Blocking autocrine VEGF signaling by sunitinib,an anti-cancer drug,promotes embryonic stem cell self-renewal and somatic cell reprogramming

Maintaining the self-renewal of embryonic stem cells (ESCs) could be achieved by activating the extrinsic signaling, i.e., the use of leukemia inhibitory factor (LIF), or blocking the intrinsic differentiation pathways, i.e., the use of GSK3 and MEK inhibitors (2i). Here we found that even in medium supplemented with LIF, mESCs still tend to differentiate toward meso-endoderm lineages after long-term culture and the culture spontaneously secretes vascular endothelial growth factors (VEGFs). Blocking VEGF signaling with sunitinib, an anti-cancer drug and a receptor tyrosine kinase (RTK) inhibitor mainly targeting VEGF receptors (VEGFRs), is capable of maintaining the mESCs in the undifferentiated state without the need for feeder cells or LIF. Sunitinib facilitates the derivation of mESCs from blastocysts, and the mESCs maintained in sunitinib-containing medium remain pluripotent and are able to contribute to chimeric mice. Sunitinib also promotes iPSC generation from MEFs with only Oct4. Knocking down VEGFR2 or blocking it with neutralizing antibody mimicks the effect of sunitinib, indicating that blocking VEGF/VEGFR signaling is indeed beneficial to the self-renewal of mESCs. We also found that hypoxia-inducible factor alpha (HIF1α) and endoplasmic reticulum (ER) stress are involved in the production of VEGF in mESCs. Blocking both pathways inhibits the expression of VEGF and prevents spontaneous differentiation of mESCs. Interestingly, LIF may also exert its effect by downregulating HIF1α and ER stress pathways and subsequent VEGF expression. These results indicate the existence of an intrinsic differentiation pathway in mESCs by activating the autocrine VEGF signaling. Blocking VEGF signaling with sunitinib or other small molecules help to maintain the mESCs in the ground state of pluripotency.     

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.     

Id2诱导Myc表达促进小鼠胚胎干细胞自我更新

小鼠胚胎干细胞(mouse embryonic stem cells,m ESCs)分离自小鼠的囊胚内细胞团,在体外具有无限的自我更新能力和多向分化的潜能,因此拥有重大的社会和经济效益.前期人们发现,DNA结合抑制因子1(inhibitor of DNA binding 1,Id1)在含血清培养条件下,可以促进m ESCs自我更新,但其家族成员,如Id2和Id3,在m ESCs中的作用尚不清楚.本课题在m ESCs里分别上调Id2和Id3基因的表达,发现它们在含血清的培养条件下均具有促进m ESCs自我更新的能力,但Id2促进自我更新的能力大于Id3.通过转录组测序技术发现Id2上调c-Myc和n-Myc基因的表达水平.最后功能性验证实验证实,只有同时干扰c-Myc和n-Myc基因的表达才能够极大地削弱Id2维持m ESCs未分化状态的作用,表明Id2主要通过诱导Myc家族成员的表达来促进m ESCs的自我更新.本研究结果将扩大人们对干细胞多能性调控网络的认识,利于干细胞未来的基础研究和安全应用.     

Construction and Validation of a Regulatory Network for Pluripotency and Self-Renewal of Mouse Embryonic Stem Cells

A 30-node signed and directed network responsible for self-renewal and pluripotency of mouse embryonic stem cells (mESCs) was extracted from several ChIP-Seq and knockdown followed by expression prior studies. The underlying regulatory logic among network components was then learned using the initial network topology and single cell gene expression measurements from mESCs cultured in serum/LIF or serum-free 2i/LIF conditions. Comparing the learned network regulatory logic derived from cells cultured in serum/LIF vs. 2i/LIF revealed differential roles for Nanog, Oct4/Pou5f1, Sox2, Esrrb and Tcf3. Overall, gene expression in the serum/LIF condition was more variable than in the 2i/LIF but mostly consistent across the two conditions. Expression levels for most genes in single cells were bimodal across the entire population and this motivated a Boolean modeling approach. In silico predictions derived from removal of nodes from the Boolean dynamical model were validated with experimental single and combinatorial RNA interference (RNAi) knockdowns of selected network components. Quantitative post-RNAi expression level measurements of remaining network components showed good agreement with the in silico predictions. Computational removal of nodes from the Boolean network model was also used to predict lineage specification outcomes. In summary, data integration, modeling, and targeted experiments were used to improve our understanding of the regulatory topology that controls mESC fate decisions as well as to develop robust directed lineage specification protocols.     

Murine Embryonic Stem Cell Plasticity Is Regulated through Klf5 and Maintained by Metalloproteinase MMP1 and Hypoxia

Mouse embryonic stem cells (mESCs) are expanded and maintained pluripotent in vitro in the presence of leukemia inhibitory factor (LIF), an IL6 cytokine family member which displays pleiotropic functions, depending on both cell maturity and cell type. LIF withdrawal leads to heterogeneous differentiation of mESCs with a proportion of the differentiated cells apoptosising. During LIF withdrawal, cells sequentially enter a reversible and irreversible phase of differentiation during which LIF addition induces different effects. However the regulators and effectors of LIF–mediated reprogramming are poorly understood. By employing a LIF-dependent ‘plasticity’ test, that we set up, we show that Klf5, but not JunB is a key LIF effector. Furthermore PI3K signaling, required for the maintenance of mESC pluripotency, has no effect on mESC plasticity while displaying a major role in committed cells by stimulating expression of the mesodermal marker Brachyury at the expense of endoderm and neuroectoderm lineage markers. We also show that the MMP1 metalloproteinase, which can replace LIF for maintenance of pluripotency, mimics LIF in the plasticity window, but less efficiently. Finally, we demonstrate that mESCs maintain plasticity and pluripotency potentials in vitro under hypoxic/physioxic growth conditions at 3% O₂ despite lower levels of Pluri and Master gene expression in comparison to 20% O₂.     

Jak2 and Tyk2 are necessary for lineage-specific differentiation, but not for the maintenance of self-renewal of mouse embryonic stem cells

As the LIF-induced Jak1/STAT3 pathway has been reported to play a crucial role in self-renewal of mESCs, we sought to determine if Jak2, which is also expressed in mESCs, might also be involved in the pathway. By employing an RNAi strategy, we established both Jak2 and Jak2/Tyk2 knockdown mESC clones. Both Jak2 and Jak2/Tyk2 knockdown clones maintained the undifferentiated state as wild-type controls, even in a very low concentration of LIF. However, we observed not only faster onset of differentiation but also differential expression of tissue-specific lineage genes for ectodermal and mesodermal, but not endodermal origins from embryoid bodies generated from both types of knockdown clones compared to the wild-type. Furthermore, the reduced level of Jak2 caused differentiation of mESCs in the presence of LIF when the Wnt pathway was activated by LiCl treatment. Taken together, we demonstrated that Jak2 and Tyk2 are not involved in LIF-induced STAT3 pathway for self-renewal of mESCs, but play a role in early lineage decision of mESCs to various differentiated cell types.     

Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity

The TET family of FE(II) and 2-oxoglutarate-dependent enzymes (Tet1/2/3) promote DNA demethylation by converting 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), which they further oxidize into 5-formylcytosine and 5-carboxylcytosine. Tet1 is robustly expressed in mouse embryonic stem cells (mESCs) and has been implicated in mESC maintenance. Here we demonstrate that, unlike genetic deletion, RNAi-mediated depletion of Tet1 in mESCs led to a significant reduction in 5hmC and loss of mESC identity. The differentiation phenotype due to Tet1 depletion positively correlated with the extent of 5hmC loss. Meta-analyses of genomic data sets suggested interaction between Tet1 and leukemia inhibitory factor (LIF) signaling. LIF signaling is known to promote self-renewal and pluripotency in mESCs partly by opposing MAPK/ERK-mediated differentiation. Withdrawal of LIF leads to differentiation of mESCs. We discovered that Tet1 depletion impaired LIF-dependent Stat3-mediated gene activation by affecting Stat3's ability to bind to its target sites on chromatin. Nanog overexpression or inhibition of MAPK/ERK signaling, both known to maintain mESCs in the absence of LIF, rescued Tet1 depletion, further supporting the dependence of LIF/Stat3 signaling on Tet1. These data support the conclusion that analysis of mESCs in the hours/days immediately following efficient Tet1 depletion reveals Tet1's normal physiological role in maintaining the pluripotent state that may be subject to homeostatic compensation in genetic models.     

Nanog overcomes reprogramming barriers and induces pluripotency in minimal conditions

Induced pluripotency requires the expression of defined factors and culture conditions that support the self-renewal of embryonic stem (ES) cells. Small molecule inhibition of MAP kinase (MEK) and glycogen synthase kinase 3 (GSK3) with LIF (2i/LIF) provides an optimal culture environment for mouse ES cells and promotes transition to naive pluripotency in partially reprogrammed (pre-iPS) cells. Here we show that 2i/LIF treatment in clonal lines of pre-iPS cells results in the activation of endogenous Nanog and rapid downregulation of retroviral Oct4 expression. Nanog enables somatic cell reprogramming in serum-free medium supplemented with LIF, a culture condition which does not support induced pluripotency or the self-renewal of ES cells, and is sufficient to reprogram epiblast-derived stem cells to naive pluripotency in serum-free medium alone. Nanog also enhances reprogramming in cooperation with kinase inhibition or 5-aza-cytidine, a small molecule inhibitor of DNA methylation. These results highlight the capacity of Nanog to overcome multiple barriers to reprogramming and reveal a synergy between Nanog and chemical inhibitors that promote reprogramming. We conclude that Nanog induces pluripotency in minimal conditions. This provides a strategy for imposing naive pluripotency in mammalian cells independently of species-specific culture requirements.     

Soft substrates promote homogeneous self-renewal of embryonic stem cells via downregulating cell-matrix tractions

Maintaining undifferentiated mouse embryonic stem cell (mESC) culture has been a major challenge as mESCs cultured in Leukemia Inhibitory Factor (LIF) conditions exhibit spontaneous differentiation, fluctuating expression of pluripotency genes, and genes of specialized cells. Here we show that, in sharp contrast to the mESCs seeded on the conventional rigid substrates, the mESCs cultured on the soft substrates that match the intrinsic stiffness of the mESCs and in the absence of exogenous LIF for 5 days, surprisingly still generated homogeneous undifferentiated colonies, maintained high levels of Oct3/4, Nanog, and Alkaline Phosphatase (AP) activities, and formed embryoid bodies and teratomas efficiently. A different line of mESCs, cultured on the soft substrates without exogenous LIF, maintained the capacity of generating homogeneous undifferentiated colonies with relatively high levels of Oct3/4 and AP activities, up to at least 15 passages, suggesting that this soft substrate approach applies to long term culture of different mESC lines. mESC colonies on these soft substrates without LIF generated low cell-matrix tractions and low stiffness. Both tractions and stiffness of the colonies increased with substrate stiffness, accompanied by downregulation of Oct3/4 expression. Our findings demonstrate that mESC self-renewal and pluripotency can be maintained homogeneously on soft substrates via the biophysical mechanism of facilitating generation of low cell-matrix tractions.     

Complete basis set,hybrid-DFT study and NBO interpretation of conformational analysis of 2-methoxytetrahydropyran and its thiopyran and selenopyran analogues in relation to the anomeric effect

2-Methoxytetrahydropyran (1), -thiopyran (2) and -selenopyran (3) have been chosen as model compounds to investigate the origin of the anomeric effect (AE). The impacts of the hyperconjugation, electrostatic and steric interactions on the conformational preferences of compounds 1–3 have been analysed by means of complete basis set-4, hybrid-density functional theory (B3LYP/6-311+G**) based methods and natural bond orbital (NBO) interpretation. Both levels of theory showed that the axial conformations of compounds 1–3 are more stable than their equatorial conformations. The Gibbs free energy difference (G _eq–G _ax) values (i.e. ΔG _eq–ax) between the axial and equatorial conformations increase from compound 1 to compound 2 but decrease from compound 2 to compound 3. Based on the NBO results obtained, the AE associated with the electron delocalisation [i.e. Σ(endo-AE_eq + exo-AE_eq) ? Σ(endo-AE_ax + exo-AE_ax)] increase slightly from compound 1 to compound 2 but decrease from compound 2 to compound 3. Similar trend is also observed for the differences between the calculated total steric exchange energy values [i.e. Δ(TSEE)_eq–ax]. On the other hand, the calculated differences between the dipole moment values of the axial and equatorial conformations [i.e. Δ(μ_eq–μ_ax)] decrease from compound 1 to compound 3. These findings led to the proposal that the AE associated with the electron delocalisation (the hyperconjugation effect) is more significant for the explanation of the conformational preferences of compounds 1–3 than the electrostatic model. The correlations between the AE associated with the electron delocalisation, bond orders, TSEE, ΔG _eq–ax, dipole–dipole interactions, structural parameters and conformational behaviours of compounds 1–3 have been investigated.     

Discovery of a novel and potent class of anti-HIV-1 maturation inhibitors with improved virology profile against gag polymorphisms

A new class of betulin-derived α-keto amides was identified as HIV-1 maturation inhibitors. Through lead optimization, GSK8999 was identified with IC₅₀ values of 17 nM, 23 nM, 25 nM, and 8 nM for wild type, Q369H, V370A, and T371A respectively. When tested in a panel of 62 HIV-1 isolates covering a diversity of CA-SP1 genotypes including A, AE, B, C, and G using a PBMC based assay, GSK8999 was potent against 57 of 62 isolates demonstrating an improvement over the first generation maturation inhibitor BVM. The data disclosed here also demonstrated that the new α-keto amide GSK8999 has a mechanism of action consistent with inhibition of the proteolytic cleavage of CA-SP1.     

Insulin-Stimulated Glycogen Synthesis in Cultured Hepatoma Cells: Differential Effects of Inhibitors of Insulin Signaling Molecules

Abstract

In rat HTC hepatoma cells overexpressing human insulin receptors, insulin stimulated glycogen synthesis by 55–70%. To study postreceptor signaling events leading to insulin-stimulated glycogen synthesis in these cells, we have employed pathway-specific chemical inhibitors such as LY294002, rapamycin and PD98059 to inhibit phosphatidylinositol-3-kinase (PI3K), p70 ribosomal S6 kinase and mitogen-activated protein kinase (MAPK) kinase/MAPK, respectively. LY294002 (50 μM) completely abolished insulin-stimulated glycogen synthesis whereas rapamycin (2–20 nM) partially inhibited it. Neither LY294002 nor rapamycin significantly affected the basal glycogen synthesis. However, PD98059 (100 μM) significantly inhibited the basal glycogen synthesis without affecting insulin-stimulated glycogen synthesis. In these cells, insulin at 100 nM decreased glycogen synthase kinase 3α (GSK3α) activity by 30–35%. LY294002, but neither rapamycin nor PD98059, abolished insulin-induced inactivation of GSK3α. These data suggest that insulin-stimulated glycogen synthesis in rat HTC hepatoma cells is mediated mainly by PI3K-dependent mechanism. In these cells, inactivation of GSK3α, downstream of PI3K, may play a role in insulin-stimulated glycogen synthesis.