BMP Induces Cochlin Expression to Facilitate Self-renewal and Suppress Neural Differentiation of Mouse Embryonic Stem Cells

BMP4 maintains self-renewal of mouse embryonic stem cells (ESCs) in collaboration with LIF. Here, we report the identification of a novel key BMP target gene, cochlin (Coch) in mouse ESCs. Coch can be significantly up-regulated by BMP4 specifically in ESCs but not in somatic differentiated cells, and this up-regulation is dependent on the BMP signaling mediators Smad1/5 and Smad4. Overexpression of Coch can partially substitute BMP4 to promote self-renewal of mouse ESCs together with LIF, whereas knockdown of Coch impairs self-renewal marker gene expression even in the presence of both BMP4 and LIF. Further studies showed that COCH could mimic BMP4 in repressing neural differentiation of mouse ESCs upon LIF withdrawal and the inhibitory effect of BMP4 on neural differentiation is compromised by Coch knockdown. Taken together, our data suggest that COCH is a part of the downstream target network of BMP signaling and serves as another important effector to fine-tune mouse ESC fates.     

小鼠胚胎干细胞定向分化为神经干细胞过程中microRNA的变化

目的用生物芯片技术分析胚胎干细胞定向分化为神经干细胞过程中microRNA(miRNA)的表达变化,筛选调控的分化的miRNA,研究分化调控机制。方法胚胎干细胞在含LIF培养基中培养3d后,采用经典5步培养方法定向诱导向神经干细胞分化,采用nestin作为神经干细胞标记进行鉴定,送检胚胎干细胞及神经干细胞,提取总RNA以及小分子RNA,经荧光标记后与miRNA基因芯片杂交,获得胚胎干细胞诱导前后miRNA表达谱。结果1)胚胎干细胞在含LIF培养过程中保持未分化状态,Oct-4、碱性磷酸酶表达阳性;2)经典五步法诱导胚胎干细胞定向分化为神经干细胞,nestin阳性细胞为85%;3)通过基因微阵列分析,有90个miRNA的改变显著,其中68个表达上调,22个表达下调。结论miRNA可能对胚胎干细胞定向分化为神经干细胞过程起到关键作用。     

Convergent Regulation of Neuronal Differentiation and Erk and Akt Kinases in Human Neural Progenitor Cells by Lysophosphatidic Acid,Sphingosine 1-Phosphate,and LIF: Specific Roles for the LPA1 Receptor

The bioactive lysophospholipids lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) have diverse effects on the developing nervous system and neural progenitors, but the molecular basis for their pleiotropic effects is poorly understood. We previously defined LPA and S1P signaling in proliferating human neural progenitor (hNP) cells, and the current study investigates their role in neuronal differentiation of these cells. Differentiation in the presence of LPA or S1P significantly enhanced cell survival and decreased expression of neuronal markers. Further, the LPA receptor antagonist Ki16425 fully blocked the effects of LPA, and differentiation in the presence of Ki16425 dramatically enhanced neurite length. LPA and S1P robustly activated Erk, but surprisingly both strongly suppressed Akt activation. Ki16425 and pertussis toxin blocked LPA activation of Erk but not LPA inhibition of Akt, suggesting distinct receptor and G-protein subtypes mediate these effects. Finally, we explored cross talk between lysophospholipid signaling and the cytokine leukemia inhibitory factor (LIF). LPA/S1P effects on neuronal differentiation were amplified in the presence of LIF. Similarly, the ability of LPA/S1P to regulate Erk and Akt was impacted by the presence of LIF; LIF enhanced the inhibitory effect of LPA/S1P on Akt phosphorylation, while LIF blunted the activation of Erk by LPA/S1P. Taken together, our results suggest that LPA and S1P enhance survival and inhibit neuronal differentiation of hNP cells, and LPA1 is critical for the effect of LPA. The pleiotropic effects of LPA may reflect differences in receptor subtype expression or cross talk with LIF receptor signaling.     

Cytokines that signal through the leukemia inhibitory factor receptor-beta complex in the nervous system

Cytokines that signal through the leukemia inhibitory factor (LIF) receptor, such as LIF and ciliary neuronotrophic factor, have a wide range of roles within both the developing and mature nervous system. They play a vital role in the differentiation of neural precursor cells into astrocytes and can prevent or promote neuronal differentiation. One of the conundrums regarding signalling through the LIF receptor is how it can have multiple, often conflicting roles in different cell types, such as enhancing the differentiation of astrocytes while inhibiting the differentiation of some neuronal cells. Factors that can modulate signal transduction downstream of cytokine signalling, such as "suppressor of cytokine signalling" proteins, which inhibit the JAK/STAT but not the mitogen-activated protein kinase pathway, may therefore play an important role in determining how a given cell will respond to cytokine signalling. This review discusses the general effects of cytokine signalling within the nervous system. Special emphasis is placed on differentiation of neural precursor cells and the role that regulation of cytokine signalling may play in how a given precursor cell responds to cytokine stimulation.     

Gene expression in human neural stem cells: effects of leukemia inhibitory factor

Human neural precursor cells grown in culture provide a source of tissue for drug screening, developmental studies and cell therapy. However, mechanisms underlying their growth and differentiation are poorly understood. We show that epidermal growth factor (EGF) responsive precursors derived from the developing human cortex undergo senescence after 30-40 population doublings. Leukemia inhibitory factor (LIF) increased overall expansion rates, prevented senescence and allowed the growth of a long-term self renewing neural stem cell (ltNSCctx) for up to 110 population doublings. We established basal gene expression in ltNSCctx using Affymetrix oligonucleotide microarrays that delineated specific members of important growth factor and signaling families consistently expressed across three separate lines. Following LIF withdrawal, 200 genes showed significant decreases. Protein analysis confirmed LIF-regulated expression of glial fibrillary acidic protein, CD44, and major histocompatibility complex I. This study provides the first molecular profile of human ltNSCctx cultures capable of long-term self renewal, and reveals specific sets of genes that are directly or indirectly regulated by LIF.     

LIF promotes neurogenesis and maintains neural precursors in cell populations derived from spiral ganglion stem cells

Background  

Stem cells with the ability to form clonal floating colonies (spheres) were recently isolated from the neonatal murine spiral ganglion. To further examine the features of inner ear-derived neural stem cells and their derivatives, we investigated the effects of leukemia inhibitory factor (LIF), a neurokine that has been shown to promote self-renewal of other neural stem cells and to affect neural and glial cell differentiation.     

Regulation of neurokine receptor signaling and trafficking

Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) are neurally active cytokines, or neurokines. LIF signals through a receptor consisting of gp130 and the low affinity LIF receptor (LIFR), while the CNTF receptor consists of gp130, LIFR, and the low affinity CNTF receptor (CNTFR). Ser1044 of the LIFR is phosphorylated by Erk1/2 MAP kinase. Stimulation of neural cells with growth factors which strongly activate Erk1/2 decreases LIF-mediated signal transduction due to increased degradation of the LIFR as a consequence of Erk1/2-dependent phosphorylation of the receptor at Ser1044.     

LIF and BMP signaling generate separate and discrete types of GFAP-expressing cells

Bone morphogenetic protein (BMP) and leukemia inhibitory factor (LIF) signaling both promote the differentiation of neural stem/progenitor cells into glial fibrillary acidic protein (GFAP) immunoreactive cells. This study compares the cellular and molecular characteristics, and the potentiality, of GFAP(+) cells generated by these different signaling pathways. Treatment of cultured embryonic subventricular zone (SVZ) progenitor cells with LIF generates GFAP(+) cells that have a bipolar/tripolar morphology, remain in cell cycle, contain progenitor cell markers and demonstrate self-renewal with enhanced neurogenesis - characteristics that are typical of adult SVZ and subgranular zone (SGZ) stem cells/astrocytes. By contrast, BMP-induced GFAP(+) cells are stellate, exit the cell cycle, and lack progenitor traits and self-renewal--characteristics that are typical of astrocytes in the non-neurogenic adult cortex. In vivo, transgenic overexpression of BMP4 increases the number of GFAP(+) astrocytes but depletes the GFAP(+) progenitor cell pool, whereas transgenic inhibition of BMP signaling increases the size of the GFAP(+) progenitor cell pool but reduces the overall numbers of astrocytes. We conclude that LIF and BMP signaling generate different astrocytic cell types, and propose that these cells are, respectively, adult progenitor cells and mature astrocytes.     

Impact of IFN-β and LIF overexpression on human adipose-derived stem cells properties

Adipose-derived stem cells (ADSCs) are a subset of mesenchymal stem cells that their therapeutic effects in various diseases make them an interesting tool in cell therapy. In the current study, we aimed to overexpress interferon-β (IFN-β) and leukemia inhibitory factor (LIF) cytokines in human ADSCs to evaluate the impact of this overexpression on human ADSCs properties. Here, we designed a construct containing IFN-β and LIF and then, transduced human adipose-derived stem cells (hADSCs) by this construct via a lentiviral vector (PCDH-513B). We assessed the ability of long-term expression of the transgene in transduced cells by western blot analysis and enzyme-linked immunosorbent assay techniques on Days 15, 45, and 75 after transduction. For the evaluation of stem cell properties, flow cytometry and differentiation assays were performed. Finally, the MTT assay was done to assess the proliferation of transduced cells compares to controls. Our results showed high-efficiency transduction with highest expression rates on Day 75 after transduction which were 70 pg/ml for IFN-β and 77.9 pg/ml for LIF in comparison with 25.60 pg/ml and 27.63 pg/ml, respectively, in untransduced cells (p = .0001). Also, transduced cells expressed a high level of ADSCs surface markers and successfully differentiated into adipocytes, chondrocytes, neural cells, and osteocytes besides the preservation rate of proliferation near untreated cells (p = .88). All in all, we successfully constructed an hADSC population stably overexpressed IFN-β and LIF cytokines. Considering the IFN-β and LIF anti-inflammatory and neuroprotective effects as well as immune-regulatory properties of hADSCs, the obtained cells of this study could be subjected for further evaluations in experimental autoimmune encephalomyelitis mice model.     

Differential response of neuronal cells to a fusion protein of ciliary neurotrophic factor/soluble CNTF-receptor and leukemia inhibitory factor.

Ciliary neurotrophic factor (CNTF) displays neurotrophic activities on motor neurons and neural cell populations both in vivo and in vitro. On target cells lacking intrinsic expression of specific receptor alpha subunits cytokines of the IL-6 family only act in the presence of their specific agonistic soluble receptors. Here, we report the construction and expression of a CNTF/soluble CNTF-receptor (sCNTF-R) fusion protein (Hyper-CNTF) with enhanced biological activity on cells expressing gp130 and leukemia inhibitory factor receptor (LIF-R), but not membrane-bound CNTF-R. At the cDNA level, the C-terminus of the extracellular domain of human CNTF-R (amino acids 1-346) was linked via a single glycine residue to the N-terminus of human CNTF (amino acids 1-186). Recombinant Hyper-CNTF protein was expressed in COS-7 cells. Hyper-CNTF efficiently induced dose-dependent STAT3 phosphorylation and proliferation of BAF-3 cells stably transfected with gp130 and LIF-R cDNAs. While on BAF3/gp130/LIF-R cells, Hyper-CNTF and LIF exhibited similar biological responses, the activity of Hyper-CNTF on pheochromocytoma cells (PC12 cells) was quite distinct from that of LIF. In contrast to LIF, Hyper-CNTF stimulated neurite outgrowth of PC12 cells in a time- and dose-dependent manner correlating with the ability to phosphorylate MAP kinases. These data indicate that although LIF and Hyper-CNTF use the same heterodimeric receptor complex of gp130 and LIFR, only Hyper-CNTF induces neuronal differentiation. The therapeutic potential of Hyper-CNTF as a superagonistic neurotrophin is discussed.     

Leukemia Inhibitory Factor Is an Autocrine Survival Factor for Schwann Cells

Abstract: Schwann cells play a major role in promoting nerve survival and regeneration after injury. Their activities include providing neurotrophic factors and increasing the production of extracellular matrix components and cell surface adhesion molecules to promote axon regeneration. Following nerve transection, leukemia inhibitory factor (LIF) is up-regulated by Schwann cells at the injury site. LIF receptors are also up-regulated at the nerve injury site, but their cellular localization and function have not been fully characterized. We demonstrate that Schwann cells express mRNAs for LIF and the LIF receptor components LIF receptor subunit β and glycoprotein 130 in vitro. We also show that although LIF is not required for the genesis of Schwann cells, it can potentiate the survival of differentiated Schwann cells in the context of neuregulin support. Not only does exogenous LIF promote survival under these conditions, but addition of the soluble LIF receptor (LIF binding protein) and anti-LIF antibodies significantly reduced cell survival, suggesting that LIF exerts autocrine effects. These results suggest that Schwann cell survival following nerve injury is potentially modulated by LIF.     

Oncostatin M is a differentiation factor for myeloid leukemia cells.

Oncostatin M (OSM) is a 28-kDa glycoprotein produced by stimulated macrophages and T lymphocytes that inhibits the proliferation of a number of different cell lines derived from solid tumors. Analysis of both amino acid sequence and gene structure has demonstrated that OSM is a member of a cytokine family that includes leukemia inhibitory factor (LIF), IL-6, and granulocyte colony-stimulating factor (G-CSF). We demonstrate that, like LIF, IL-6 and G-CSF, OSM can induce the differentiation of the myeloblastic M1 murine leukemia cells into macrophage-like cells. The morphologic and functional changes induced by OSM are more similar to those observed with LIF and IL-6 than those induced with G-CSF. OSM can also induce the differentiation of the histiocytic U937 human leukemia cells in the presence of granulocyte-macrophage CSF, a property shared with LIF and IL-6. In murine M1 cells, binding of labeled OSM is completely inhibited by excess LIF or OSM, reflecting the binding of OSM to the high affinity form of the murine LIF receptor. In contrast, the binding of labeled OSM to human U937 leukemia cells is inhibited by OSM, but the inhibition by LIF is significantly less. These results suggest that, in human leukemia cells, OSM may act through the LIF receptor and an OSM-specific receptor. The existence of an OSM-specific receptor was confirmed by both growth inhibition and competition binding assays on A375 human melanoma cells. The growth of human A375 cells was inhibited by OSM and IL-6 but not LIF or G-CSF. Neither LIF, G-CSF, nor IL-6 could compete with the binding of labeled OSM to A375 cells.     

Leukaemia Inhibitory Factor derived from rat colon carcinoma cells increases host susceptibility to tumour growth

We have tested Leukaemia Inhibitory Factor (LIF) production by 12 rat colon tumour clones isolated from a single cell line that display various degrees of tumorigenicity A highly significant relationship was found between levels of soluble LIF produced by the clones and their in vivo tumorigenicity. Such results suggested a role for LIF as a tumour facilitating agent. To test this hypothesis, the highly tumorigenic and LIF producing PROb clone was transfected with the LIF cDNA in antisense orientation in order to decrease LIF production. Conversely, REGb, a low LIF producer that is rejected by syngeneic animals, as well as nude mice, was transfected with the LIF cDNA to increase its production. PROb cells transfected with antisense cDNA were shown to have decreased LIF production along with decreased tumorigenicity. LlF-transfected REGb cells expressing high LIF levels still regressed in syngeneic rats, but could form progressive tumours in nude mice. We did not detect LIF receptors on PROb or REGb cells and their in vitro proliferation was not modified by the addition of exogenous LIF. Therefore, LIF was not an autocrine growth regulator for PROb and REGb cells. Instead, LIF appears to facilitate in vivo tumour growth, without being an immunosuppressive factor sufficient on its own to allow growth of immunogenic cells in fully immunocompetent hosts.     

Leukaemia inhibitory factor (LIF) is functionally linked to axotrophin and both LIF and axotrophin are linked to regulatory immune tolerance

Axotrophin (axot) is a newly characterised stem cell gene and mice that lack axotrophin are viable and fertile, but show premature neural degeneration and defective development of the corpus callosum. By comparing axot+/+, axot+/- and axot-/- littermates, we now show that axotrophin is also involved in immune regulation. Both T cell proliferation and T cell-derived leukaemia inhibitory factor (LIF) were suppressed by axotrophin in a gene-dose-dependent manner. Moreover, a role for axotrophin in the feedback regulation of LIF is implicated. This is the first evidence that fate determination mediated by LIF maybe qualified by axotrophin.