Role of Lef1 in sustaining self-renewal in mouse embryonic stem cells

Embryonic stem cells (ESCs) can 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 regenerative medicine,the molecular mechanisms controlling ESC behavior are incompletely understood.Previously,activation of the canonical Wnt signaling pathway has been shown to contribute to mouse ESC self-renewal.Here we report that ectopic expression of Lef1,a component of the Wnt signaling pathway,has a positive effect on the self-renewal of mouse ESCs.Lef1 up-regulates Oct4 promoter activity and physically interacts with Nanog,two key components of the ESC pluripotency machinery.Moreover,siRNA for Lef1 induced mouse ESC differentiation.Our results thus suggest that in response to Wnt signaling Lef1 binds to stabilized β-catenin and helps maintain the undifferentiated status of ESCs through modulation of Oct4 and Nanog.     

The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells

Embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocysts grow infinitely while maintaining pluripotency. Leukemia inhibitory factor (LIF) can maintain self-renewal of mouse ES cells through activation of Stat3. However, LIF/Stat3 is dispensable for maintenance of ICM and human ES cells, suggesting that the pathway is not fundamental for pluripotency. In search of a critical factor(s) that underlies pluripotency in both ICM and ES cells, we performed in silico differential display and identified several genes specifically expressed in mouse ES cells and preimplantation embryos. We found that one of them, encoding the homeoprotein Nanog, was capable of maintaining ES cell self-renewal independently of LIF/Stat3. nanog-deficient ICM failed to generate epiblast and only produced parietal endoderm-like cells. nanog-deficient ES cells lost pluripotency and differentiated into extraembryonic endoderm lineage. These data demonstrate that Nanog is a critical factor underlying pluripotency in both ICM and ES cells.     

Nanog基因的功能与调控研究进展

胚胎干细胞的无限增殖能力和亚全能性决定了它在再生医学、新药开发及发育生物学基础研究中具有巨大的应用前景。探索维持胚胎干细胞亚全能性的因子及其网络的调控功能成为胚胎干细胞生物学研究的热点。已研究发现多个与维持胚胎干细胞亚全能性相关的基因如Oct4, Nanog, Sox2等,其中Nanog是2003年5月末发现的一个基因,它对维持胚胎干细胞亚全能性起关键性作用,能够独立于L1F/Stat3维持ICM和胚胎干细胞的亚全能性。几年来,Nanog的生物学功能及其与 Oct4, Sox2等亚全能性维持基因之间的相互作用关系已有较为深入的研究,并发现多个调控Nanog表达的转录因子,从而进一步明晰Nanog与已知调控胚胎发育的信号通路之间的关系。本文在综述Nanog基因的表达特征和功能的基础上、重点探讨Nanog基因表达调控以及Oct4, Sox2等亚全能性维持基因之间的相互作用关系,并对未来的研究趋势予以展望。     

Self-renewal and differentiation of mouse embryonic stem cells as measured by Oct4 expression: the role of the cAMP/PKA pathway

In this study we examined the role of the cAMP/protein kinase A (PKA) pathway in affecting IOUD2 ES cell self-renewal and differentiation, Oct4 expression, and cell proliferation. Forskolin, the adenylate cyclase agonist, alone had no effect on ES cell self-renewal. However, when cells were treated with the differentiation-inducing agent retinoic acid, forskolin significantly promoted ES cell self-renewal. Effectively, forskolin rescued cells from a pathway of differentiation. Culturing ES cells in the presence of the phosphodiesterase inhibitor IBMX had no effect on ES cell self-renewal but did increase cell proliferation. In the presence of 100 μM IBMX without LIF, 10 μM forskolin significantly increased ES cell self-renewal. The cell permeable cAMP analog 8-Br-cAMP (1 and 5 mM) promoted ES cell differentiation in the presence of LIF, while in the absence of LIF, it promoted ES cell self-renewal. The effect of the PKA specific inhibitors H89 and KT5720 on Oct4 expression was, again, LIF-dependent. In the presence of LIF, these inhibitors decreased Oct4 expression, while they increased Oct4 expression in the absence of LIF. In general, ES cells maintained on a self-renewal pathway through the presence of LIF show little effect from altered cAMP signaling except at higher levels. However, in strict contrast, when ES cell are on a differentiation pathway through exposure to retinoic acid or the removal of LIF, altering cAMP levels can rescue the self-renewal process promoting Oct4 expression. This study clearly shows that the cAMP/PKA pathway plays a role in ES cell self-renewal pathways. This work was partly funded by the Millennium Research Fund National University of Ireland Galway.     

Glycogen synthase kinase 3 (GSK3) inhibitor, SB-216763, promotes pluripotency in mouse embryonic stem cells

Canonical Wnt/β-catenin signaling has been suggested to promote self-renewal of pluripotent mouse and human embryonic stem cells. Here, we show that SB-216763, a glycogen synthase kinase-3 (GSK3) inhibitor, can maintain mouse embryonic stem cells (mESCs) in a pluripotent state in the absence of exogenous leukemia inhibitory factor (LIF) when cultured on mouse embryonic fibroblasts (MEFs). MESCs maintained with SB-216763 for one month were morphologically indistinguishable from LIF-treated mESCs and expressed pluripotent-specific genes Oct4, Sox2, and Nanog. Furthermore, Nanog immunostaining was more homogenous in SB-216763-treated colonies compared to LIF. Embryoid bodies (EBs) prepared from these mESCs expressed early-stage markers for all three germ layers, and could efficiently differentiate into cardiac-like cells and MAP2-immunoreactive neurons. To our knowledge, SB-216763 is the first GSK3 inhibitor that can promote self-renewal of mESC co-cultured with MEFs for more than two months.     

Calcineurin-NFAT signaling critically regulates early lineage specification in mouse embryonic stem cells and embryos

Self-renewal and pluripotency are hallmarks of embryonic stem cells (ESCs). However, the signaling pathways that trigger their transition from self-renewal to differentiation remain elusive. Here, we report that calcineurin-NFAT signaling is both necessary and sufficient to switch ESCs from an undifferentiated state to lineage-specific cells and that the inhibition of this pathway can maintain long-term ESC self-renewal independent of leukemia inhibitory factor. Mechanistically, this pathway converges with the Erk1/2 pathway to regulate Src expression and promote the epithelial-mesenchymal transition (EMT), a process required for lineage specification in response to differentiation stimuli. Furthermore, calcineurin-NFAT signaling is activated when the earliest differentiation event occurs in mouse embryos, and its inhibition disrupts extraembryonic lineage development. Collectively, our results demonstrate that the NFAT and Erk1/2 cascades form a signaling switch for early lineage segregation in mouse ESCs and provide significant insights into the regulation of the balance between ESC self-renewal and early lineage specification.