Leukemia inhibitory factor: a biological perspective

The notion that a single hormone may exert a broad range of effects has become well established. As such, leukemia inhibitory factor (LIF) is a prime example. LIF was initially described, purified, and genetically cloned on the basis of its ability to induce the differentiation and suppress the clonogenicity of the monocytic leukemia cell line, M1. Subsequently, it has become apparent that in vitro LIF inhibits the differentiation of pluripotential ES cells, stimulates the synthesis of hepatic acute-phase proteins, induces a switch in neurotransmitter phenotype from adrenergic to cholinergic, suppresses adipocyte lipoprotein lipase activity, and results in an increase in bone resorption. Moreover, elevation of LIF levels in vivo has a number of patho-physiological consequences, many of which parallel those effects observed in vitro. The challenge that lies ahead is to determine whether other sites of LIF action exist and to define more clearly the physiological role LIF plays in vivo. A major mechanism of cell-cell communication is by the production and secretion of polypeptide hormones by one cell type, which act either systemically or locally, via interaction with specific receptors on the surface of responsive cells. Recently, it has become apparent that hormones initially described and named, on the basis of a specific action, in many cases exert a spectrum of effects on a broad range of cell types. Moreover, the effects exerted are often mimicked closely by other hormones. Hormones that act in a pleiotropic manner are, for example, transforming growth factor-beta (TGF-beta), the various fibroblast growth factors (FGFs), interleukin-6 (IL-6), and leukemia inhibitory factor (LIF). This review will focus on the various biological effects ascribed to LIF.     

Combined action of stem cell factor,leukemia inhibitory factor,and cAMP on in vitro proliferation of mouse primordial germ cells

In the present paper we investigated the effects of stem cell factor/mastocyte growth factor (SCF/MGF), leukemia inhibitory factor/differentiating inhibitory activity (LIF/DIA) (two growth factors known to affect primordial germ cell growth in vitro) and forskolin (FRSK) (an activator of adenylate cyclase in many cell types) alone or in combination on the survival and proliferation of primordial germ cells (PGCs) obtained from 8.5, 10.5, and 11.5 days post coitum (dpc) mouse embryos and cultured without pre-formed cell feeder layers. The results showed that both at 1 and 3 days of culture the addition of 100 ng/ml SCF, 20 μM FRSK, or in some instances 20 ng/ml LIF alone caused a significant increase of PGC number as compared with controls. The highest effects were obtained when SCF and/or LIF were used together with FRSK. Moreover, we found that FRSK elevated cAMP levels in purified 11.5 dpc PGCs and that this compound, but not SCF and LIF, stimulated PGC proliferation, as assessed by 5-bromo-2′-deoxyuridin (BrdU) incorporation. These results suggest a mechanism of combined action of cAMP with SCF and/or LIF in the control of proliferation of mouse PGCs in vitro. © 1993 Wiley-Liss, Inc.     

Leukemia inhibitory factor (LIF) concentration modulates embryonic stem cell self-renewal and differentiation independently of proliferation

A major limitation of the widespread use of stem cells in a variety of biotechnological applications is the relatively low level of knowledge about how to maintain these cells in vitro without losing the long-term multilineage growth properties required for their clinical utility. An experimental and theoretical framework for predicting and controlling the outcome of stem cell stimulation by exogenous cytokines would thus be useful. An emerging theme from recent hematopoietic stem cell (HSC)-expansion studies is that a net gain in HSC numbers requires the maintenance of critical signaling ligand(s) above a threshold level. These ligand-receptor complex thresholds can be maintained, for example, by high concentrations of soluble cytokines or by cytokine presentation on cell surfaces. According to such a model, when the relevant ligand-receptor interaction falls below this threshold level, the probability of a differentiation response is increased; otherwise, self-renewal is favored. Taking advantage of the ability of the cytokine leukemia inhibitory factor (LIF) to maintain embryonic stem (ES) cell pluripotentiality at high concentrations, we are testing this model by investigating critical parameters in the control of ES cell responses. We have developed quantitative assays of ES cell differentiation by measuring cell-surface alkaline phosphatase activity, cell-surface stage specific embryonic antigen (SSEA)-1 expression, and the ability of ES cells to form embryoid bodies. Examination of ES cell responses over a range of LIF concentrations shows that LIF supplementation has little effect on ES cell-growth rate but significantly alters the probability of a cell undergoing a self-renewal vs. a differentiation division. In vitro culture parameters such as inoculum cell density, medium exchange, as well as cell-intrinsic processes such as autocrine secretion are shown to affect this decision. In addition to yielding new information on stem cell regulation by exogenous factors, these studies provide important clues about culture of these cells and should stimulate further investigations into the mechanistic basis of stem cell differentiation control.     

Formation of germ-line chimeras from embryonic stem cells maintained with recombinant leukemia inhibitory factor

Murine embryonic stem (ES) cells can be maintained as stem cells in vitro only in the presence of feeder cells or a soluble factor produced by a number of cell lines. We have previously demonstrated that leukemia inhibitory factor (LIF) is the molecule which prevents ES cell differentiation in culture. In this report we demonstrate that recombinant LIF can substitute for feeder cells in maintaining the full developmental potential of ES cells. The totipotent D3 ES cell line, previously isolated and maintained on growth-arrested primary embryo fibroblasts, was transferred to media supplemented with 1000 U/ml (10 ng/ml) recombinant LIF. In the presence of LIF the ES cells were maintained for over 2 months as undifferentiated cells in the absence of any feeder cells. When injected into blastocysts the ES cells which had been maintained in LIF-supplemented media efficiently formed germ-line chimeras.     

New approach for the establishment of mouse early embryonic stem cells and induction of their differentiation

Eleven early embryonic stem (EES) cell lines were established using a new novel method. Two cell stage embryos from the ddY mouse strain were cultured in alpha-MEM supplemented with 10% fetal calf serum (FCS) and embryotrophic factors (ETFs) and allowed to develop to the trilaminal germ disc embryonic stage. Only small round cells (EES cells) were isolated by the colony isolating technique and subsequently cultured in the same medium containing the ETFs and leukemia inhibitory factors (LIF-10 ng/ml). The newly established embryonic stem (ES) cells isolated from inner cell mass of blastocysts differentiated from two cell stage embryo in culture. The EES and ES cell lines were maintained in an undifferentiated state using Ham's F12 medium supplemented with 10% FCS and 1 ng/ml of LIF. The EES cells maintained their normal genetic and morphological features as well as their potential to differentiate into a broad spectrum of cell types as well as their ability to contribute to all cell lineages in chimeric mice. Moreover, these cell lines changed and differentiated into various kinds of cells by removing LIF and by the addition of ETFs to the vitro culture system. All 11 EES cell lines and 3 ES cell lines formed embryoid bodies; however, cell line EES-4 formed tube-like structures which extended, anastomosed with each other, and finally formed networks when the LIF were absent. Primitive germ organ-like structures composed of 3 germ layers were recognized in the cultures following the administration of ETFs. In conclusion, the new method devised by us is a novel, easy and reliable technique for establishing EES cell lines.     

Effect of leukemia inhibitory factor on bovine embryos produced in vitro under chemically defined conditions

The objective of these experiments was to assess putative embryotrophic effects of leukemia inhibitory factor (LIF) on bovine preimplantation development in chemically defined media. Recombinant human LIF was added to embryo culture media at a concentration of 100 ng/ml. When added for culture of morulae LIF had no positive effect on the proportion of embryos reaching the blastocyst stage. However, LIF significantly reduced development to the blastocyst stage when added for culture of 4-cell stage embryos (P<0.05). In contrast, a positive effect was found for progression of blastocyst development. In vitro blastocyst hatching rates were significantly improved in the presence of LIF (P<0.02). Number of total cells and of inner cell mass (ICM) cells were increased in LIF-treated blastocysts. In vitro survival of frozen-thawed blastocysts was not improved by adding LIF to morula stage embryos before cryopreservation. The pregnancy rate after direct transfer of cryopreserved LIF-treated embryos was not different from that for untreated control embryos. Data indicate that addition of LIF has no major beneficial effect on bovine embryos produced in these chemically defined conditions.     

Human amniotic epithelial cells maintain mouse spermatogonial stem cells in an undifferentiated state due to high leukemia inhibitor factor (LIF) expression

Spermatogonial stem cells (SSCs), like other stem cells, have unique properties: prolonged proliferation, self-renewal, generation of differentiated progeny, and maintenance of developmental potential. Long-term cultivation of normal SSCs into stable cell lines, and maintaining SSCs in an undifferentiated state capable of self-renewal, is a major challenge. Here, we compare the effect of leukemia inhibitory factor (LIF) expression on mouse SSCs isolated from testicular tissue cultured under different conditions. We found that human amniotic epithelial cells (hAECs) with high LIF expression (LIF(high)) feeder cells allowed mouse SSCs to maintain a high level of AP activity when cultured long term. Expression of some important stem cell markers was higher in mouse SSCs cultured on hAECs (LIF(high)) compared to those cultured on hAECs (LIF(low)). Taken together, these results suggest that LIF expression could be a crucial component for feeder cells to maintain mouse SSCs in an undifferentiated, proliferative state capable of self-renewal.     

Osteoblasts display receptors for and responses to leukemia-inhibitory factor

Specific binding of leukemia-inhibitory factor (LIF) to osteoblasts, but not multinucleated osteoclasts, was demonstrated by receptor autoradiography by using cells isolated from newborn rat long bones. The clonal rat osteogenic sarcoma cells, UMR 106-06, which have several phenotypic properties of osteoblasts, expressed 300 LIF receptors per cell, with an apparent KD of 60 pM. Treatment of calvarial osteoblasts or UMR 106-01 cells with LIF resulted in a dose-dependent inhibition of plasminogen activator (PA) activity. Both calvarial osteoblasts and osteogenic sarcoma cells were shown by Western blotting and reverse fibrin autography to produce plasminogen activator inhibitor-1 (PAI-1), the production of which was increased by LIF treatment. Northern blot analysis revealed that LIF treatment resulted in a rapid (peak 1 hour), dose-dependent increase in mRNA for PAI-1. LIF treatment of the preosteoblast cell line, UMR 201, enhanced the alkaline phosphatase response of these cells to retinoic acid. Each of the osteoblast-like cell types (calvarial osteoblasts, UMR 106-06, and UMR 201) was shown to produce LIF by bioassay and, by using the polymerase chain reaction (PCR), was shown to express low levels of mRNA for LIF. These data establish that cells of the osteoblast lineage are targets for LIF action. The reported anabolic effects of this cytokine on bone formation in vivo could be related to inhibition of protease activity. LIF may be an important paracrine modulator in bone, or perhaps an autocrine one, based on the evidence for its production by osteoblasts and osteoblast-like cells.     

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.     

Oncostatin M binds the high-affinity leukemia inhibitory factor receptor.

Oncostatin M (OSM) is a glycoprotein cytokine that was recently demonstrated to be structurally and functionally related to the leukemia inhibitory factor (LIF). We have investigated the binding of each cytokine to a variety of cellular receptors including those on solid tumor lines, leukemic cells, endothelial cells, macrophages, and cells transfected with the recently cloned low-affinity LIF receptor, and to a soluble form of the LIF receptor. LIF is incapable of binding either high- or low-affinity OSM receptors, yet OSM is capable of binding the high-affinity but not the low-affinity LIF receptor. Since the presence of high-affinity LIF receptors correlates with the biological activity of LIF on a wide range of target cells, we predict that OSM should have similar effects on LIF-responsive cells.     

Glial cell line-derived neurotrophic factor alters the growth characteristics and genomic imprinting of mouse multipotent adult germline stem cells

This study evaluated the essentiality of glial cell line-derived neurotrophic factor (GDNF) for in vitro culture of established mouse multipotent adult germline stem (maGS) cell lines by culturing them in the presence of GDNF, leukemia inhibitory factor (LIF) or both. We show that, in the absence of LIF, GDNF slows the proliferation of maGS cells and result in smaller sized colonies without any change in distribution of cells to different cell-cycle stages, expression of pluripotency genes and in vitro differentiation potential. Furthermore, in the absence of LIF, GDNF increased the expression of male germ-line genes and repopulated the empty seminiferous tubule of W/W^v mutant mouse without the formation of teratoma. GDNF also altered the genomic imprinting of Igf2, Peg1, and H19 genes but had no effect on DNA methylation of Oct4, Nanog and Stra8 genes. However, these effects of GDNF were masked in the presence of LIF. GDNF also did not interfere with the multipotency of maGS cells if they are cultured in the presence of LIF. In conclusion, our results suggest that, in the absence of LIF, GDNF alters the growth characteristics of maGS cells and partially impart them some of the germline stem (GS) cell-like characteristics.     

Inhibition of differentiation by leukemia inhibitory factor distinguishes two induction pathways in P19 embryonal carcinoma cells

The ability of leukemia inhibitory factor (LIF) to block differentiation of P19 embryonal carcinoma (EC) cells under a variety of induction conditions was determined. LIF inhibits differentiation under several conditions which lead to endodermal and mesodermal cell lineages including skeletal and cardiac muscle. In contrast, LIF does not block differentiation when cells are induced under conditions which lead to neuro-ectodermal cell types including neurons and astroglial cells. These studies demonstrate that P19 EC cell differentiation can be divided into LIF sensitive and insensitive pathways which correlate with differentiation of endodermal/mesodermal and neuro-ectodermal cell types, respectively. The effect of LIF on mRNA levels for several genes which have previously been implicated in mediating differentiation in P19 EC cells was determined. LIF has no effect on the mRNA levels for retinoic acid receptor (RAR) alpha, RAR beta, RAR gamma, jun A, jun D, c-fos, or fra-1. In contrast LIF stimulates jun B mRNA expression by a factor of four to six under all induction conditions.     

The ribosomal S6 kinases, cAMP-responsive element-binding, and STAT3 proteins are regulated by different leukemia inhibitory factor signaling pathways in mouse embryonic stem cells

Mouse embryonic stem (ES) cells remain "pluripotent" in vitro in the continuous presence of leukemia inhibitory factor (LIF). In the absence of LIF, ES cells are irreversibly committed to differentiate into various lineages. In this study we have set up an in vitro assay based on the anti-apoptotic activity of LIF to distinguish pluripotent from "differentiation-committed" ES cells. We have examined the phosphorylation profiles of known (STAT3 and ERKs) and identified new (ribosomal S6 kinases (RSKs) and cAMP-responsive element-binding protein (CREB)) LIF-regulated targets in ES and in ES-derived neuronal cells. We have demonstrated that although STAT3, a crucial player in the maintenance of ES cell pluripotency, is induced by LIF in all cell types tested, the LIF-dependent activation of RSKs is restricted to ES cells. We have shown that LIF-induced phosphorylation of RSKs in ES cells is dependent on ERKs, whereas STAT3 phosphorylation is not mediated by any known MAPK activities. Our results also demonstrate that the LIF-dependent phosphorylation of CREB is partially under the control of the RSK2 kinase.     

Regulation of vasoactive intestinal peptide expression in sympathetic neurons in culture and after axotomy: The role of cholinergic differentiation factor/leukemia inhibitory factor

Vasoactive intestinal peptide (VIP) expression increases in sympathetic neurons when they are grown in dissociated cell or explant cultures and when they are axotomized in vivo. In dissociated cell culture, the magnitude of the VIP increase was reduced when nonneuronal cells were removed and medium conditioned by ganglionic nonneuronal cells increased VIP in neuron-enriched cultures. Antiserum Against cholinergic differentiation factor (also leukemia inhibitory factor; CDF/LIF), but not against ciliary neurotrophic factor, immunoprecipitated this activity. Medium conditioned by sympathetic ganglion explants also contained a VIP-stimulatory molecule that was immunoprecipitated by CDF/LIF antiserum, and CDF/LIF antiserum partially blocked VIP induction in explants. CDF/LIF mRNA was increased in dissociated cell cultures, in ganglion explants and in vivo after axotomy. Our results suggest that CDF/LIF released from ganglionic nonneuronal cells plays an important role in regulating VIP after axotomy. 1994 John Wiley & Sons, Inc.