In the three decades since pluripotent mouse embryonic stem (ES) cells were first described 1,2 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 extracts [1][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.Reprints and permissions information is available at www.nature.com/reprints.Correspondence and requests for materials should be addressed to Q.L.Y. (qying@keck.usc.edu) or A.S. (ags39@cscr.cam.ac.uk). Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. We found that, in combination with LIF, either inhibitor replaces the requirement for serum/BMP and supports robust long-term ES-cell propagation (Supplementary Information). Lineage commitment does not occur despite a reduced expression of inhibitorof-differentiation proteins. In contrast, ES cells plated without LIF in either PD184352 or SU5402 progressively degenerate and cannot be maintained even though differentiation is suppressed. To reduce off-target side effects we tried low doses of PD184352 and SU5402 together (PS). In PS we find that undifferentiated ES cells expand through multiple passages (Fig. 1a, b). Differentiation is constrained, although occasional neural rosettes emerge. This result, observed with several independent ES cell lines, suggests that the minimal requirements for ES-cell self-renewal may be to deflect commitment signals emanating from FGF receptor and ERK signalling. However, apoptosis is relatively high in PS, especially immediately after passage, and cells survive poorly at clonal density, which is indicative of collateral compromise to cell growth and viability. Author ContributionsES-cell propagation has been reported to be enhanced by an indirubin entity, 6-bromoindirubin-3′-oxime (BIO), that inhibits glycogen synthase kinase-3 (GSK3) 4 . ...

Glycogen synthase kinase 3 (GSK-3) is a multifunctional serine/threonine kinase found in all eukaryotes. The enzyme is a key regulator of numerous signalling pathways, including cellular responses to Wnt, receptor tyrosine kinases and G-protein-coupled receptors and is involved in a wide range of cellular processes, ranging from glycogen metabolism to cell cycle regulation and proliferation. GSK-3 is unusual in that it is normally active in cells and is primarily regulated through inhibition of its activity. Another peculiarity compared with other protein kinases is its preference for primed substrates,that is, substrates previously phosphorylated by another kinase. Several recent advances have improved our understanding of GSK-3 regulation in multiple pathways. These include the solution of the crystal structure of GSK-3, which has provided insight into GSK-3's penchant for primed substrates and the regulation of GSK-3 by serine phosphorylation, and findings related to the involvement of GSK-3 in the Wnt/β-catenin and Hedgehog pathways. Finally, since increased GSK-3 activity may be linked to pathology in diseases such as Alzheimer's disease and non-insulin-dependent diabetes mellitus,several new GSK-3 inhibitors, such as the aloisines, the paullones and the maleimides, have been developed. Although they are just starting to be characterized in cell culture experiments, these new inhibitors hold promise as therapeutic agents.

Signalling by the Wnt family of secreted lipoproteins plays essential roles in development and disease 1 . The canonical Wnt/β-catenin pathway requires a single-span transmembrane receptor, LDL receptor related protein 6 (LRP6) 2-4 , whose phosphorylation at multiple PPPSP motifs is induced upon Wnt stimulation and critical for signal transduction 5 . The kinase responsible for LRP6 phosphorylation has not been identified. Here we provide biochemical and genetic evidence for a 'dual-kinase' mechanism for LRP6 phosphorylation and activation. Surprisingly, glycogen synthase kinase 3 (GSK3), which is known for its inhibitory role in Wnt signalling via promoting β-catenin phosphorylation and degradation, mediates LRP6 phosphorylation and activation. We demonstrate that Wnt induces sequential phosphorylation of LRP6 by GSK3 and casein kinase 1 (CK1), and this dual-phosphorylation promotes the engagement of LRP6 with the scaffolding protein Axin. We further show that a membrane-associated form of GSK3, contrary to cytosolic GSK3, stimulates Wnt signalling and Xenopus axis duplication. Our results identify two key kinases mediating Wnt coreceptor activation, reveal an unexpected and intricate logic of Wnt/β-catenin signalling, and illustrate GSK3 as a bona fide switch dictating both on and off states of a pivotal regulatory pathway.Canonical Wnt signalling operates through regulating phosphorylation and degradation of the transcription co-activator β-catenin 1,6 . Without Wnt stimulation, β-catenin is assembled into the Axin complex, in which β-catenin is sequentially phosphorylated by CK1 and GSK3 and earmarked for degradation 7-9 . Wnt stimulation leads to inhibition of β-catenin phosphorylation/degradation. This signal transduction is initiated at the plasma membrane by two distinct receptors, a Frizzled serpentine receptor and LRP6 or LRP5, which together may form a Wnt-induced Frizzled-LRP6 (or LRP5) complex 3,6,10-12 . While the mechanism by which this receptor pair initiates signalling remains to be understood, Wnt-induced LRP6 phosphorylation at a PPPSP motif, which is reiterated five times in LRP5/6 cytoplasmic domain (Fig. 1a), plays a critical role 5 . (For simplicity we use PPPSP to represent PPPSP or PPPTP). Indeed, LRP6 mutants lacking these motifs or harbouring substitutions at the S/T residues are inactive in signalling 5,13 . Conversely, a single PPPSP motif upon transfer to a heterologous receptor is sufficient to initiate β-catenin signalling 5 . As the phosphorylated PPPSP motif mediates LRP5/6-Axin interaction 5,14 , we proposed a model 5 in which Wnt-induced LRP6 Figs. 1 and 2). In vivo GSK3 overexpression promoted LRP6 PPPSP phosphorylation ( Fig. 1b) whereas LiCl and SB216763 prevented Wnt-induced LRP6 PPPSP phosphorylation (Fig. 1c). Importantly, Wnt-induced LRP6 phosphorylation was abolished in mouse embryo fibroblasts (MEFs) that harbour genetic deletions of Gsk3α and Gsk3β genes ( Fig. 1d). These data show that GSK3 is involved in LRP6 PPPSP phosphorylation.GSK3 phosphorylation of m...

In mammalian cells, glycogen synthase kinase-3 (GSK-3) exists as two homologs, GSK-3alpha and GSK-3beta, encoded by independent genes, which share similar kinase domains but differ substantially in their termini. Here, we describe the generation of an allelic series of mouse embryonic stem cell (ESC) lines with 0-4 functional GSK-3 alleles and examine GSK-3-isoform function in Wnt/beta-catenin signaling. No compensatory upregulation in GSK-3 protein levels or activity was detected in cells lacking either GSK-3alpha or GSK-3beta, and Wnt/beta-catenin signaling was normal. Only in cells lacking three or all four of the alleles was a gene-dosage effect on beta-catenin/TCF-mediated transcription observed. Indeed, GSK-3alpha/beta double-knockout ESCs displayed hyperactivated Wnt/beta-catenin signaling and were severely compromised in their ability to differentiate, but could be rescued to normality by re-expression of functional GSK-3. The rheostatic regulation of GSK-3 highlights the importance of considering the contributions of both homologs when studying GSK-3 functions in mammalian systems.