Differentiation of embryonic stem cells into cardiomyocytes induced by leukemia inhibitory factor (LIF)

An experimental model of mouse embryonic stem cell (ESC) differentiation into cells with contractile activity (similar to that of cardiomyocytes) without embryoid body formation has been obtained. The main factor inducing ESC differentiation along the cardiomyocyte pathway is recombinant cytokine LIF added in the course of long-term culturing. The contractile cells respond positively to treatment with isoproterenol, a cardioactive drug, which is evidence for the presence in these cells of β-adrenoreceptors characteristic of terminally differentiated mammalian cardiomyocytes.     

High and low affinity receptors for human interleukin for DA cells/leukemia inhibitory factor on human cells. Molecular characterization and cellular distribution.

Radioiodinated recombinant human interleukin DA (HILDA)/leukemia inhibitory factor (LIF) purified from conditioned medium of Chinese hamster ovary transfected cells enabled the identification of specific receptor sites on a variety of human cell types. Using low concentrations (up to 500 pM) of the ligand iodinated at a high specific radioactivity, high affinity receptors (equilibrium dissociation constant Kd in the range of 30-100 pM) were first demonstrated. They were expressed at low levels by human peripheral blood monocytes but not by lymphocytes, NK cells, granulocytes, and platelets. The myelomonocytic cell line THP1 as well as the T lymphoma cell line HSB2 and the lymphoblastoid B cell line DAB were also receptor-negative. In contrast, most of the non-lymphoid tumoral cell lines tested, including melanomas, neuroblastomas, and carcinomas, expressed high affinity HILDA/LIF receptors at variable levels (Bmax from 20 to 600 sites/cell). The kinetics of HILDA/LIF high affinity binding to the choriocarcinoma JAR cell line were characterized at 4 degrees C with association and dissociation rate constants of k1 = 2.2 10(9) M-1 min-1 and k-1 = 0.0084 min-1, respectively, corresponding to a steady-state dissociation constant k1/k-1 = 3.8 pM. The subsequent use of higher concentrations of HILDA/LIF labeled at a lower specific radioactivity enabled the identification of a low affinity component on several cell lines (Kd in the range of 1-4 nM; Bmax from 1,000 to 5,000 sites/cell). On JAR cells, this low affinity component was characterized by association and dissociation rate constants at 4 degrees C of k1 = 7.3 10(7) M-1 min-1 and k-1 = 0.19 min-1, respectively (k-1/k1 = 2.6 nM). Affinity cross-linking of HILDA/LIF to JAR cells showed two cross-linked species under both reducing and nonreducing conditions corresponding to receptor species of 120 and 250 kDa, respectively. Whereas both bands had similar intensities under high affinity conditions, the higher band predominated under low affinity conditions. Our data suggest that the 250-kDa chain could constitute the low affinity binding component whereas the association of both 250- and 120-Da subunits would form the high affinity structure.     

Human differentiation-stimulating factor (leukemia inhibitory factor, human interleukin DA) gene maps distal to the Ewing sarcoma breakpoint on 22q

The human gene encoding differentiation-stimulating factor (D-factor) has previously been isolated and shown to be identical to leukemia inhibitory factor (LIF). We have determined a fine structure map of approximately 20-kb surrounding the D-factor/LIF gene. Southern blot analysis using a somatic cell hybrid panel shows that the gene maps to chromosome 22. D-factor/LIF was further sublocalized to 22q11.2----q13.1, distal to a Ewing sarcoma (ES) breakpoint, using a second somatic cell hybrid panel. Probes to the 5' and 3' regions of the locus and the cDNA were used to screen for restriction fragment length polymorphisms, but none were detected. Analysis by pulsed field gel electrophoresis suggests that D-factor/LIF is not near the ES breakpoint.     

Rat leukocyte inhibitory factor (LIF): Similarities to human LIF and generation by cells from rats with collagen-induced arthritis

Rat lymph node cells (LNC) produce a mediator which exhibits functional and physicochemical similarities to human leukocyte inhibitory factor (LIF). Measurement of LIF can be used to quantify cellular sensitivity in rats to foreign protein antigens or to a heterologous antigen in vitro. Concanavalin A-induced rat LIF has a molecular weight of 100,000-60,000 daltons and retains activity after heating to 56 °C for 30 min. Rat LIF is not synthesized in the presence of puromycin and appears to be a protein, since it is inactivated by treatment with chymotrypsin. Moreover, the activity of rat LIF is susceptible to the serine esterase inhibitor, diisopropylphosphofluoridate, but it is resistant to neuraminidase treatment. Finally, rat LIF preferentially inhibits the migration of rat and human polymorphonuclear leukocytes but not that of rat or guinea pig peritoneal exudate cells enriched for macrophages. Except for the more dispersed size of rat LIF, these properties are analogous to those described for human LIF. LIF activity is generated, in an antigen-specific manner, by LNC sensitized to ovalbumin or purified protein derivative of tuberculin when cultured with the antigen used for sensitization. LNC from rats rendered arthritic by prior intradermal (id) injection of native chick type II collagen in incomplete Freund's adjuvant produce LIF in response to this heterologous antigen. These studies delineate a new assay for cellular sensitivity in rats and provide additional evidence that cellular reactivity to type II collagen is present in this animal model of arthritis.     

Isolation of embryonic stem (ES) cells in media supplemented with recombinant leukemia inhibitory factor (LIF)

The isolation of pluripotent murine embryonic stem (ES) cells has previously been achieved by coculturing the ES cells with fibroblast feeder cells. In this report we demonstrate that ES cell lines can be isolated from murine 129/Sv He blastocysts in the absence of feeder cells in culture medium supplemented with recombinant leukemia inhibitory factor (LIF). Three of the ES cell lines (MBL-1, MBL-2, and MBL-3) were isolated by directly explanting blastocysts, whilst two ES cell lines (MBL-4 and MBL-5) were isolated from blastocysts pretreated by immunosurgery. Three of the ES cell lines contained the Y chromosome (MBL-1, MBL-2, and MBL-5) with a high proportion of the cells displaying a normal diploid karyotype with a modal chromosome number of 40. All of the ES cell lines tested expressed the stem cell markers ECMA-7 and alkaline phosphatase, which were lost on removal of LIF when the ES cells differentiated into a variety of cell types. The full developmental potential of the ES cells was determined by injecting cells from two of the independently derived ES cell lines, MBL-1 and MBL-5, into C57BL/6J blastocysts. A high proportion of the pups born were chimeric as judged by coat pigmentation. Subsequent breeding established that the ES cells had contributed to the germ line. These results demonstrate that feeder cells are not essential for the isolation of pluripotent ES cell lines.     

Characterization and NH2-terminal amino acid sequence of natural human interleukin for DA cells: leukemia inhibitory factor. Differentiation inhibitory activity secreted by a T lymphoma cell line

Six human T lymphomas and an NK-like cell line were tested for their ability to produce HILDA, one of the two human growth promoting activities for the DA1.a cells. Among them, the HSB2 cell line turned out to be the only one secreting significant HILDA activity (200-400 units/ml) after activation with 50 nM phorbol myristate acetate. Subclones of the HSB2 cell line were obtained by limiting dilution experiments. One of them (2B3) was found to secrete 1,000-5,000 units/ml of HILDA after phorbol myristate acetate activation in the presence of 10% fetal calf serum and 200-500 units/ml in serum-free conditions. 2B3-HILDA was purified from serum-free conditioned medium by a four-step procedure including fast flow cationic exchange at pH 6, concanavalin A chromatography, reverse-phase high performance liquid chromatography and gel-filtration high performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified radiolabeled cytokine revealed a single band of Mr 43,000, which co-electrophoresed with the biological activity. The NH2-terminal amino acid sequence of the first residues of the protein were determined and found to be similar to the equivalent residues deduced from the molecularly cloned cytokine. Isoelectric point determination revealed some charge heterogeneity of HILDA, which focused to pH 8.5-9 after neuraminidase treatment. Carbohydrate content of the cytokine was studied by deglycosylation experiments which showed that the O-linked oligosaccharides represented 2,000-3,000 and that the N-linked sugars account for half of the apparent molecular weight of HILDA.     

Modulation of PDGF mediated osteoblast chemotaxis by leukemia inhibitory factor LIF

Cell migration is a key event in tissue repair and remodeling. PDGF, a growth factor for multiple target cells, has been shown to be a potent chemoattractant for a variety of mesenchymal cells. However, it is likely that PDGF-mediated cell migration will be influenced by other cytokines that can be produced during physiological and pathological conditions. Leukemia inhibitory factor (LIF), a cytokine that is produced by a variety of cells including osteoblasts, may promote bone formation, but the mechanism is not known. Since osteoblasts are responsible for laying down new matrix during skeletal remodeling, in this report we have examined whether PDGF or LIF influences the migration of osteoblasts. Among several cytokines and growth factors tested, only PDGF was able to elicit a major chemotactic (directed migration) and a minor chemokinetic (random-migration) response in osteoblasts. LIF alone was not active in either chemotaxis or chemokinesis but when included with PDGF it caused a reduction in chemokinesis. Further, pretreatment of osteoblasts with LIF caused an increase in PDGF-driven chemotaxis. Finally, osteoblasts exposed briefly to LIF synthesized a higher level of non-collagenous proteins upon further treatment with PDGF. These observations are consistent with a role for LIF in promoting bone formation, both by influencing directional migration of osteoblasts and in laying down new matrix. © 1996 Wiley-Liss, Inc.     

Differentiation of embryonic stem cells into cardiomyocytes with the help of cytokine LIF (leukemia inhibitory factor)

An experimental model of differentiated mouse embryonic stem cells with retractive activity similar to that of cardiomyocytes without preliminary formation of embryoid bodies was obtained. The basic factor that induced in vitro embryonic cell differentiation into cardial type is the recombinant cytokine LIF under prolonged cultivation. The positive reaction of the cells with retractive activity to isoproterenol indicates the presence of the beta-adrenergic receptor activity characteristic only for terminal differentiated mammalian cardiomyocytes.     

Extensive mannose phosphorylation on leukemia inhibitory factor (LIF) controls its extracellular levels by multiple mechanisms

In addition to soluble acid hydrolases, many nonlysosomal proteins have been shown to bear mannose 6-phosphate (Man-6-P) residues. Quantification of the extent of mannose phosphorylation and the relevance to physiological function, however, remain poorly defined. In this study, we investigated the mannose phosphorylation status of leukemia inhibitory factor (LIF), a previously identified high affinity ligand for the cation-independent mannose 6-phosphate receptor (CI-MPR), and we analyzed the effects of this modification on its secretion and uptake in cultured cells. When media from LIF-overexpressing cells were fractionated using a CI-MPR affinity column, 35-45% of the total LIF molecules were bound and specifically eluted with free Man-6-P thus confirming LIF as a bona fide Man-6-P-modified protein. Surprisingly, mass spectrometric analysis of LIF glycopeptides enriched on the CI-MPR column revealed that all six N-glycan sites could be Man-6-P-modified. The relative utilization of these sites, however, was not uniform. Analysis of glycan-deleted LIF mutants demonstrated that loss of glycans bearing the majority of Man-6-P residues leads to higher steady-state levels of secreted LIF. Using mouse embryonic stem cells, we showed that the mannose phosphorylation of LIF mediates its internalization thereby reducing extracellular levels and stimulating embryonic stem cell differentiation. Finally, immunofluorescence experiments indicate that LIF is targeted directly to lysosomes following its biosynthesis, providing another mechanism whereby mannose phosphorylation serves to control extracellular levels of LIF. Failure to modify LIF in the context of mucolipidosis II and its subsequent accumulation in the extracellular space may have important implications for disease pathogenesis.     

Differential hormonal regulation of leukemia inhibitory factor (LIF) in rabbit and mouse uterus

Leukemia inhibitory factor (LIF) has been shown to be essential for the implantation of mouse blastocysts. The present study was designed to determine how LIF protein was hormonally regulated in rabbit and mouse uterus using immunohistochemistry. In unmated rabbits, LIF protein was at a low level in the uterine epithelium and glands, and up-regulated by progesterone alone or estradiol-17β and progesterone combined. Estradiol-17β alone had no apparent effect. In ovariectomized mice, the level of LIF protein was very low in the uterine epithelium and glands, and was up-regulated by estradiol-17β alone or estradiol-17β and progesterone combined. Progesterone alone had no apparent effect. These results suggest that LIF protein is differentially regulated in rabbit and mouse uterus by progesterone and estrogen, respectively. This would explain the high level of LIF protein observed in uterine epithelium and glands prior to blastocyst implantation in the two species with different hormonal requirements for implantation. © 1996 Wiley-Liss, Inc.     

Cellular cooperation in mitogen-induced human leukocyte inhibitory factor (LIF) synthesis.

Interactions between human T and B lymphocytes and between lymphocyte subpopulations and accessory cells in lymphokine synthesis were investigated. The cells were stimulated with leukoagglutinin (LA), concanavalin A (Con A), protein A (prot A) and anti-β₂-microglobulin (anti-β₂m). The presence of leukocyte inhibitory factor (LIF) in the culture supernatants was tested by the agarose-migration method. The results indicated that monocytes augmented LIF synthesis of T cells but suppressed that of B cells. Monocyte-helper effect was mediated by both cell-cell contact and soluble factors. In addition, T lymphocytes were found to augment B-cell LIF production. B lymphocytes enhanced Con A- but suppressed LA-induced LIF production by T cells. T-cell/B-cell collaboration was based on a direct cell-cell contact and no soluble factors were found.     

LncRNA882 regulates leukemia inhibitory factor (LIF) by sponging miR-15b in the endometrial epithelium cells of dairy goat

Despite the fact that long noncoding RNAs (lncRNAs) play roles in almost all biological processes, little is known about their biological function in the endometrium during the formation of endometrial receptivity. In this study, a comprehensive analysis of lncRNAs in goat endometrial tissues on Day 5 (prereceptive endometrium, PE) and Day 15 (receptive endometrium, RE) of pregnancy was performed by using RNA-Seq. As a result, 668 differentially expressed lncRNAs (DELs) were found between the PE and RE. Further study showed that lncRNA882, regulated by estrogen (E2) and progestin (P4), could act as competing endogenous RNAs (ceRNAs) for miR-15b, which inhibited the expression of transforming growth factor-b-activated kinase 1 binding protein 3 (TAB3) and then indirectly regulated the level of leukemia inhibitory factor (LIF). This was helpful for the formation of endometrial receptivity in dairy goats. In conclusion, we elucidated the endometrium lncRNA profiles of PE and RE in dairy goats; lncRNA882 acted as a ceRNA for miR-15b and then indirectly regulated the level of LIF in goat endometrial epithelium cells. Thus, this study helped us to better understand the molecular regulation of endometrial receptivity in dairy goats.     

Down-regulation of interleukin 6 receptors of mouse myelomonocytic leukemic cells by leukemia inhibitory factor.

We examined the effect of leukemia inhibitory factor (LIF) on the expression of interleukin 6 receptors (IL-6R) on mouse myelomonocytic leukemic M1 cells. Binding studies using 125I-labeled human and murine IL-6 revealed that LIF caused a decrease in IL-6 binding to M1 cells. The decrease became evident within 1 h, and the maximum decrease was observed at 3-6 h. Scatchard plot analysis revealed that M1 cells had a single class of high affinity receptors for IL-6 and that LIF-induced decrease in IL-6 binding was due to a decrease in the number of IL-6R on the cell surface and not to changes in their affinity. The affinity of IL-6R on M1 cells to human IL-6 (Kd = 2.25 nM) was about 10-fold lower than that to murine IL-6 (Kd = 200 pM). The amount of IL-6 secreted into culture media by M1 cells that were treated with LIF for up to 12 h was not enough to cause receptor down-regulation. Northern blot analysis demonstrated that IL-6R mRNA was down-regulated by LIF treatment, and similar regulation was also observed when the cells were treated with IL-6. The time course of the IL-6R mRNA level was similar to that of IL-6R expression on the cell surface, suggesting that the main mechanism responsible for the loss of high affinity IL-6R was the regulation of IL-6R mRNA. Although the half-life of IL-6R on the cell surface was about 30 min, the addition of LIF reduced it to 16 min, suggesting the existence of an additional mechanism responsible for the loss of high affinity IL-6R on the cell surface.     

Cell cycle synchronization of leukemia inhibitory factor (LIF)-dependent porcine-induced pluripotent stem cells and the generation of cloned embryos

Nuclear transfer (NT) from porcine iPSC to create cloned piglets is unusually inefficient. Here we examined whether such failure might be related to the cell cycle stage of donor nuclei. Porcine iPSC, derived here from the inner cell mass of blastocysts, have a prolonged S phase and are highly sensitive to drugs normally used for synchronization. However, a double-blocking procedure with 0.3 μM aphidicolin for 10 h followed by 20 ng/ml nocodazole for 4 h arrested 94.3% of the cells at G₂/M and, after release from the block, provided 70.1% cells in the subsequent G₁ phase without causing any significant loss of cell viability or pluripotent phenotype. Nuclei from different cell cycle stages were used as donors for NT to in vitro-matured metaphase II oocytes. G₂/M nuclei were more efficient than either G₁ and S stage nuclei in undergoing first cleavage and in producing blastocysts, but all groups had a high incidence of chromosomal/nuclear abnormalities at 2 h and 6 h compared with non-synchronized NT controls from fetal fibroblasts. Many G₂ embryos extruded a pseudo-second polar body soon after NT and, at blastocyst, tended to be either polyploid or diploid. By contrast, the few G₁ blastocysts that developed were usually mosaic or aneuploid. The poor developmental potential of G₁ nuclei may relate to lack of a G₁/S check point, as the cells become active in DNA synthesis shortly after exit from mitosis. Together, these data provide at least a partial explanation for the almost complete failure to produce cloned piglets from piPSC.     

Production of a 26,000-dalton interleukin 1 inhibitor by human monocytes is regulated by granulocyte-macrophage colony-stimulating factor

An interleukin 1 (IL 1) inhibitor is secreted into culture medium by a human promyelocytic cell line, H-161, upon stimulation with (PMA) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF). Since the morphological characteristics of this cell line were macrophage-like, human monocytes were tested for their ability to produce similar activity using the same induction conditions. Upon induction of adherent peripheral blood monocytes with rhGM-CSF and/or PMA, an IL 1 antagonistic activity was found in the cell supernatants, as determined by IL 1 receptor binding assay, using the murine EL-4.6.1C10 cell line as the cell target. Most of the inhibition of IL 1 binding induced by PMA or by PMA/rhGM-CSF was shown to be caused by IL 1, since it was neutralized by a mixture of anti-IL 1 alpha/beta antibodies and was active in the murine thymocyte proliferation assay (LAF). The activity induced by GM-CSF alone was not neutralized by anti-IL 1 alpha/beta antibodies and showed no LAF activity. The IL 1 inhibitor activity was induced by rhGM-CSF with a D50 around 40 pg/ml. The activity was produced for more than 3 wk in the presence of GM-CSF; removal of GM-CSF was followed by a rapid decrease of IL 1 antagonistic activity. The specific binding of biosynthetically labeled IL 1 inhibitor to target cells (EL-4.6.1C10) showed a protein of 26 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This molecule shares biological and physical characteristics with the urinary IL 1 inhibitor and the promyelocytic H-161-derived IL 1 inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)