In the three decades since pluripotent mouse embryonic stem (ES) cells were first described1,2 they have been derived and maintained by using various empirical combinations of feeder cells, conditioned media, cytokines, growth factors, hormones, fetal calf serum, and serum extracts1,2,3,4,5,6,7. Consequently ES-cell self-renewal is generally considered to be dependent on multifactorial stimulation of dedicated transcriptional circuitries, pre-eminent among which is the activation of STAT3 by cytokines (ref. 8). Here we show, however, that extrinsic stimuli are dispensable for the derivation, propagation and pluripotency of ES cells. Self-renewal is enabled by the elimination of differentiation-inducing signalling from mitogen-activated protein kinase. Additional inhibition of glycogen synthase kinase 3 consolidates biosynthetic capacity and suppresses residual differentiation. Complete bypass of cytokine signalling is confirmed by isolating ES cells genetically devoid of STAT3. These findings reveal that ES cells have an innate programme for self-replication that does not require extrinsic instruction. This property may account for their latent tumorigenicity. The delineation of minimal requirements for self-renewal now provides a defined platform for the precise description and dissection of the pluripotent state.
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We thank D. Alessi for discussion and advice on GSK3 signalling; A. Rizzino, S. Meloche and R. Kemler for Fgf4, Erk2 and Ecadherin targeted ES cells, respectively; F. Watt for the ΔNhLef1 construct; N. Shpiro and R. Marquez for synthesizing PD184352, CHIR99021 and PD0325901; B. Amati and G. Faga for advice on Myc immunoblotting; J. Vrana for fluorescence-activated cell sorting support; and C. Manson, K. Savill and colleagues for mouse husbandry. This research was funded by the Medical Research Council and the Biotechnology and Biological Sciences Research Council of the UK, the Canadian Institutes of Health Research, and by the European Commission Framework VI project EuroStemCell. P.C. is a Royal Society Research Professor, and A.S. is a Medical Research Council Professor.
Author Contributions Q.L.Y. and A.S. conceived the study; Q.L.Y., J.W. and J.N. designed, executed and interpreted experiments; L.B.M. generated CBA ES cells; B.D. and J.W. generated and provided GSK3 mutant ES cells; P.C. contributed expert advice and inhibitors; and A.S. wrote the paper.
The file contains Supplementary Figure 1 a-k with Legends, Supplementary Table and details of embryo transfers and crosses and Supplementary Tables of antibodies and primers. (PDF 1160 kb)
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Ying, Q., Wray, J., Nichols, J. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008) doi:10.1038/nature06968
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