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

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Abstract

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.

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Acknowledgements

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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