Addition Of Leukemia Inhibitory Factor Research Articles

Leukemia inhibitory factor shortens primary cilia by upregulating C-C motif chemokine 2 in human neural stem/progenitor cells.

Primary cilia on neural stem/progenitor cells (NSPCs) play an important role in determining cell fate, although the regulatory mechanisms involved in the ciliogenesis remain largely unknown. In this study, we analyzed the effect of the leukemia inhibitory factor (LIF) for the primary cilia in immortalized human NSPCs. LIF withdrawal elongated the primary cilia length, whereas the addition of LIF shortened it. Microarray gene expression analysis revealed that differentially expressed genes (DEGs) associated with LIF treatment were related with the multiple cytokine signaling pathways. Among the DEGs, C-C motif chemokine 2 (CCL2) had the highest ranking and its increase in the protein concentration in the NSPCs-conditioned medium after the LIF treatment was confirmed by ELISA. Interestingly, we found that CCL2 was a negative regulator of cilium length, and LIF-induced shortening of primary cilia was antagonized by CCL2-specific antibody, suggesting that LIF could influence cilia length via upregulating CCL2. The shortening effect of LIF and CCL2 on primary cilia was also observed in SH-SY5Y cells. The results of the study suggested that the LIF-CCL2 axis may well be a regulator of NSPCs and its primary cilia length, which could affect multiple cellular processes, including NSPC proliferation and differentiation.

Effects of insulin-like growth factor 1, leukemia inhibitory factor, and basic fibroblast growth factor on goat oocyte maturation and early embryonic development in vitro

In this study, we investigated the effects of insulin-like growth factor 1 (IGF-1), leukemia inhibitory factor (LIF), and basic fibroblast growth factor (bFGF) on the in vitro maturation of goat oocytes and early embryonic development of in vitro-fertilized (IVF) oocytes. We also aimed to prepare an efficient in vitro maturation culture system for goat oocytes as a technical basis for improving both the quality of goat oocytes and the efficiency of embryo production in vitro. The experimental results revealed that the addition of bFGF (40 ng/mL), LIF (20 ng/mL), and IGF-1 (20 ng/mL) to the maturation medium improved the maturation rate of goat oocytes. Compared with the control group, the in vitro maturation rate of oocytes (83.0% vs. 70.93%, P < 0.05) and the development rate of IVF blastocysts (45.88% vs. 35.81%, P < 0.05) in the bFGF, LIF, and IGF-1 (FLI) group were significantly increased. The total number of developing blastocysts (114.40 vs. 83.20, P < 0.05) and the ratio of inner cell mass cells (39.50 vs. 25.33, P < 0.05) were also significantly higher in the FLI group than in the control group. Evaluation of maternal gene expression revealed that FLI could significantly increase the expression levels of DNMT1, ZAR1, and NLRP5 and cumulus cell expansion genes PTX3, TNFAIP6, PTGS2, and HAS2 in mature oocytes (P < 0.01). We indirectly proved that bFGF, LIF, and IGF-1 support goat oocyte maturation and early embryonic development. In summary, this study established a reliable and efficient in vitro goat oocyte maturation culture system using bFGF, LIF, and IGF-1. The results of this study can serve as a technical basis for improving the quality of goat oocytes matured in vitro, potential of early embryonic development, IVF of goat embryos, and in vitro production of other livestock.

Leukemia inhibitory factor enhances the development and subsequent blastocysts quality of yak oocytes in vitro.

Leukemia inhibitory factor (LIF) is a multipotent cytokine of the IL-6 family which plays a critical role in the maturation and development of oocytes. This study evaluated the influence of LIF on the maturation and development ability of yak oocytes, and the quality of subsequent blastocysts under in vitro culture settings. Different concentrations of LIF (0, 25, 50, and 100 ng/mL) were added during the in vitro culture of oocytes to detect the maturation rate of oocytes, levels of mitochondria, reactive oxygen species (ROS), actin, and apoptosis in oocytes, mRNA transcription levels of apoptosis and antioxidant-related genes in oocytes, and total cell number and apoptosis levels in subsequent blastocysts. The findings revealed that 50 ng/mL LIF could significantly increase the maturation rate (p < 0.01), levels of mitochondria (p < 0.01) and actin (p < 0.01), and mRNA transcription levels of anti-apoptotic and antioxidant-related genes in yak oocytes. Also, 50 ng/mL LIF could significantly lower the generation of ROS (p < 0.01) and apoptosis levels of oocytes (p < 0.01). In addition, blastocysts formed from 50 ng/mL LIF-treated oocytes showed significantly larger total cell numbers (p < 0.01) and lower apoptosis rates (p < 0.01) than the control group. In conclusion, the addition of LIF during the in vitro maturation of yak oocytes improved the quality and the competence of maturation and development in oocytes, as well as the quality of subsequent blastocysts. The result of this study provided some insights into the role and function of LIF in vitro yak oocytes maturation, as well as provided fundamental knowledge for assisted reproductive technologies in the yak.

Layer-Number-Dependent Effects of Graphene Oxide on the Pluripotency of Mouse Embryonic Stem Cells Through the Regulation of the Interaction Between the Extracellular Matrix and Integrins.

IntroductionEmbryonic stem cells (ESCs) possess great application prospects in biological research and regenerative medicine, so it is important to obtain ESCs with excellent and stable cellular states during in vitro expansion. The feeder layer culture system with the addition of leukemia inhibitory factor (LIF) is currently applied in ESC cultures, but it has a series of disadvantages that could influence the culture efficiency and quality of the ESCs. With the development of nanotechnology, many studies have applied nanomaterials to optimize the stem cell culture system and regulate the fate of stem cells. In this study, we investigated the layer-number-dependent biofunction of graphene oxide (GO) on the pluripotency of ESCs from mice (mESCs).MethodsSingle-layer GO (SGO) and multi-layer GO (MGO) were characterized and their effects on the cytotoxicity and self-renewal of mESCs were detected in vitro. The differentiation potentials of mESCs were identified through the formation of embryoid bodies and teratomas. The regulatory mechanism of GO was verified by blocking the target receptors on the surface of mESCs using antibodies.ResultsBoth SGO and MGO were biocompatible with mESCs, but only MGO effectively sustained their self-renewal and differentiation potential. In addition, GO influenced the cellular activities of mESCs by regulating the interactions between extracellular matrix proteins and integrins.ConclusionThis work demonstrates the layer-number-dependent effects of GO on regulating the cell behavior of mESCs and reveals the extracellular regulatory mechanism of this process.

1 Effect of cytokine-supplemented maturation medium on bovine somatic cell nuclear transfer embryo development

Invitro maturation is an important process in the invitro production of embryos. It has been shown recently that 3 cytokines: fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1), increase the efficiency of IVM, blastocyst production, and invivo development following somatic cell nuclear transfer (SCNT) in pigs (Yuan et al. 2017 PNAS 114, E5796-E5804). This study was designed to assess the effect of these cytokines on IVM in bovine oocytes, their consecutive development to blastocyst, and pregnancy rate when used in SCNT. Cumulus–oocyte complexes (COCs) were retrieved from abattoir-derived ovaries, matured for 21h in either standard maturation medium [TCM-199 (Gibco/Life Technologies), 10% fetal bovine serum, 0.5µgmL−1 FSH, 5µgmL−1 LH, 100UmL−1 penicillin/streptomycin] or maturation medium supplemented with 20ngmL−1 human LIF, 20ngmL−1 IGF1, and 40ngmL−1 FGF2. After IVM, the first polar body and metaphase plate were removed from MII oocytes. Fibroblast cells were injected in the perivitelline space and fused with enucleated oocytes in 0.28M sorbitol fusion medium (0.1mM calcium, 0.5mM magnesium, 0.5mM HEPES, 1gmL−1 bovine serum albumin) by a single pulse of 1.75 kV/cm for 22ms. Reconstructed embryos were activated by exposure to 5μM ionomycin for 5min, followed by 4h of incubation in 2mM 6-(dimethylamino)purine and 10μgmL−1 cycloheximide. Cleavage and blastocyst rates were assessed at Day 2 and Day 7, respectively. Blastocysts were transferred to oestrus synchronized recipients and initial pregnancy rates assessed at Day 40 after embryo transfer. Statistical analysis was performed using one-way ANOVA or chi-squared test. Data are presented as mean±s.e.m. The MII rate was significantly higher in maturation medium supplemented with cytokines compared with control medium (80.2±2.33%, n=885 vs. 66.8±1.82%, n=822; P&amp;lt;0.05, 7 replicates, one-way ANOVA). No statistical difference was found in the cleavage rate of SCNT embryos between treatment and control groups (94.2±1.34%, n=259 vs. 90.9±1.22%, n=208; P&amp;gt;0.05, 8 replicates, one-way ANOVA), respectively. However, a significant increase in blastocyst rate was observed in the treatment group compared with the control group (40.6±5.1%, n=446 vs. 24.3±2.9%, n=300; P&amp;lt;0.05, 8 replicates, one-way ANOVA). SCNT embryos derived from the treatment group also resulted in a significant increase in initial pregnancy rates (50.3±20.9%, n=48 vs. 29.0±20.6%, n=31; P&amp;lt;0.05, 4 replicates, chi-squared). Full-term pregnancy rates are pending. In conclusion, addition of FGF2, LIF, and IGF1 to maturation medium improves bovine IVM and SCNT blastocyst development and initial pregnancy rates. The effect on full-term pregnancy success is yet to be determined. This research was supported by UAES project (1343) and RFBR (18-29-07089).

Human brain microvascular endothelial cells produce CXCL9 after IFN gamma stimulation through JAK2/STAT1 Activation

BackgroundEmergent large vessel occlusion (ELVO) is the deadliest form of stroke and is caused by a blockage within a major a cerebral artery, usually the middle cerebral artery. This condition triggers edema, water movement into the brain, thus pressuring the brain producing massive damage resulting in disability and death. Leukemia inhibitory factor (LIF), a neuroprotective and anti‐inflammatory cytokine, decreases neurodegeneration and increases survival in a rat model of ELVO. Previously we have reported IFNγ is the primary inflammatory mediator in this stroke model and treatment with LIF decreases its expression. This study examined the presence of IFNγ on human endothelial cells by measuring the expression of the IFNγ‐induced CXCL9 and determine LIF's ability to block IFNγ signaling.MethodsFor cells under normoxia conditions, cells were plated and allowed to mature for 7 days, then stimulated with 1 ng/ml IFNγ. At time points 6, 24, 48 and 72 hours after stimulation the supernatant was taken and used for ELISA to determine the amount of CXCL9 released. For OGD conditions, cells were allowed to grow for a week and given glucose free media and placed in oxygen free chamber for 6 hours. The cells were then perfused with media with glucose and 1 ng/ml IFNγ. The supernatant was collected the same time points previously mentioned and CXCL9 was determined as stated previously. To determine the effects inhibitors had on CXCL9 release after being treated with inhibitors to JAK2 and STAT1 under normoxic conditions, cells were treated with IFNy the same as above along coupled with 50 μM and 100 M of fludarabine and AG490.ResultsExposing cerebral microvascular endothelial cells to 1 ng/ml IFNγ significantly induced expression of CXCL9 under both normoxia and oxygen‐glucose deprivation (OGD). Inhibitors to JAK2 and STAT1 significantly blocked the induction of CXCL9. Under normoxic conditions, the addition of LIF (200 ng/ml) with IFNγ significantly augmented the increase in CXCL9 in endothelial cells exposed to OGD.ConclusionCerebral microvascular endothelial cells are a major source of CXCL9, which is upregulated by IFNγ and LIF in a JAK2/STAT1‐dependent manner. The LIF signal transduction pathway in these cells has previously been shown to be dependent upon kinases such as ERK and Akt. CXCL9 is known to break down the blood‐brain barrier, which leads to edema. One potential explanation for this induction of CXCL9 is to open the blood‐brain barrier and allow for anti‐inflammatory leukocytes to enter the ischemic brain and promote repair after stroke.Support or Funding InformationThis project was funded by NINDS Award # 5R0INS091146‐04This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Optimization of In Vitro Culture Conditions of Sturgeon Germ Cells for Purpose of Surrogate Production.

Simple SummaryThe sturgeon is among the most ancient of actinopterygian fishes. Most species of sturgeon are listed as critically endangered due to habitat alteration caused by damming of rivers, pollution and overharvesting. Germ cell transplant is a useful tool to save these endangered species. To expand germ cell populations and sustain the supply for long periods for transplant, we established basal culture conditions for sturgeon germ cells. Germ cell mitotic activity has been enhanced by eliminating gonad somatic cells, supplementing with growth factor and using an alternative to fetal bovine serum. The optimal condition identified was purified germ cells cultured in serum-free medium supplemented with leukemia inhibitory factor (LIF) and glial cell line-derived neurotrophic factor (GDNF) at 21 °C. Cultured sterlet germ cells showed development after transplant into Russian sturgeon. The study provided useful information for sturgeon germ cell culture.To expand germ cell populations and provide a consistent supply for transplantation, we established basal culture conditions for sturgeon germ cells and subsequently increased their mitotic activity by eliminating gonad somatic cells, supplementing with growth factor, and replacing fetal bovine serum (FBS). The initial basal culture conditions were Leibovitz’s L-15 medium (pH 8.0) supplemented with 5% FBS (p < 0.001) at 21 °C. Proliferation of germ cells was significantly enhanced and maintained for longer periods by elimination of gonad somatic cells and culture under feeder-cell free conditions, with addition of leukemia inhibitory factor and glial-cell-derived neurotrophic factor (p < 0.001). A serum-free culture medium improved germ cell proliferation compared to the L-15 with FBS (p < 0.05). Morphology remained similar to that of fresh germ cells for at least 40 d culture. Germline-specific gene expression analysis revealed no significant changes to germ cells before and after culture. Sterlet Acipenser ruthenus germ cells cultured more than 40 days showed development after transplant into Russian sturgeon Acipenser gueldenstaedtii. Polymerase chain reaction showed 33.3% of recipient gonads to contain sterlet cells after four months. This study developed optimal culture condition for sturgeon germ cells. Germ cells after 40 d culture developed in recipient gonads. This study provided useful information for culture of sturgeon germ cells.

Bioenergetic Changes Underline Plasticity of Murine Embryonic Stem Cells.

Murine embryonic stem cells (mESCs) are endowed by a time-dependent window of plasticity during their early commitment steps. Indeed, while mESCs deprived of leukemia inhibitory factor (LIF) for 24 hours revert to their naive pluripotent state after subsequent LIF readdition, cells deprived of LIF for 48 hours are no longer efficient in reverting, upon LIF addition, and undergo irreversible differentiation. We investigated undisclosed bioenergetic profiles of early mESC-derived committed cells versus their undifferentiated states in order to reveal specific bioenergetic changes associated with mESC plasticity. Multiparametric bioenergetic analysis revealed that pluripotent (+LIF) and reversibly committed cells (-LIF24h) are energetically flexible, depending on both oxidative phosphorylation (OXPHOS) and glycolysis. They exhibit high mitochondrial respiration in the presence of the main energetic substrates and can also rely on glycolysis in the presence of OXPHOS inhibitor. Inhibition of the glycolysis or mitochondrial respiration does not change drastically the expression of pluripotency genes, which remain well expressed. In addition, cells treated with these inhibitors keep their capacity to differentiate efficiently upon embryoid bodies formation. Transition from metabolically active mESCs to irreversibly committed cells is associated with a clear change in mitochondrial network morphology, to an increase of adenosine triphosphate (ATP) produced from glycolysis and a decline of ATP turnover and of the mitochondrial activity without change in the mitochondrial mass. Our study pointed that plasticity window of mESCs is associated with the bivalent energetic metabolism and potency to shift to glycolysis or OXPHOS on demand. LIF removal provokes glycolytic metabolic orientation and consecutive loss of the LIF-dependent reversion of cells to the pluripotent state. Stem Cells 2019;37:463-475.

177 Increasing in vitro embryonic development through improved oocyte maturation in cattle oocytes

In vitro maturation (IVM) is an important process in the in vitro production of embryos. It has been recently shown that 3 cytokines: fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1) have increased the efficiency of IVM, blastocyst production, and in vivo development in pig (Yuan et al. 2017 Proc. Natl. Acad. Sci. USA 114, E5796-E5804). In vitro maturation in medium supplemented with cytokines doubled the blastocyst rate and quadrupled the litter size when transferred. It was observed that the addition of cytokines to IVM medium had an effect on the regulation of pMAPK1/3, cumulus cell expansion, and transzonal projections in cumulus-oocyte complexes (COC). This study was designed to assess the effect of these 3 cytokines on IVM in bovine oocytes and their consecutive development to blastocyst. Intracellular glutathione level (GSH), frequently used as an indicator of metaphase II (MII) oocyte quality, was also evaluated. The COC were retrieved from abattoir-derived ovaries and matured for 21h in either our standard maturation medium [TCM-199 (Gibco/Life Technologies, Grand Island, NY, USA), containing 10% fetal bovine serum, 0.5µg mL−1 FSH, 5µg mL−1 LH, and 100U mL−1 penicillin/streptomycin] or maturation medium supplemented with 20ng mL−1 human LIF, 20ng mL−1 human IGF1, and 40ng mL−1 human FGF2. After IVM, COC were placed in fertilization medium and incubated with frozen-thawed sperm for 20h. Cumulus cells were removed from fertilized COC and cultured in SOF culture medium at 38.5°C in 5% CO2/humidified air. Cleavage and blastocyst rates were assessed at 48h and Day 8 post-IVF, respectively. To assess GSH level, MII oocytes were incubated in 20 µM CellTracker Blue CMF2HC (Thermo Fisher Scientific, Waltham, MA, USA) and observed under blue fluorescent light. All statistical analysis was performed using one-way ANOVA and data are presented as mean±s.e.m. The MII rate, assessed by the presence of the first polar body, was significantly higher in the maturation medium supplemented with cytokines compared with the control medium (167/202; 82.4±2.02% v. 136/198; 68.8±1.1%; P&amp;lt;0.05, 4 replicates). For IVF, no statistical difference was found in the cleavage rate between oocytes matured in the medium supplemented with cytokines compared with control medium (351/473; 74.3±4.86% v. 358/573; 63.9±4.03%; P&amp;gt;0.05, 5 replicates), respectively. However, a significant increase in blastocyst rate was observed in the cytokine-containing medium (64/351; 17.7±2.06%) compared with the control group (42/358; 11.0±1.96%; P&amp;lt;0.05, 5 replicates). Furthermore, our preliminary data indicate an increase in GSH in MII oocytes matured in the cytokine-containing medium. In conclusion, the addition of FGF2, LIF, and IGF1 to maturation media improves bovine IVM efficiency and quality of the MII oocytes, leading to a greater blastocyst development rate. Supported by RFBR (18-29-07089) and UAES (1343).

Leukemia Inhibitory Factor Increases Survival of Pluripotent Stem Cell-Derived Cardiomyocytes.

Leukemia inhibitory factor (LIF) is a growth factor with pleiotropic biological functions. It has been reported that LIF acts at different stages during mesoderm development. Also, it has been shown that LIF has a cytoprotective effect on neonatal murine cardiomyocytes (CMs) in culture, but little is known about the role of LIF during human cardiogenesis. Thus, we analyzed the effects of LIF on human pluripotent stem cells (PSC) undergoing cardiac differentiation. We first showed that LIF is expressed in the human heart during early development. We found that the addition of LIF within a precise time window during the in vitro differentiation process significantly increased CMs viability. This finding was associated to a decrease in the expression of pro-apoptotic protein Bax, which coincides with a reduction of the apoptotic rate. Therefore, the addition of LIF may represent a promising strategy for increasing CMs survival derived from PSCs.

Maternal effect gene expression in porcine metaphase II oocytes and embryos in vitro: effect of epidermal growth factor, interleukin-1β and leukemia inhibitory factor.

Maternal effect genes (MEG) play a crucial role in early embryogenesis. In vitro culture conditions may affect MEG expression in porcine oocytes and embryos. We investigated whether in vitro culture medium supplementation with epidermal growth factor (EGF), IL-1β or LIF (leukemia inhibitory factor) affects the mRNA level of ZAR-1 (zygote arrest 1), NPM2 (nucleoplasmin 2) and DPPA3 (developmental associated protein 3) in porcine MII oocytes and embryos. Cumulus-oocyte complexes (COCs) were matured in NCSU-37 medium (control) or in NCSU-37 with EGF 10 ng/ml, IL-1β 10 ng/ml or LIF 50 ng/ml. After maturation for 44-46 h, MII oocytes were preserved for the analysis of MEG mRNA levels (experiment 1). In experiment 2, COCs were fertilized, and the presumptive zygotes were cultured in the same groups. Then, 2-, 4-, 8-cell embryos, morulae and blastocysts were collected for the analysis of MEG mRNA levels. LIF addition to the maturation medium increased MII oocyte numbers (P < 0.05), while EGF and IL-1β did not affect oocyte maturation. Medium supplementation with EGF resulted in lower DPPA3 mRNA levels in MII oocytes and in 2- and 4-cell embryos versus control embryos (P < 0.05). LIF treatment increased DPPA3 mRNA levels in morulae and blastocysts (P < 0.05). Culture with EGF and IL-1β decreased ZAR-1 and NPM2 mRNA levels in 2-cell embryos (P < 0.05). The inclusion of EGF or IL-1β in the porcine in vitro production system influences ZAR-1, NPM2 and DPPA3 mRNA in MII oocytes and embryos but not beyond the 4-cell stage. LIF stimulates oocyte maturation and affects DPPA3 mRNA in porcine morulae and blastocysts in vitro.

Biophysical regulation of mouse embryonic stem cell fate and genomic integrity by feeder derived matrices.

For maintaining pluripotency, mouse embryonic stem cells (mESCs) are typically grown on mitotically inactivated mouse embryonic fibroblasts (MEFs). While the role of MEF conditioned media (MEFCM) and leukemia inhibitory factor (LIF) in regulating mESC pluripotency has led to culturing of mESCs on LIF/MEFCM supplemented gelatin-coated substrates, the role of physical interactions between MEFs and mESCs in regulating mESC pluripotency remains to be fully understood. Here, we address this question by characterizing the physicochemical properties of MEF derived matrices (MEFDMs), and probing their role in regulating mESC fate. We show that MEFDM composition and stiffness-dictated by MEF contractility-regulates mESC pluripotency by modulating mESC contractility through integrin-mediated mechanoadaptation. While baseline mESC pluripotency is maintained at early time points, activation of mESC contractility by LPA leads to drop in pluripotency levels. In contrast, addition of blebbistatin and LIF independently increases pluripotency by suppressing mechanoadaptation, highlighting the role of mechanoadaptation in regulating pluripotency and illustrating the role of LIF as a mechano-inhibitor in mESCs. Long-term culture of mESCs on MEFDMs under LIF-free conditions triggers loss of pluripotency, and induces ligand-dependent expression of the osteogenic transcription factor Runx2. Maintenance of genomic integrity (euploidy) on MEFDMs but not on gelatin-coated substrates, combined with the ability of MEFDMs in supporting LIF-free expansion and differentiation of mESCs, illustrates the suitability of MEFDMs for clinical and regenerative medicine applications.

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.

Novel cell lines isolated from mouse embryonic stem cells exhibiting de novo methylation of the E-cadherin promoter.

Mouse embryonic stem cells (mESCs) and epiblast stem cells represent the naïve and primed pluripotent states, respectively. These cells self-renew via distinct signaling pathways and can transition between the two states in the presence of appropriate growth factors. Manipulation of signaling pathways has therefore allowed the isolation of novel pluripotent cell types such as Fibroblast growth factor, Activin and BIO-derived stem cells and IESCs. However, the effect of cell seeding density on pluripotency remains unexplored. In this study, we have examined whether mESCs can epigenetically regulate E-cadherin to enter a primed-like state in response to low cell seeding density. We show that low density seeding in the absence of leukaemia inhibitory factor (LIF) induces decreased apoptosis and maintenance of pluripotency via Activin/Nodal, concomitant with loss of E-cadherin, Signal transducer and activator of transcription phosphorylation, and chimera-forming ability. These cells, E-cadherin negative proliferating stem cells (ENPSCs) can be reverted to a naïve phenotype by addition of LIF or forced E-cadherin expression. However, prolonged culture of ENPSCs without LIF leads to methylation of the E-cadherin promoter (ENPSC(M)), which cannot be reversed by LIF supplementation, and increased histone H3K27 and decreased H3K4 trimethylation. Transcript analysis of ENPSC(M) revealed a primed-like phenotype and their differentiation leads to enrichment of neuroectoderm cells. The generation of ENPSCs is similar to tumorigenesis as ENPSCs exhibit transcript alterations associated with neoplasia, hyperplasia, carcinoma, and metastasis. We therefore describe a novel cell model to elucidate the role of E-cadherin in pluripotency and to investigate epigenetic regulation of this gene during mESC differentiation and tumor metastasis.

Somatostatin receptor type 2 contributes to the self-renewal of murine embryonic stem cells.

The roles of G-protein coupled receptors (GPCRs) in stem cell biology remain unclear. In this study, we aimed to identify GPCRs that might contribute to the self-renewal of mouse embryonic stem cells (mESCs). The expression levels of pluripotent genes and GPCR gene were detected in E14 mESCs using PCR array and RT-PCR. Immunofluorescent staining was used to examine the expression of pluripotent markers and the receptor translocation. Western blot analysis was used to detect phosphorylation of signal proteins. Knock-down of receptor was conducted to confirm its role in pluripotency maintenance. In leukemia inhibitory factor (LIF)-free medium, mESCs lost the typical morphology of pluripotency, accompanied by markedly decreases in expression of somatostatin receptor type 2 (SSTR2), as well as the pluripotency biomarkers Oct4, Sox2, Rex1 and Nanog. Addition of the SSTR2 agonist octreotide or seglitide (0.1-30 μmol/L) in LIF-free medium dose-dependently promoted the self-renewal of mESCs, whereas the SSTR2 antagonist S4 (0.03-3 μmol/L) dose-dependently blocked octreotide-induced self-renewal. Knock-down of SSTR2 significantly decreased the self-renewal of mESCs even in the presence of LIF. Addition of LIF (1000 U/mL) or octreotide (1 μmol/L) in LIF-free medium significantly increased both phosphorylation and nuclear ocalization of STAT3. The activation of SSTR2 contributes to the self-renewal of mESCs via activation of the STAT3 pathway.

Influence of Basement Membrane Proteins and Endothelial Cell-Derived Factors on the Morphology of Human Fetal-Derived Astrocytes in 2D

Astrocytes are the most prevalent type of glial cell in the brain, participating in a variety of diverse functions from regulating cerebral blood flow to controlling synapse formation. Astrocytes and astrocyte-conditioned media are widely used in models of the blood-brain barrier (BBB), however, very little is known about astrocyte culture in 2D. To test the hypothesis that surface coating and soluble factors influence astrocyte morphology in 2D, we quantitatively analyzed the morphology of human fetal derived astrocytes on glass, matrigel, fibronectin, collagen IV, and collagen I, and after the addition soluble factors including platelet-derived growth factor (PDGF), laminin, basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF). Matrigel surface coatings, as well as addition of leukemia inhibitory factor (LIF) to the media, were found to have the strongest effects on 2D astrocyte morphology, and may be important in improving existing BBB models. In addition, the novel set of quantitative parameters proposed in this paper provide a test for determining the influence of compounds on astrocyte morphology, both to screen for new endothelial cell-secreted factors that influence astrocytes, and to determine in a high-throughput way which factors are important for translation to more complex, 3D BBB models.

Development of a serum‐free defined system employing growth factors for preantral follicle culture

This study was conducted to evaluate if mouse preantral follicles can yield developmentally competent oocytes following culture in serum-free, defined medium. Donor follicles were obtained from 14-day-old B6CBAF1 mice, and cultured in α-MEM-Glutamax medium. The replacement of fetal bovine serum with knockout serum replacement (KSR) did not significantly reduce follicle growth or oocyte maturation in vitro, although it significantly reduced the development of oocytes after activation. Regardless of the replacement medium, follicle growth was not influenced by the addition of leukemia inhibitory factor (LIF). The addition of 100 ng/ml stem cell factor (SCF) to the KSR-supplemented serum-free medium significantly stimulated follicle development, which further improved blastocyst formation after oocyte activation. On Day 3 of culture, a significant increase in Bmp7 expression was detected in the SCF-containing medium compared with the serum-containing medium, whereas Gdf9 and Amh were increased in the serum-containing medium. A significant increase in estradiol production was detected under serum-free conditions, but minimal progesterone secretion was detected throughout the culture period. In conclusion, serum-free media can be used to optimize ovarian follicle cultures, and the addition of SCF is beneficial for deriving developmentally competent oocytes through follicle culture.

Glycoprotein 130 promotes human blastocyst development in vitro

To investigate the efficacy of leukemia inhibitory factor (LIF) and/or glycoprotein 130 (gp130) on in vitro growth of human embryos. Laboratory study. University hospital-based IVF clinic. A total of 164 frozen embryos that survived thawing were cultured in media supplemented with LIF and/or gp130 or control media. Morphological development was evaluated by light microscopy. Protein expression profiles of single blastocysts were evaluated using matrix-assisted laser desorption/ionization time of flight-based intact cell mass spectrometry. Embryo development and protein content. Addition of gp130 to culture media improved blastocyst formation (73% vs. 43%). Addition of LIF to the culture media did not improve embryo development. Protein fingerprint spectra were obtained that revealed significant intensity changes for multiple molecular species including thymosin beta-10, thymosin beta-4, histone H2A, histone H2B, histone H4, ubiquitin, ubiquitin-T, and acyl-CoA binding protein. Glycoprotein 130, but not LIF, seems to be beneficial for preimplantation embryo development, implicating a physiological role in regulating preimplantation development in humans and thus ought to be included in culture media designed for embryo culture to the blastocyst stage. Furthermore, these findings highlight the great potential of matrix-assisted laser desorption/ionization time of flight mass spectrometry and intact cell mass spectrometry as a versatile tool in reproductive medicine research.

Traitement par aide ventilatoire du stridor nocturne dans l’atrophie multisystématisée

The study of germ cell-specific gene regulation in vitro is challenging. Here we report that the promoter of the oocyte-specific gene, Gdf9, is active in a population of cultured murine embryonic stem cells (ES) which have a phenotype resembling oocytes. The promoter region of the murine Gdf9 coupled to enhanced green fluorescent protein (eGFP) was stably transfected into XX mouse ES cells. eGFP was expressed only in oocytes of chimeric mice generated from the transfected XX ES cells. The transfected ES cells were examined when cultured on feeder layers or as embryoid bodies. Large eGFP-positive cells, surrounded by a structure resembling a zona pellucida appeared transiently in cultures of the ES cells on feeder layers. Surprisingly, they were detectable on days 1 and 2 of culture but virtually absent on day 3. Addition of leukemia inhibitory factor (LIF) to the media significantly increased the number of eGFP-positive cels resembling oocytes. Quantitative-time PCR demonstrated a parallel increase in Gdf9 and Zp3 mRNA with changes in the abundance of eGFP-positive cells. In embryoid body cultures, eGFP-positive cells appeared transiently and then re-appeared in regional clusters after 30–45 days of culture. These findings demonstrate that a population of cultured murine ES cells contain the transcriptional machinery to drive expression of an oocyte-specific gene, and that those cells phenotypically resemble oocytes.

300 DEVELOPMENTAL STAGE OF BOVINE BLASTOCYSTS AND THE ADDITION OF LEUKEMIA INHIBITORY FACTOR (LIF) AND BASIC FIBROBLAST GROWTH FACTOR (bFGF) DURING CULTURE IN THE ESTABLISHMENT OF EMBRYONIC STEM CELLS-LIKE COLONIES

The pluripotent stem cells have the capacity to generate cells of 3 germ layers (ectoderm, mesoderm, and endoderm). The greatest example of pluripotent stem cells are the cells of the inner cell mass (ICM) of the blastocyst, called embryonic stem cells (ESC). Several studies have been conducted to determine the best culture conditions of bovine blastocysts for the establishment of ESCbov. It is known that basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF) suppress differentiation in ESC from mice and, but although LIF is important for the growth of ESC in humans, bFGF is not important in preventing differentiation (Prelle et al. 1994 J. Reprod. Fertil.). Because there are no reports in the literature concerning the use of LIF and bFGF in the bovine, the objectives of this study were to use bovine blastocysts in different developmental stages to establish colonies of ESCbov, comparing the action of bFGF and LIF in maintaining the pluripotency of ESCbov during culture. For this, we used bovine blastocysts (IVP) cultured in basal medium with addition of 2.5% FCS; the ICM of early blastocysts, expanded blastocysts, and hatched blastocysts were mechanically removed using 2 insulin needles. The ICM cells were plated in 6 wells containing medium specific for ESC, subdivided into 3 groups: Group 1 (medium + LIF), Group 2 (medium + bFGF), and Group 3 (medium + bFGF + LIF) over a monolayer of mitotically inactivated bovine fibroblasts (20 × 104 cells/1.9 cm2 per well). To confirm the pluripotency of the cells in culture, the colonies were marked with Oct-4, Nanog, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 antibodies. The cells were also immunostained for cytokeratin, α fetoprotein, and vimentin to detect spontaneous differentiation. Adhesion and growth of ICM to the monolayer was observed only when blastocysts (n = 160) were used. This adherence began about 2 days after plating. None of the ICM obtained from early blastocysts (n = 300) resulted in colony formation, and the ICM of hatched blastocysts obtained (n = 45) showed a tendency to detach from the monolayer and form cystic embryoid bodies. The use of LIF was fundamental in the establishment of colonies; however, bFGF was not a significant factor in the cultivation of ESC. The colonies presented imunofluorescence for Oct-4, Nanog, SSEA-3, and TRA-1-81, but were negative for SSEA-1, SSEA-4, and TRA-1-60. The cells in primary culture were negative for α-fetoprotein and vimentin, but some colonies showed positive staining for cytokeratin, demonstrating the contamination of the culture with trophoblastic cells. However, after the first passage, cells presented spontaneous differentiation for mesodermal and endodermal tissues. The addition of LIF and bFGF did not reduce spontaneous differentiation. The expanded blastocysts are the most adequate embryos for derivation and establishment of ESC-like colonies in the bovine. The addition of LIF is important for maintaining cells in culture; however, neither LIF nor bFGF prevented spontaneous differentiation after the first passage.