Presence Of LIF Research Articles

Isolation of pristine MXene from Nb₄AlC₃ MAX phase: a first-principles study.

Synthesis of pristine MXene sheets from MAX phase is one of the foremost challenges in getting a complete understanding of the properties of this new technologically important 2D-material. Efforts to exfoliate Nb4AlC3 MAX phase always lead to Nb4C3 MXene sheets, which are functionalized and have several Al atoms attached. Using the first-principles calculations, we perform an intensive study on the chemical transformation of MAX phase into MXene sheets by inserting HF, alkali atoms and LiF in Nb4AlC3 MAX phase. Calculated bond-dissociation energy (BDE) shows that the presence of HF in MAX phase always results in functionalized MXene, as the binding of H with MXene is quite strong while that with F is weak. Insertion of alkali atoms does not facilitate pristine MXene isolation due to the presence of chemical bonds of almost equal strength. In contrast, weak Li-MXene and strong Li-F bonding in Nb4AlC3 with LiF ensured strong anisotropy in BDE, which will result in the dissociation of the Li-MXene bond. Ab initio molecular dynamics calculations capture these features and show that at 500-650 K, the Li-MXene bond indeed breaks leaving a pristine MXene sheet behind. The approach and insights developed here for chemical exfoliation of layered materials bonded by chemical bonds instead of van der Waals can promote their experimental realization.

WNT/β-catenin signaling affects cell lineage and pluripotency-specific gene expression in bovine blastocysts: prospects for bovine embryonic stem cell derivation.

Despite many attempts, true bovine embryonic stem cells (bESC) still remain elusive. The WNT pathway has been associated with stem cell control in vertebrates and its role in pluripotency maintenance has been proven for several mammalian species, including rodents and primates. Thus, we have aimed to investigate the effect of WNT activation on pluripotency marker gene expression in the inner cell mass (ICM) and the trophectoderm (TE) and to study the derivation potential of primary bESC lines from blastocysts obtained in the presence of the glycogen synthase kinase 3 inhibitor (GSK3i). WNT activity clearly exerted a positive effect on pluripotency gene expression in developing bovine embryos, manifested by upregulation of OCT4, NANOG, REX1, SOX2, c-MYC, and KLF4 in the ICM and downregulation of CDX2 in the TE. However, the prolonged exposition of preimplantation embryos to the GSK3i resulted in reduced potential to form primary ESC-like colonies. The results of bESC derivation experiments allowed us to speculate that the derived cell lines may share features of both naïve and primed ESCs. Similar to mouse epiblast stem cells and human ESCs, the derived lines grew as flat monolayer colonies intolerant to passaging as single cells. JAK/STAT signaling was indispensable for proper colony formation and proliferation, yet LIF alone was inefficient to support self-renewal. Concomitant with the naïve state of mouse ESCs, WNT activity supported by LIF had beneficial effects on cell culture propagation, survival after passage, morphology, and pluripotency-related marker gene expression. Moreover, colonies derived in the presence of LIF and GSK3i maintained KLF4 transcription over several passages, whereas EpiSCs virtually do not express KLF4.

Differentiate or Die: 3-Bromopyruvate and Pluripotency in Mouse Embryonic Stem Cells.

BackgroundPluripotent embryonic stem cells grown under standard conditions (ESC) have a markedly glycolytic profile, which is shared with many different types of cancer cells. Thus, some therapeutic strategies suggest that pharmacologically shifting cancer cells towards an oxidative phenotype, using glycolysis inhibitors, may reduce cancer aggressiveness. Given the metabolic parallels between cancer and stemness would chemotherapeutical agents have an effect on pluripotency, and could a strategy involving these agents be envisioned to modulate stem cell fate in an accessible manner? In this manuscript we attempted to determine the effects of 3-bromopyruvate (3BrP) in pluripotency. Although it has other intracellular targets, this compound is a potent inhibitor of glycolysis enzymes thought to be important to maintain a glycolytic profile. The goal was also to determine if we could contribute towards a pharmacologically accessible metabolic strategy to influence cell differentiation.Methodology/Principal FindingsMouse embryonic stem cells (mESC) grown under standard pluripotency conditions (in the presence of Leukemia Inducing Factor- LIF) were treated with 3BrP. As a positive control for differentiation other mESCs were grown without LIF. Overall our results demonstrate that 3BrP negatively affects pluripotency, forcing cells to become less glycolytic and with more active mitochondria. These changes in metabolism are correlated with increased differentiation, even under pluripotency conditions (i.e. in the presence of LIF). However, 3BrP also significantly impaired cell function, and may have other roles besides affecting the metabolic profile of mESCs.Conclusions/FindingsTreatment of mESCs with 3BrP triggered a metabolic switch and loss of pluripotency, even in the presence of LIF. Interestingly, the positive control for differentiation allowed for a distinction between 3BrP effects and changes associated with spontaneous differentiation/loss of pluripotency in the absence of LIF. Additionally, there was a slight differentiation bias towards mesoderm in the presence of 3BrP. However, the side effects on cellular function suggest that the use of this drug is probably not adequate to efficiently push cells towards specific differentiation fates.

Effect of LIF-withdrawal on acetylcholine synthesis in the embryonic stem cell line CGR8 is not mediated by STAT3, PI3Ks or cAMP/PKA pathways

Acetylcholine (ACh) acts as a local cellular signaling molecule and is widely expressed in nature, including mammalian cells and embryonic stem cells. The murine embryonic stem cell line CGR8 synthesizes and releases substantial amounts of ACh. Particularly during early differentiation – a period associated with multiple alterations in geno-/phenotype functions – synthesis and release of ACh are increased by 10-fold. In murine stem cells second messengers of the STAT-3, PI3K and cAMP/PKA pathways are involved in maintaining self-renewal and pluripotency. The present experiments were designed to test whether blockers of these signaling pathways enhance ACh cell content in the presence of LIF, i.e. when CGR8 is pluripotent. NSC74859, an inhibitor of STAT-3, affected neither the proliferation rate nor ACh cell content, whereas the more sensitive STAT-3 inhibitor FLLL31 reduced the proliferation rate and increased ACh cell content by about 3-fold. The PI3K inhibitor LY294002 reduced the proliferation rate but did not modify the ACh cell content, whereas the PKA inhibitor H89 produced effects comparable to FLLL31. Interestingly, in control experiments a strong inverse correlation was found between cell density and ACh cell content, which could explain the 3-fold increase in the ACh cell content observed in the presence of FLLL31 and H89. Forskolin, a PKA activator, had no effect. In conclusion, it appears unlikely that the 10-fold increase in ACh cell content induced by LIF removal, i.e. during early differentiation, is mediated by second messengers of the STAT-3, PI3K and cAMP/PKA pathways. However, the PI3K pathway appears to be involved in control of the inverse relation between cell density and ACh cell content, because this correlation was significantly attenuated in the presence of LY294002.

Novel role of mitochondrial manganese superoxide dismutase in STAT3 dependent pluripotency of mouse embryonic stem cells.

Leukemia Inhibitory Factor (LIF)/Signal transducer and activator of transcription 3 (STAT3) signaling pathway maintains the stemness and pluripotency of mouse embryonic stem cells (mESCs). Detailed knowledge on key intermediates in this pathway as well as any parallel pathways is largely missing. We initiated our study by investigating the effect of small molecule Curcumin on various signalling pathways essential for self-renewal. Curcumin sustained the LIF independent self-renewal of mESCs and induced pluripotent stem cells (miPSCs) in a STAT3 activity dependent manner. Gene expression analysis showed LIF/STAT3 and redox signaling components to be majorly modulated. Amongst ROS genes, expression of Manganese Superoxide Dismutase (MnSOD) specifically relied on STAT3 signaling as evidenced by STAT3 inhibition and reporter assay. The silencing of MnSOD, but not Cu-ZnSOD expression, resulted in the loss of mESC pluripotency in presence of LIF, and the overexpression of MnSOD is sufficient for maintaining the expression of pluripotent genes in the absence of STAT3 signaling. Finally, we demonstrate MnSOD to stabilize the turnover of pluripotent proteins at the post-translational level by modulating proteasomal activity. In conclusion, our findings unravel a novel role of STAT3 mediated MnSOD in the self-renewal of mESCs.

Physical interaction between MPP8 and PRC1 complex and its implication for regulation of spermatogenesis

Epigenetic modifications such as DNA methylation and histone H3 lysine 27 methylation (H3K27me) are repressive marks that silence gene expression. The M phase phosphoprotein (MPP8) associates with proteins involved in both DNA methylation and histone modifications, and therefore, is a potential candidate to mediate crosstalk between repressive epigenetic pathways. Here, by performing immunohistochemical analyses we demonstrate that MPP8 is expressed in the rodent testis, especially in spermatocytes, suggesting a role in spermatogenesis. Interestingly, we found that MPP8 physically interacts with PRC1 (Polycomb Repressive Complex 1) components which are known to possess essential function in testis development by modulating monoubiquitination of Histone H2A (uH2A) and trimethylation of Histone H3 Lysine 27 (H3K27me3) residues. Knockdown analysis of MPP8 in HeLa cells resulted in derepression of a set of genes that are normally expressed in spermatogonia, spermatids and mature sperm, thereby indicating a role for this molecule in silencing testis-related genes in somatic cells. In addition, depletion of MPP8 in murine ES cells specifically induced expression of genes involved in mesoderm differentiation, such as Cdx2 and Brachyury even in the presence of LIF, which implicated that MPP8 might be required to repress differentiation associated genes during early development. Taken together, our results indicate that MPP8 could have a role for silencing genes that are associated with differentiation of the testis and the mesoderm by interacting with epigenetic repressors modules such as the PRC1 complex.

Establishment of trophoblast stem cells under defined culture conditions in mice.

The inner cell mass (ICM) and trophoblast cell lineages duet early embryonic development in mammals. After implantation, the ICM forms the embryo proper as well as some extraembryonic tissues, whereas the trophoectoderm (TE) exclusively forms the fetal portion of the placenta and the trophoblast giant cells. Although embryonic stem (ES) cells can be derived from ICM in cultures of mouse blastocysts in the presence of LIF and/or combinations of small-molecule chemical compounds, and the undifferentiated pluripotent state can be stably maintained without use of serum and feeder cells, defined culture conditions for derivation and maintenance of undifferentiated trophoblast stem (TS) cells have not been established. Here, we report that addition of FGF2, activin A, XAV939, and Y27632 are necessary and sufficient for derivation of TS cells from both of E3.5 blastocysts and E6.5 early postimplantation extraembryonic ectoderm. Moreover, the undifferentiated TS cell state can be stably maintained in chemically defined culture conditions. Cells derived in this manner expressed TS cell marker genes, including Eomes, Elf5, Cdx2, Klf5, Cdh1, Esrrb, Sox2, and Tcfap2c; differentiated into all trophoblast subtypes (trophoblast giant cells, spongiotrophoblast, and labyrinthine trophoblast) in vitro; and exclusively contributed to trophoblast lineages in chimeric animals. This delineation of minimal requirements for derivation and self-renewal provides a defined platform for precise description and dissection of the molecular state of TS cells.

On the Electrodeposition of Niobium from 1-Butyl-1-Methylpyrrolidinium Bis(trifluoromethylsulfonyl)imide at Conductive Diamond Substrates

This work relates to the electrochemical deposition of Nb from a liquid solution in which the ionic liquid 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide ([BMP][TFSA]) is used as solvent. Conductive diamond was employed as substrate, while the experiments were carried out with NbF5 in the presence of either NaF or LiF. Cyclic voltammetries were used to characterise the electrochemical behaviour of the system. Deposition experiments were carried out at fixed potentials, corresponding to the voltammetric peaks of the cathodic scans. The surfaces were characterised by scanning electron microscope-energy dispersive X-ray (SEM-EDX) analysis and the results used in combination with the voltammetries to establish a possible mechanism for the Nb electrodeposition process. The electroreduction of Nb(V) to Nb(0) was found to proceed through different steps, involving the formation of the Nb(II) carbide and other nonstoichiometric carbide phases. The effect of Na and Li ions on the electrochemical process was studied: although three voltammetric peaks were detected in both cases, an appreciable depolarisation of the cathodic reactions was observed in the presence of Li ions. XRD analysis confirmed that a coating of metallic Nb was obtained at −1.5 V and 125 °C in the presence of LiF.

Chromatin Accessibility at a STAT3 Target Site Is Altered Prior to Astrocyte Differentiation

DNA demethylation of astrocyte-specific gene promoters and STAT3 activation in neural precursor cells (NPCs) are essential for astrogliogenesis in the developing brain. To date, it remains unclear whether DNA methylation is the sole epigenetic determinant responsible for suppressing astrocyte-specific genes. Here, we used mouse embryonic stem cells (TKO ESCs) that lacked all 3 DNA methyltransferase genes, Dnmt1, Dnmt3a, and Dnmt3b, and thereby exhibit complete demethylation of the astrocyte-specific glial fibrillary acidic protein (Gfap) gene promoter. We found that although the Gfap promoter was demethylated, STAT3 failed to bind to its cognate element to induce Gfap transcription, whereas it induced transcription of a different target gene, Socs3. Moreover, although the Gfap promoter region containing the STAT3-binding site (GSBS) is enriched with transcription-repressive histone modifications, such as methylation of H3 at lysine 9 (H3K9me3) and H3K27me3, the reduction of these modifications in TKO ESCs was not sufficient for binding of STAT3 at GSBS. Furthermore, GSBS was digested by micrococcal nuclease in late-gestational NPCs that express GFAP upon LIF stimulation, but not in cells that show no expression of GFAP even in the presence of LIF, indicating that STAT3 can access GSBS in the former cells. We further showed that expression of NF-1A, which is known to potentiate differentiation of mid-gestational NPCs into astrocytes, increased its accessibility. Taken together, our results suggest that chromatin accessibility of GSBS plays a critical role in the regulation of Gfap expression.

TGF-β-Superfamily Signaling Regulates Embryonic Stem Cell Heterogeneity: Self-Renewal as a Dynamic and Regulated Equilibrium

Embryonic stem cells dynamically fluctuate between phenotypic states, as defined by expression levels of genes such as Nanog, while remaining pluripotent. The dynamic phenotype of stem cells is in part determined by gene expression control and dictated by various signaling pathways and transcriptional regulators. We sought to define the activities of two TGF-β-related signaling pathways, bone morphogenetic protein (BMP) and Nodal signaling, in modulating mouse embryonic stem (ES) cell heterogeneity in undifferentiated culture conditions. Both BMP and Nodal signaling pathways were seen to be active in distinct Nanog subpopulations, with subtle quantitative differences in activity. Pharmacological and genetic modulation of BMP or Nodal signaling strongly influenced the heterogeneous state of undifferentiated ES cells, as assessed by dynamic expression of Nanog reporters. Inhibition of Nodal signaling enhanced BMP activity, which through the downstream target Id factors, enhanced the capacity of ES cells to remain in the Nanog-high epigenetic state. The combined inhibition of Nodal and BMP signaling resulted in the accumulation of Nanog-negative cells, even in the presence of LIF, uncovering a shared role for BMP and Nodal signaling in maintaining Nanog expression and repression of differentiation. These results demonstrate a complex requirement for both arms of TGF-β-related signaling to influence the dynamic cellular phenotype of undifferentiated ES cells in serum-based media, and that differing subpopulations of ES cells in heterogeneous culture have distinct responses to these signaling pathways. Several pathways, including BMP, Nodal, and FGF signaling, have important regulatory function in defining the steady-state distribution of heterogeneity of stem cells.

3-O-Sulfated Heparan Sulfate Recognized by the Antibody HS4C3 Contribute to the Differentiation of Mouse Embryonic Stem Cells via Fas Signaling

Maintenance of self-renewal and pluripotency in mouse embryonic stem cells (mESCs) is regulated by the balance between several extrinsic signaling pathways. Recently, we demonstrated that heparan sulfate (HS) chains play important roles in the maintenance and differentiation of mESCs by regulating extrinsic signaling. Sulfated HS structures are modified by various sulfotransferases during development. However, the significance of specific HS structures during development remains unclear. Here, we show that 3-O-sulfated HS structures synthesized by HS 3-O-sulfotransferases (3OSTs) and recognized by the antibody HS4C3 increase during differentiation of mESCs. Furthermore, expression of Fas on the cell surface of the differentiated cells also increased. Overexpression of the HS4C3-binding epitope in mESCs induced apoptosis and spontaneous differentiation even in the presence of LIF and serum. These data showed that the HS4C3-binding epitope was required for differentiation of mESCs. Up-regulation of the HS4C3-binding epitope resulted in the recruitment of Fas from the cytoplasm to lipid rafts on the cell surface followed by activation of Fas signaling. Indeed, the HS4C3-binding epitope interacted with a region that included the heparin-binding domain (KLRRRVH) of Fas. Reduced self-renewal capability in cells overexpressing 3OST resulted from the degradation of Nanog by activated caspase-3, which is downstream of Fas signaling, and was rescued by the inhibition of Fas signaling. We also found that knockdown of 3OST and inhibition of Fas signaling reduced the potential for differentiation into the three germ layers during embryoid body formation. This is the first demonstration that activation of Fas signaling is mediated by an increase in the HS4C3-binding epitope and indicates a novel signaling pathway for differentiation in mESCs.

Glycogen synthase kinase 3 (GSK3) inhibitor 6-bromoindirubin-3'-oxime (BIO) promotes the proliferation of mouse male germline stem cells (mGSCs) under serum- and feeder-free conditions

The 6-bromoindirubin-3'-oxime (BIO), as one of glycogen synthase kinase 3 (GSK3) inhibitors, is a key regulator of many signaling pathways with the capacity to maintain the pluripotency of human and mouse embryonic stem cells (ESCs). Whether BIO can maintain the pluripotency of mouse male germline stem cells (mGSCs) remains unclear at present. In this study, with BIO and retinol (RE) both in Knock-out serum replacement (KSR) medium, we got a relatively optimal feeder- and serum-free system for mGSCs in vitro. After continuous culturing, the proliferation efficiency of undifferentiated mGSCs and differentiation capacity of mGSC-induced embryoid bodies (EBs) were examined as well. Results show that in the presence of LIF or retinol, BIO significantly increased the number of mGSCs and alkaline phosphatase (AP) positive colonies, and the mitosis index through the BrdU assay. BIO also increased the expression of pluripotent markers including Oct4, Nanog, PCNA and c-Myc analysed by real-time polymerase chain reaction (PCR). The mGSCs cultured in RE medium containing BIO can form embryoid bodies (EBs), which consist of three embryonic germ layers analysed by immunofluorescence. When these cells were transplanted into infertile mice, they could well repair the capacity of making male germ cells. Our results demonstrate the combined treatment of BIO and RE could significantly promote the proliferation of mouse mGSC colonies and maintain the undifferentiated status.

B-box and SPRY Domain Containing Protein (BSPRY) is Associated with the Maintenance of Mouse Embryonic Stem Cell Pluripotency and Early Embryonic Development

Mouse embryonic stem (ES) cells consist of heterogeneous populations with differing abilities to proliferate and differentiate. We previously demonstrated that the expression level of platelet endothelial cell adhesion molecule 1 (PECAM1)/CD31 was positively correlated with the undifferentiated state of mouse ES cells. In order to screen for a novel gene(s) involved in ES cell pluripotency, we performed an oligo microarray analysis and identified that B-box and SPRY domain containing protein (BSPRY) was expressed at high levels in PECAM1-positive cells. Two splice isoforms of BSPRY, BSPRY-1 and BSPRY-2, were expressed in undifferentiated ES cells and in blastocysts. Knockdown of BSPRY-1/2 in ES cells significantly reduced the number of undifferentiated colonies and caused increased expression of primitive ectoderm marker gene Fgf5. The overexpression of BSPRY-2 reciprocally increased the number of undifferentiated ES cells in the presence of LIF. Similarly, injection of BSPRY-1/2 siRNAs into 2-cell embryos caused developmental retardation and degeneration of embryos, and a significant decrease in the number of cells, especially in the inner cell mass (ICM), was observed at the blastocyst stage. Furthermore, microinjection of a BSPRY-1 expression vector into pronuclear stage embryos resulted in an increase in the hatching blastocysts rate after 120 h of culture. These results suggest that BSPRY-1 and BSPRY-2 are associated with both ES cell pluripotency and early embryonic development.

Isolation of pluripotent stem cells from human third molar dental pulp.

Potent stem/progenitor cells have been isolated from normal human dental pulps, termed dental pulp stem cells (DPSCs). However, no study has described the presence of stem cell populations in human dental pulp from the third molar with embryonic phenotypes. The dental pulp tissue was cultured in media with the presence of LIF, EGF, and PDGF. In the present study, we describe a new population of pluripotent stem cells that were isolated from dental pulp (DPPSC). These cells are SSEA-4(+), Oct4(+), Nanog(+), FLK-1(+), HNF3beta(+), Nestin(+), Sox2(+), Lin28(+), c-Myc(+), CD13(+), CD105(+), CD3(-), CD45(-), CD90(low), CD29(+), CD73(low), STRO-1(low) and CD146(-). We have investigated by SEM analysis and q-RT-PCR the capacity of DPPSCs to 3D differentiate in vitro using the Cell Carrier 3D glass scaffold into tissues that have similar characteristics to embryonic mesoderm and endoderm layers. These data would support the use of these cells, which are derived from an easily accessible source and can be used in future regeneration protocols for many tissue types that differentiate from the three embryonic layers.

Glass-ceramics based on spodumene–enstatite system from natural raw materials

Glasses of compositions (wt%) corresponding to 50–90 spodumene and 50–10 enstatite were prepared depending on natural raw materials and Li 2CO 3 as small ingredient. The crystallization behavior was studied using DTA and XRD. The effect of addition of the nucleating agents (LiF) to a selected glass was also examined. Polarizing microscope and dilatometeric techniques were used to study the microstructures and the thermal expansion coefficient, respectively. XRD showed that the crystalline materials are mainly enstatite, clinoenstatite, β-eucryptite ss and β-spodumene ss. The shift of the glass composition to that of enstatite results in increased bulk crystallization. The presence of LiF does not greatly affect the mineral assemblages yet it improves the thermal expansion coefficients (TEC) of the crystalline samples and promotes the initial crystallization. Low and even negative values, ranging from −5.91 × 10 −7/K to 2.48 × 10 −7/K in the temperature range 293–573 K and 300–737 K, respectively, could be obtained, which make such materials applicable for thermally stable articles.

Effect of 1 wt% LiF additive on the densification of nanocrystalline Y 2O 3 ceramics by spark plasma sintering

Densification of nanocrystalline cubic yttria (nc-Y 2O 3) powder, with 18 nm crystal size and 1 wt% LiF as a sintering additive was investigated. Specimens were fabricated by spark plasma sintering at 100 MPa, within the temperature range of 700–1500 °C. Sintering at 700 °C for 5 and 20 min resulted in 95% and 99.7% dense specimens, with an average grain size of 84 and 130 nm, respectively. nc-Y 2O 3 without additive was only 65% dense at 700 °C for 5 min. The presence of LiF at low sintering temperatures facilitated rapid densification by particle sliding and jamming release. Sintering at high temperatures resulted in segregation of LiF to the grain boundaries and its entrapment as globular phase within the fast growing Y 2O 3 grains. The sintering enhancement advantage of LiF was lost at high SPS temperatures.

SAF-A Has a Role in Transcriptional Regulation ofOct4in ES Cells Through Promoter Binding

Methodologies to reprogram somatic cells into patient-specific pluripotent cells, which could potentially be used in personalized drug discovery and cell replacement therapies, are currently under development. Oct4 activation is essential for successful reprogramming and pluripotency of embryonic stem (ES) cells, albeit molecular details of Oct4 activation are not completely understood. Here we report that endogenous SAF-A is involved in regulation of Oct4 expression, binds the Oct4 proximal promoter in ES cells, and dissociates from the promoter upon early differentiation induced by LIF withdrawal. Depletion of SAF-A decreases Oct4 expression even in the presence of LIF, and results in an increase of the mesodermal marker Brachyury. The overexpression of wild-type human SAF-A rescues the mouse knock-down phenotype and results in increased Oct4 level. We also demonstrate that endogenous SAF-A interacts with the C-terminal domain (CTD) of endogenous RNA polymerase II and that the interaction is independent of CTD phosphorylation and mRNA. Moreover, we show that SAF-A exist in complexes with transcription factors Sox2 and Oct4 as well as STAT3 in ES cells. The number of endogenous SAF-A:Oct4 and SAF-A:Sox2 complexes decreases upon LIF depletion. These discoveries allow us to propose a model for activation of Oct4 transcription.

Mechanisms for the enhanced hydrogen desorption performance of the TiF4-catalyzed Na2LiAlH6 used for hydrogen storage

A dramatic enhancement in the dehydrogenation/hydrogenation kinetics of Na2LiAlH6 was achieved by adding 5 wt% TiF4. The starting temperature for dehydrogenation of the sample with TiF4 added was lowered by 50 °C with respect to the pristine sample. In-depth investigations revealed that the addition of TiF4 to Na2LiAlH6 not only enhanced the dehydrogenation kinetics, but also altered the dehydrogenation thermodynamics. The apparent activation energy and the desorption enthalpy change of the Na2LiAlH6 with 5 wt% TiF4 added were calculated to be ca. 143.6 ± 2.7 kJ mol−1 and 57.3 ± 2.3 kJ mol−1 of H2 by using Kissinger's approach and the van't Hoff equation, both lower than those of the pristine sample, respectively. We firmly believe that both Ti and F play critical roles in the dehydrogenation/hydrogenation process of the Na2LiAlH6 sample with added TiF4 due to the formation of Ti–Al clusters, the substitution of F− for H− and the presence of LiF. All these are responsible for the significant improvement of hydrogen storage performances of the Na2LiAlH6 sample with the addition of TiF4. This understanding provides us with insights into the dehydrogenation kinetic mechanisms of Na2LiAlH6 used for hydrogen storage and directs the optimization of high-performance catalysts for the hydrogen storage reaction of the alanates.

AMPK Regulates Nanog Gene Expression Via p53 in Mouse Embryonic Stem Cells.

Abstract 2537Poster Board II-514High glucose level is usually required for embryonic stem cell (ESC) culture but culture medium without glucose is a rather favorable condition to enhance differentiation of ESCs. However, the exact molecular relationship of how glucose metabolism affects ESC pluripotency remains unclear. AMP-activated protein kinase (AMPK), a key regulator for controlling the metabolism of glucose and fatty acids, is activated in response to a high intracellular AMP to ATP ratio (ATP-exhausting stress). We hypothesized that AMPK activity is associated with maintenance of ESC pluripotency and tested this hypothesis utilizing aminoimidazole carboxamide ribonucleotide (AICAR) and Compound C, respectively as an activator and a selective inhibitor of AMPK. Here, we demonstrate that in the mouse ESC line, R1, in the presence of LIF, AICAR significantly reduced protein expression of Nanog and SSEA-1 (Nanog: 365+/−21 vs. 270+/−25; SSEA-1: 13195+/−707 vs. 7572+/−507, mean fluorescence intensity (MFI), +/-SD, control vs. AICAR (0.5 mM, 24h treatment), n=3 expts, p<0.01). In contrast, Compound C enhanced protein expression of SSEA-1 and Nanog (Nanog: 365+/−21 vs. 555+/−35; SSEA-1: 13195+/−707 vs. 15864+/−894, mean fluorescence intensity (MFI), +/-SD, control vs. AICAR (0.005 mM, 24h treatment), n=3 expts, p<0.02). This indicates that AMPK has a positive effect on protein expression of Nanog and SSEA-1. We extended our study to investigate whether increased Nanog protein expression level via AMPK activity resulted from enhanced stabilization of Nanog protein by measuring the half-life of nanog protein following treatment of mouse ESCs with cycloheximide, a protein synthesis inhibitor. Nanog protein stability was not affected by AMPK activity. Next, we examined whether AMPK activator or inhibitor could change Nanog and Oct-4 mRNA levels. AICAR reduced Nanog mRNA expression by 40%, but did not influence Oct-4 mRNA expression. AMPK induces cell cycle arrest or apoptosis under ATP-exhausting stresses by phosphorylating and activating the p53 tumor suppressor, which could repress Nanog gene expression. Activated AMPK induced phosphorylation of p53 at serine 15 and 392 without disturbing LIF induced STAT3 signaling in this mouse ESC line (Fig. 1). Thus, we postulate that activated AMPK might inhibit Nanog gene activity by phosphorylation of p53. Taken together, our data suggest that AMPK activity in response to metabolic ATP and AMP levels may play a primary role in controlling ESC pluripotency through p53-mediated regulation of Nanog gene expression. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Singh et al. reply

Replying to: H. F. Jørgensen, Z.-F. Chen, M. Merkenschlager & A. G. Fisher , 10.1038/nature07783 ; N. J. Buckley, R. Johnson, Y.-M. Sun & L. W. Stanton , 10.1038/nature07784 (2009) In contrast to the comments made by Jørgensen 1 and Buckley 2, our experiments showed that REST maintains the self-renewal and pluripotency of mouse embryonic stem cells (mESCs)3. Two recent papers support our work: ref. 4 indicated that REST is indeed in the network that regulates ESC self-renewal and pluripotency and ref. 5 showed that mESCs with lower REST levels derived from a mouse model of Down’s syndrome have decreased levels of self-renewal markers and a higher propensity towards differentiation, even when cultured in the presence of LIF. We note that Buckley and Stanton also recently concluded that REST is part of the Oct4–Sox2–Nanog regulatory network and has “a key role in the maintenance of the ESC phenotype”6. We proposed that REST represses a set of microRNAs that potentially target self-renewal genes. At least one of them, miR-21, represses self-renewal, probably by destabilizing the messenger RNAs of Sox2 and/or Nanog (not Tbx3 or c-Myc as suggested by Buckley et al.2). In this model, changes in the cellular environment that counter this function of REST or stimulate the mRNA levels of Sox2 or Nanog could minimize the effect of REST. For this reason, in our study we used mESCs with a low passage number, and we cultured them without feeder cells to avoid possible contributions of the feeder cells or an adaptive response to high passage.