Rev. Cell Dev. Biol. 17, 435–462 (2001).

    CAS  Article  Google Scholar 

  2. 2

    Rossant, J. Stem cells from the mammalian blastocyst. Stem Cells 19, 477–482 (2001).

    CAS  Article  Google Scholar 

  3. 3

    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).

    CAS  Article  Google Scholar 

  4. 4

    Evans, M.J. & Kaufman, M.H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981).

    CAS  Article  Google Scholar 

  5. 5

    Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    CAS  Article  Google Scholar 

  6. 6

    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).

    CAS  Article  Google Scholar 

  7. 7

    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).

    CAS  Article  Google Scholar 

  8. 8

    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).

    CAS  Article  Google Scholar 

  9. 9

    Sato, N. et al. Molecular signature of human embryonic stem cells and its comparison with the mouse. Dev. Biol. 260, 404–413 (2003).

    CAS  Article  Google Scholar 

  10. 10

    Meijer, L. et al. GSK-3 selective inhibitors derived from Tyrian purple indirubins. Chem. Biol. (in the press).

  11. 11

    Xu, C. et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 19, 971–974 (2001).

    CAS  Article  Google Scholar 

  12. 12

    Okamoto, K. et al. A novel octamer binding transcription factor is differentially expressed in mouse embryonic cells. Cell 60, 461–472 (1990).

    CAS  Article  Google Scholar 

  13. 13

    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).

    CAS  Article  Google Scholar 

  14. 14

    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).

    CAS  Article  Google Scholar 

  15. 15

    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).

    CAS  Article  Google Scholar 

  16. 16

    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).

    CAS  Article  Google Scholar 

  17. 17

    Brivanlou, A.H. & Darnell, J.E., Jr. Signal transduction and the control of gene expression. Science 295, 813–818 (2002).

    CAS  Article  Google Scholar 

  18. 18

    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).

    CAS  Article  Google Scholar 

  19. 19

    Moon, R.T., Bowerman, B., Boutros, M. & Perrimon, N. The promise and perils of Wnt signaling through β-catenin. Science 296, 1644–1646 (2002).

    CAS  Article  Google Scholar 

  20. 20

    Doble, B.W. & Woodgett, J.R. GSK-3: tricks of the trade for a multi-tasking kinase. J. Cell Sci. 116, 1175–1186 (2003).

    CAS  Article  Google Scholar 

  21. 21

    Korinek, V. et al. Constitutive transcriptional activation by a β-catenin-Tcf complex in APC−/− colon carcinoma. Science 275, 1784–1787 (1997).

    CAS  Article  Google Scholar 

  22. 22

    Tetsu, O. & McCormick, F. Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 398, 422–426 (1999).

    CAS  Article  Google Scholar 

  23. 23

    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).

    CAS  Article  Google Scholar 

  24. 24

    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).

    CAS  Article  Google Scholar 

  25. 25

    Molenaar, M. et al. XTcf-3 transcription factor mediates β-catenin-induced axis formation in Xenopus embryos. Cell 86, 391–399 (1996).

    CAS  Article  Google Scholar 

  26. 26

    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).

    CAS  Article  Google Scholar 

  27. 27

    Chambers, I. et al. Functional expression cloning of nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113, 643–655 (2003).

    CAS  Article  Google Scholar 

  28. 28

    Mitsui, K. et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113, 631–642 (2003).

    CAS  Article  Google Scholar 

  29. 29

    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).

    CAS  Article  Google Scholar 

  30. 30

    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).

    CAS  Article  Google Scholar 

  31. 31

    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).

    CAS  Article  Google Scholar 

  32. 32

    Niwa, H. Molecular mechanism to maintain stem cell renewal of ES cells. Cell Struct. Funct. 26, 137–148 (2001).

    CAS  Article  Google Scholar 

  33. 33

    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).

    CAS  Article  Google Scholar 

  34. 34

    Huelsken, J. et al. Requirement for β-catenin in anterior-posterior axis formation in mice. J. Cell Biol. 148, 567–578 (2000).

    CAS  Article  Google Scholar 

  35. 35

    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).

    CAS  Article  Google Scholar 

  36. 36

    Lloyd, S., Fleming, T.P. & Collins, J.E. Expression of Wnt genes during mouse preimplantation development. Gene Expr. Patterns 3, 309–312 (2003).

    CAS  Article  Google Scholar 

  37. 37

    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).

    CAS  Article  Google Scholar 

  38. 38

    Kubo, F., Takeichi, M. & Nakagawa, S. Wnt2b controls retinal cell differentiation at the ciliary marginal zone. Development 130, 587–598 (2003).

    CAS  Article  Google Scholar 

  39. 39

    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).

    CAS  Article  Google Scholar 

  40. 40

    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).

    CAS  Article  Google Scholar 

  41. 41

    Chenn, A. & Walsh, C.A. Regulation of cerebral cortical size by control of cell cycle exit in neural precursors. Science 297, 365–369 (2002).

    CAS  Article  Google Scholar 

  42. 42

    Reya, T. et al. A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 423, 409–414 (2003).

    CAS  Article  Google Scholar 

  43. 43

    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).

    CAS  Google Scholar 

  44. 44

    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).

    CAS  Article  Google Scholar 

  45. 45

    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).

    CAS  Article  Google Scholar 

  46. 46

    Ding, S. et al. Synthetic small molecules that control stem cell fate. Proc. Natl. Acad. Sci. USA 100, 7632–7637 (2003).

    CAS  Article  Google Scholar 

  47. 47

    Willert, K. et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423, 448–452 (2003).

    CAS  Article  Google Scholar 

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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.

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Correspondence to Ali H Brivanlou.

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Sato, N., Meijer, L., Skaltsounis, L. et al. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 10, 55–63 (2004).

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