Human Leukemia Inhibitory Factor Research Articles

A mechanistic PK/PD model of AZD0171 (anti-LIF) to support Phase II dose selection.

AZD0171 (INN: Falbikitug) is being developed as a humanized monoclonal antibody (mAb), immunoglobulin G subclass 1 (IgG1), which binds specifically to the immunosuppressive human cytokine leukemia inhibitory factor (LIF) and inhibits downstream signaling by blocking recruitment of glycoprotein 130 (gp130) to the LIF receptor (LIFR) subunit (gp190) and the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and is intended to treat adult participants with advanced solid tumors. LIF is a pleiotropic cytokine (and a member of the IL-6 family of cytokines) involved in many physiological and pathological processes and is highly expressed in a subset of solid tumors, including non-small cell lung cancer (NSCLC), colon, ovarian, prostate, and pancreatic cancer. The aim of this work was to develop a mechanistic PK/PD model to investigate the effect of AZD0171 on tumor LIF levels, predict the level of downstream signaling complex (LIF:LIFR:gp130) inhibition, and examine the dose-response relationship to support dose selection for a Phase II clinical study. Modeling results show that tumor LIF is inhibited in a dose-dependent manner with >90% inhibition for 95% of patients at the Phase II clinical dose of 1500 mg Q2W.

Computer-assisted engineering of stable human leukemia inhibitory factor

Due to their capacity for long-term expansion and differentiation potential, embryonic stem cells (ESC) can be selected as seed cells for generating cultured meat. Leukemia inhibitory factor (LIF) is a pleiotropic cytokine with a wide range of activities, including maintaining ESC in a totipotent state in vitro, which is an essential additive component of the ESC proliferation medium. However, owing to the low thermal stability of wide-type human LIF (hLIF-WT), the cost was increased by repeatedly added and storage loss. Here, we tackle this problem using a computer-assisted protein engineering strategy to create a unique modified hLIF with three mutations displaying unprecedented stability and enhanced biological function. The molecule holds considerable promise for stem cell research and the cultured meat field.

Increased Anti-Inflammatory Therapeutic Potential and Progenitor Marker Expression of Corneal Mesenchymal Stem Cells Cultured in an Optimized Propagation Medium.

There is a huge unmet need for new treatment modalities for ocular surface inflammatory disorders (OSIDs) such as dry eye disease and meibomian gland dysfunction. Mesenchymal stem cell therapies may hold the answer due to their potent immunomodulatory properties, low immunogenicity, and ability to modulate both the innate and adaptive immune response. MSC-like cells that can be isolated from the corneal stroma (C-MSCs) offer a potential new treatment strategy; however, an optimized culture medium needs to be developed to produce the ideal phenotype for use in a cell therapy to treat OSIDs. The effects of in vitro expansion of human C-MSC in a medium of M199 containing fetal bovine serum (FBS) was compared to a stem cell medium (SCM) containing knockout serum replacement (KSR) with basic fibroblast growth factor (bFGF) and human leukemia inhibitory factor (LIF), investigating viability, protein, and gene expression. Isolating populations expressing CD34 or using siRNA knockdown of CD34 were investigated. Finally, the potential of C-MSC as a cell therapy was assessed using co-culture with an in vitro corneal epithelial cell injury model and the angiogenic effects of C-MSC conditioned medium were evaluated with blood and lymph endothelial cells. Both media supported proliferation of C-MSC, with SCM increasing expression of CD34, ABCG2, PAX6, NANOG, REX1, SOX2, and THY1, supported by increased associated protein expression. Isolating cell populations expressing CD34 protein made little difference to gene expression, however, knockdown of the CD34 gene led to decreased expression of progenitor genes. C-MSC increased viability of injured corneal epithelial cells whilst decreasing levels of cytotoxicity and interleukins-6 and -8. No pro-angiogenic effect of C-MSC was seen. Culture medium can significantly influence C-MSC phenotype and culture in SCM produced a cell phenotype more suitable for further consideration as an anti-inflammatory cell therapy. C-MSC show considerable potential for development as therapies for OSIDs, acting through anti-inflammatory action.

298 Interleukin-6 Family Members Improve Pre-Implantation Bovine Blastocyst Composition

Abstract Inner cell mass (ICM) size is a predictor of bovine blastocyst quality and post-transfer pregnancy success. In vitro produced (IVP) embryos have reduced ICM cell numbers compared with in vivo produced embryos, and we propose this is one reason pregnancy success is compromised in IVP embryos. Our recent work found that human recombinant interleukin-6 (IL6) supplementation during in vitro embryo culture increased ICM cell numbers. We hypothesized that other human recombinant members of the IL6 cytokine family, specifically interleukin-11 (IL11) and leukemia inhibitory factor (LIF), will have a similar response. In vitro maturation, fertilization and culture procedures were used to produce embryos that were treated with 100 ng/mL recombinant human recombinant IL6, IL11, LIF, or carrier [control; 1% (wt/vol) SOF-BSA] beginning at day 6 post-fertilization (25 embryos/50 mL drop; 3 drops/treatment; 5 replicates). A subset of day 8 blastocysts (n = 28 to 29) was fixed and processed for cell counting. Analyses were performed by least-squares ANOVA using the general linear model. No treatment differences in overall blastocyst rates, trophectoderm (TE) cell numbers, and total cell numbers were observed at day 8. Treatment with IL6 (P = 0.09) and LIF (P = 0.06) tended to increase ICM cell numbers when compared with the control, whereas IL11 was not different from the control. Treatment with IL6 increased the ICM:TE ratio (P < 0.05) but the other cytokines did not influence this ratio. Several treatment by stage interactions were observed. In regular day 8 blastocysts (n = 10 to 14), none of the treatments affected ICM numbers or the ICM:TE ratio, but LIF increased TE number (P < 0.05) and IL6 increased total cell number (P < 0.05). When grouping expanded and hatched blastocysts together (n = 14 to 18), each treatment increased ICM cell numbers and IL6 increased the ICM:TE ratio (P < 0.05). Furthermore, both IL11 (P = 0.08) and LIF (P = 0.08) tended to increase the ICM:TE ratio. Only LIF increased total cell numbers (P < 0.05). None of the other treatments affected total cell numbers. These observations suggest that IL6, IL11, and LIF are capable of influencing ICM cell numbers in IVP bovine embryos, but they may possess different abilities in affecting TE and overall cell numbers. These findings differ from previous findings from our lab. We suspect that transitioning from bovine recombinant preparations with undefined purity and bioactivity to using purified human recombinant proteins with high bioactivity allowed us to detect these responses for each cytokine. Collectively, this work implicates several members of the IL6 cytokine family as embryokines that may be used to improve bovine IVP embryo quality. Funding for this work was provided by USDA-NIFA (2017-67015-2646 and 2021-67015-34485) and by NIH (R21-OD026516-01).

Leukemia Inhibitory Factor Impairs the Function of Peripheral γδT Cells in Patients with Colorectal Cancer

ABSTRACT Immunotherapeutic strategies are recognized as promising treatment methods for colorectal cancer (CRC). αβT cell-mediated cytotoxicity is tolerated by cancer cells with low MHC class I expression; therefore, γδT cell-based cancer immunotherapy has generated increasing interest as a potential treatment option. To enhance the potency of γδT cell-based immunotherapy, the key factors involved in the regulation of γδT cells in CRC need to be identified along with devising ways to overcome potential hurdles. In this study, we observed that leukemia inhibitory factor (LIF), the serum level of which was highly increased in those with solid tumors, could specifically attenuate the cytotoxic function of peripheral γδT cells in patients with CRC. We observed that in patients with CRC, the expression levels of perforin and granzyme were significantly decreased in the recombinant human LIF (rhLIF)-treated peripheral γδT cells, whereas that of the LIF receptor (LIFR) was higher. The regulation of the cytotoxic function of the γδT cells by rhLIF was effected mainly through the STAT3 signaling pathway, which caused an increase in the expression levels of interleukin (IL)-17, COX-2, and prostaglandin E2 (PGE2). Our results revealed that rhLIF could impair the function of γδT cells in CRC patients by reducing the cytotoxic function and increasing the expression of tumor-promoting molecules, such as IL-17, COX-2, and PGE2.

Cancer-Associated Fibroblasts Regulate the Plasticity of Breast Cancer Stemness through the Production of Leukemia Inhibitory Factor

Simple SummaryCancer-associated fibroblasts (CAFs; as components of cancer stroma) use different signaling pathways to promote tumor progression and growth, invasion, and metastasis in diverse cancers. They have crosstalk with cancer cells during tumor progression. To investigate this crosstalk, in this study, a coculture system of CAF cells isolated from breast cancer patients with breast cancer cells was used to investigate leukemia inhibitory factor (LIF) production from CAFs. LIF is a signaling molecule that activates distinct signaling pathways through which cell cycle progression, cell death, adhesion, migration, and tumorigenesis are regulated.Leukemia inhibitory factor (LIF), as a member of the interleukin-6 cytokine family, plays a complex role in solid tumors. However, the effect of LIF as a tumor microenvironment factor on plasticity control in breast cancer remains largely unknown. In this study, an in vitro investigation is conducted to determine the crosstalk between breast cancer cells and fibroblasts. Based on the results, cancer-associated fibroblasts are producers of LIF in the cocultivation system with breast cancer cells. Treatment with the CAF-CM and human LIF protein significantly promoted stemness through the dedifferentiation process and regaining of stem-cell-like properties. In addition, the results indicate that activation of LIFR signaling in breast cancer cells in the existence of CAF-secreted LIF can induce Nanog and Oct4 expression and increase breast cancer stem cell markers CD24−/CD44+. In contrast, suppression of the LIF receptor by human LIF receptor inhibition antibody decreased the cancer stem cell markers. We found that LIF was frequently overexpressed by CAFs and that LIF expression is necessary for dedifferentiation of breast cancer cell phenotype and regaining of cancer stem cell properties. Our results suggest that targeting LIF/LIFR signaling might be a potent therapeutic strategy for breast cancer and the prevention of tumor recurrence.

CRISPR/Cas9 and Transgene Verification of Gene Involvement in Unfolded Protein Response and Recombinant Protein Production in Barley Grain.

The use of plants as heterologous hosts to produce recombinant proteins has some intriguing advantages. There is, however, the potential of overloading the endoplasmic reticulum (ER) capacity when producing recombinant proteins in the seeds. This leads to an ER-stress condition and accumulating of unfolded proteins. The unfolded protein response (UPR) is activated to alleviate the ER-stress. With the aim to increase the yield of human epidermal growth factor (EGF) and mouse leukemia inhibitory factor (mLIF) in barley, we selected genes reported to have increased expression during ER-induced stress. The selected genes were calreticulin (CRT), protein disulfide isomerase (PDI), isopentenyl diphosphate isomerase (IPI), glutathione-s-transferase (GST), HSP70, HSP26, and HSP16.9. These were knocked out using CRISPR/Cas9 or overexpressed by conventional transgenesis. The generated homozygous barley lines were crossed with barley plants expressing EGF or mLIF and the offspring plants analyzed for EGF and mLIF protein accumulation in the mature grain. All manipulated genes had an impact on the expression of UPR genes when plantlets were subjected to tunicamycin (TN). The PDI knockout plant showed decreased protein body formation, with protein evenly distributed in the cells of the endosperm. The two genes, GST and IPI, were found to have a positive effect on recombinant protein production. mLIF expression was increased in a F2 homozygous GST knockout mutant background as compared to a F2 GST wild-type offspring. The overexpression of IPI in a F1 cross showed a significant increase in EGF expression. We demonstrate that manipulation of UPR related genes can have a positive effect on recombinant protein accumulation.

Differentiation Potential of Cultured Extracellular DEAD-Box Helicase 4+ Oogonial Stem Cells from Adult Human Ovaries into Somatic Lineages

The transdifferentiation potential of human oogonial stem cells (hOSCs) isolated using the antibody against extracellular DEAD-Box Helicase 4 (ecDDX4) remains undetermined. Hence, this study isolated OSCs from ovarian cortical pieces of premenopausal women using ecDDX4 antibody by magnetic activated cell sorting and expanded these cells under embryonic stem cell (ESC)-like culture conditions to inves­tigate their transdifferentiation potential. The number of ecDDX4+ cells obtained was variable in each isolation. When cultured on inactivated mouse embryonic fibroblast feeder layer with human leukemia inhibitory factor (hLIF) and basic fibroblast growth factor (bFGF) in Minimum Essential Medium, the hOSCs aggregated, forming ESC-like colonies. The average size of these cells was around 10 μm. hOSCs in culture were positive for alkaline phosphatase and further formed embryoid bodies (EBs) when grown on low attachment plates containing Essential 6 Medium without hLIF and bFGF. Subsequently, EBs differentiated into 3 germ layers, which were confirmed by staining with beta-III tubulin (TUJ1) for ectoderm, alpha-fetoprotein (AFP) for endoderm, and smooth muscle actin (SMA) for mesoderm. Further, using appropriate induction media, the EBs derived from ecDDX4+ hOSCs were differentiated into somatic lineages such as adipocytes, osteoblasts, cardiomyocytes, and neuronal precursor-like cells, which were confirmed by immunofluorescence using antibodies against specific markers for each cell type. This study corroborated the previous findings that ovaries of adult women possess germ cell progenitors that can be isolated using ecDDX4, and these cells can be manipulated as pluripotent stem cells by culturing them under ESC-like culture conditions akin to their male counterparts, the spermatogonial stem cells. Further, these cells could differentiate into somatic lineages under specific signalling environments.

Cross-reactivity of two human IL-6 family cytokines OSM and LIF explored by protein-protein docking and molecular dynamics simulation

BackgroundOncostatin M (OSM) and leukemia inhibitory factor (LIF) are two important pro-inflammatory cytokines of the interleukin-6 (IL-6) family. The two cytokines mediated signaling was recently found to be closely associated with cancer and chronic inflammation, which represent promising therapeutic targets for the treatment of many solid tumors and inflammatory disease. As the most closely related members, cross-reactivity of them may result in undesired activation of off-target cells, leading to toxicity or lack of efficacy of the therapeutic effects. However, the mechanism of the cross-reactivity of OSM and LIF is not well understood. MethodsIn this work, protein-protein docking, molecular dynamics (MD) simulations with explicit solvent and post endpoints binding free energy (BFE) analysis were carried out to further understand the structural and energetic principles of interactions between the two cytokines and the shared receptor LIFR. ResultsFor the first time, the simulation given a computational model of OSM-LIFR interaction, and provided significant insights into the mechanism of OSM and LIF cross-react with LIFR. The identified common features shared by OSM and LIF bind to LIFR involving 10 “conserved” residues (90% similarity) distributed at the binding site III comprised of AB loop, BC loop and D helix. In addition, 11 shared residues were identified in LIFR contribute 77.85% and 84.63% energies for OSM and LIF binding, which play a critical role in the formation of the two cytokine-receptor complexes. Moreover, the “nonconserved” residues at the same position of cytokines such as Asp41 in OSM and Pro51 in LIF as well as the three residues (Glu338, Asn201 and Glu260) in LIFR were also discovered. ConclusionsThese important information may facilitate the rational design of novel chemical or biological agents with less toxicity and improved efficacy.

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<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>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<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<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).

Efficient production process of bioactive recombinant human leukemia inhibitory factor in Chinese hamster ovary cells

The rhLIF is widely used as an essential factor in stem cell cultures for cell therapies. However, all the recombinant LIFs commercially available are expensive, and no commercially available rhLIF meet the standards recommended by USP for use in cell therapies. The current study reports the efficient production of N-glycosylated and bioactive rhLIF in CHO cells. The production rate of established rhLIF-expressing rCHO cells was approximately 0.85 g/l in 12-day fed-batch cultures using a 7.5 l bioreactor. The rhLIF protein was purified via a four-step purification procedure with approximately 57% recovery rate and greater than 99% purity. The purified rhLIF was N-glycosylated and biologically active with an EC50 of 0.167 ng/ml and a specific activity of 0.86 × 103 IU/mg. The purification procedure controlled the levels of process-related impurities below critical levels recommended by USP for cytokines used in cell therapies. The current work is the first production process of N-glycosylated and bioactive rhLIF, which can be applied to large-scale manufacture of GMP-grade rhLIF for use as an ancillary material in cell therapy.

Contraceptive and molecular function of a novel recombinant vaccine based human leukemia inhibitory factor on Balb/c mice: An experimental in vivo study

The functional competence of leukemia inhibitory factor (LIF), as immunocontraceptive vaccine in mice, was investigated. Balb/c mice were divided into two groups of vaccinated and controls. The recombinant human LIF (rhLIF) protein and phosphate buffer saline was emulsified with Freund's adjuvant and injected into vaccinated and control groups, respectively. Theinhibition of implantation was evaluated in mice uterine. The concentration of secreted interferon-γ (IFN-γ) and interleukin (IL)-4 were measured in cultured splenocyte of mice stimulated by rhLIF. The expressions of immune responsive gene 1 (IRG-1), cochlin (COCH), amphiregulin(Ar), and heparin-binding EGF-like growth factor (HB-EGF) genes were determined. Mice were assessed for inhibition of fertility after delivery, reversibility of immune response against rhLIF, and survival rate. Active immunization of mice with rhLIF resulted in reduction of the implantation and fertility rate up to 80.49% and 75%, respectively. All mice produced a high titer of anti-rhLIF antibodies in serums and vaginal fluids washes after 16 weeks; however, these antibodies were cleared from vaginal fluid washes after six months. A significant down-regulation in mRNA levels of IRG-1, Ar and HB-EGF was observed in vaccinated group compared to controls; however, no significant change in the expression profile of cochlin gene was detected. The results showed that rhLIF prevented pregnancy in a high percentage of female mice. Although the immunization of female Balb/c mice with rhLIF inhibited fertility and expression of genes associated with this molecule, further studies are needed to support this protein as a suitable candidate for contraceptive vaccine in mammals.

Leukemia Inhibitory Factor (LIF) Overexpression Increases the Angiogenic Potential of Bone Marrow Mesenchymal Stem/Stromal Cells.

Mesenchymal stem/stromal cells (MSCs) have the ability to secrete bioactive molecules, exerting multiple biological effects, such as tissue regeneration, reduction of inflammation, and neovascularization. The therapeutic potential of MSCs can be increased by genetic modification to overexpress cytokines and growth factors. Here we produced mouse MSCs overexpressing human leukemia inhibitory factor (LIF) to assess their proangiogenic potential in vitro and in vivo. Mouse bone marrow-derived MSCs were transduced by using a second-generation lentiviral system to express human LIF. Leukemia inhibitory factor expression was confirmed by RT-qPCR and by ELISA, allowing the quantification of the transcript and secreted protein, respectively. Flow cytometry analysis and trilineage differentiation assay showed that the MSC_LIF cell line maintained the immunophenotype and a multipotency characteristic of MSCs. The immunosuppressive activity of MSC_LIF was confirmed using a lymphoproliferation assay. Moreover, gene expression analysis demonstrated upregulation of genes coding for strategic factors in the neovascularization process, such as angiogenin, IL-8, MCP-1, and VEGF, and for the perivascular cell markers αSMA, Col4a1, SM22, and NG2. To evaluate the pro-angiogenic potential of MSC_LIF, we first tested its effects on endothelial cells obtained from umbilical vein in a scratch wound healing assay. Conditioned medium (CM) from MSC_LIF promoted a significant increase in cell migration compared to CM from control MSC. Additionally, in vitro tube formation of endothelial cells was increased by the presence of MSC_LIF, as shown in microvessel sprouting in aortic ring cultures. Finally, an in vivo Matrigel plug assay was performed, showing that MSC_LIF were more potent in promoting in vivo angiogenesis and tissue vascularization than control MSCs. In conclusion, LIF overexpression is a promising strategy to increase the proangiogenic potential of MSCs and sets precedents for future investigations of their potential applications for the treatment of ischemic diseases and tissue repair.

Production and characterization of recombinant human leukemia inhibitory factor and evaluation of anti-fertility effects of rabbit anti-rhLIF in Balb/c mice

Human leukemia inhibitory factor (hLIF) is a cytokine of interleukin-6 family. This study aimed to evaluate the recombinant production rate of active hLIF by different vector-host systems under various conditions. Moreover, a rabbit polyclonal antibody (pAb) against recombinant hLIF (rhLIF) was produced and its anti-fertility effects were explored in Balb/c mice.Four different constructs including pET22b/hLIF, pET28b/hLIF, pET32b/hLIF and pColdI/hLIF were designed and transformed into BL21-(DE3), Rosetta-(DE3), Origami-(DE3) and Shuffle T7-(DE3) host cells. The expression level and proliferative effect of rhLIF were measured by SDS-PAGE and MTT assays, respectively. Rabbit pAb to rhLIF was produced and characterized using enzyme-linked immunosorbent assay and western blot techniques. The Balb/c mice were divided into two intervention and control groups. Then, they were intraperitoneally injected by purified rabbit anti-rhLIF and non-immunized rabbit pAb, respectively. After sacrifice on day 7, the number of implantation sites was counted.The rhLIF was successfully expressed by pET32b/hLIF and pColdI/hLIF vectors in all hosts with no significant difference in the rate of their expression. The rhLIF was purified and checked for activity. The results showed that it is functionally active and the produced anti-rhLIF pAb could specifically bind to commercial rhLIF. Passive immunization results showed that anti-rhLIF antibody completely inhibited fertility in all injected Balb/c mice compared to controls.Although previous studies showed expression of rhLIF using various methods, using different vector-host systems ensures us of successful biological active expression of it. The pAb against rhLIF could be a powerful tool for inducing in vivo infertility.

In Vivo Removal of N-Terminal Fusion Domains From Recombinant Target Proteins Produced in Nicotiana benthamiana.

Plants show great potential for producing recombinant proteins in a cost-effective manner. Many strategies have therefore been employed to express high levels of recombinant proteins in plants. Although foreign domains are fused to target proteins for high expression or as an affinity tag for purification, the retention of foreign domains on a target protein may be undesirable, especially for biomedical purposes. Thus, their removal is often crucial at a certain time point after translation. Here, we developed a new strategy to produce target proteins without foreign domains. This involved in vivo removal of foreign domains fused to the N-terminus by the small ubiquitin-related modifier (SUMO) domain/SUMO-specific protease system. This strategy was tested successfully by generating a recombinant gene, BiP:p38:bdSUMO : His:hLIF, that produced human leukemia inhibitory factor (hLIF) fused to p38, a coat protein of the Turnip crinkle virus; the inclusion of p38 increased levels of protein expression. The recombinant protein was expressed at high levels in the leaf tissue of Nicotiana benthamiana. Coexpression of bdSENP1, a SUMO-specific protease, proteolytically released His:hLIF from the full-length recombinant protein in the endoplasmic reticulum of N. benthamiana leaf cells. His:hLIF was purified from leaf extracts via Ni2+–NTA affinity purification resulting in a yield of 32.49 mg/kg, and the N-terminal 5-residues were verified by amino acid sequencing. Plant-produced His:hLIF was able to maintain the pluripotency of mouse embryonic stem cells. This technique thus provides a novel method of removing foreign domains from a target protein in planta.

Derivation of primitive neural stem cells from human-induced pluripotent stem cells.

Human-induced pluripotent stem cells (hiPSCs) have facilitated studies on organ development and differentiation into specific lineages in in vitro systems. Although numerous studies have focused on cellular differentiation into neural lineage using hPSCs, most studies have initially evaluated embryoid body (EB) formation, eventually yielding terminally differentiated neurons with limited proliferation potential. This study aimed to establish human primitive neural stem cells (pNSCs) from exogene-free hiPSCs without EB formation. To derive pNSCs, we optimized N2B27 neural differentiation medium through supplementation of two inhibitors, CHIR99021 (GSK-3 inhibitor) and PD0325901 (MEK inhibitor), and growth factors including basic fibroblast growth factor (bFGF) and human leukemia inhibitory factor (hLIF). Consequently, pNSCs were efficiently derived and cultured over a long term. pNSCs displayed differentiation potential into neurons, astrocytes, and oligodendrocytes. These early NSC types potentially promote the clinical application of hiPSCs to cure human neurological disorders.

Expression and effects of leukemia inhibitory factor on nucleus pulposus degeneration.

Leukemia inhibitory factor (LIF) is a multifunctional cytokine. The present study aimed to determine the expression and effects of LIF on nucleus pulposus generation. Degenerated nucleus pulposus samples were obtained from animal models and patients with lumbar intervertebral disc herniation. Degradation scores of intervertebral discs were evaluated via magnetic resonance imaging (MRI) and histology, and the protein expression levels of LIF were detected. Furthermore, cultured primary human degenerated nucleus pulposus cells (DNPCs) were stimulated with various concentrations of recombinant human LIF protein (rhLIF), and aggrecan and collagen type II α1 (COL2α1) protein expression levels were detected by western blotting. In addition, aggrecan expression was determined by toluidine blue staining. The effects of rhLIF on proliferation and apoptosis of DNPCs were evaluated by Cell Counting Kit-8 and flow cytometry, respectively. The results revealed that the degradation scores of intervertebral discs were significantly associated with modeling time, as determined by MRI and histology. In addition, the protein expression levels of LIF were initially increased in patients with lumbar disc herniation and in rabbit models, particularly in the 2-week modeling group; however, its expression decreased with the progression of disc degeneration. Notably, LIF expression in each modeling group was higher than that in the control and 0 week modeling group. The in vitro study revealed that the protein expression levels of aggrecan and COL2α1 were significantly increased in response to rhLIF, in a dose-dependent manner, and statistical differences were identified between the treatment groups and control group. The results of toluidine blue staining were consistent with this finding. Although rhLIF had no effect on proliferation, it inhibited apoptosis of DNPCs in a concentration-dependent manner. In conclusion, LIF was upregulated during the process of intervertebral disc degeneration, and may promote the expression of extracellular matrix components. It may also be hypothesized that LIF acts as a potential protective factor by inhibiting apoptosis of DNPCs without affecting cell proliferation.

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<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>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<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).

The study of the remyelinating effect of leukemia-inhibitor factor and melatonin on the toxic cuprizone model of demyelination of murine cerebellar cells culture in vitro

The cuprizone model of toxic demyelination in vitro is widely used to study the of de- and remyelination in the CNS, as well as to address the issues of finding potential compounds that affect myelination of neuron axons.The aim of the study was to investigate the role of recombinant human leukemia inhibitory factor (rhLIF) and melatonin in remyelination, using the cuprizone demyelination model in vitro.Methods. To study the features of the demyelination and remyelination processes of neuronal axons, the culture of dissociated cerebellar cell culture of the 7-day-old FVB/N lineage mice was used. To detect the myelin sheaths, a histochemical staining with a Sudan Black B was used. To identify oligodendrocytes, immunocytochemical staining of 28-30-old-day cerebellar cells cultures for oligodendrocytes marker Olig2 was performed.Results. The direct effect of the demyelinating factor of cuprizone and remyelination agents (rhLIF and melatonin) on oligodendrocytes in vitro was confirmed. The remyelinating effect of LIF and melatonin on the restoration of myelination processes in dissociated cerebellar cell culture using histochemical and immunocytochemical staining has been revealed.Conclusions. Cuprizone-induced demyelination in vitro is associated with the death of Olig2+ oligodendrocytes and loss of myelin formation. rhLIF and melatonin are prevented the loss of oligodendrocytes and, consequently, reduced the destruction of myelin membranes.

Leukemia inhibitory factor inhibits the proliferation of gastric cancer by inducing G1-phase arrest.

Leukemia inhibitory factor (LIF), a member of the interleukin-6 cytokine family, plays a complex role in cancer. LIF inhibits the proliferation and survival of several myeloid leukemia cells but promotes tumor progression and metastasis in many solid tumors. However, the relationship between LIF and gastric cancer has not been well understood. LIF was downregulated in gastric cancer as detected by western blot analysis and immunohistochemistry (IHC). Notably, LIF was downregulated in approximately 70% (56/80) of primary gastric cancers, in which it was significantly associated with advanced clinical stage, lymph node metastasis, and poor overall survival (median 5-year survival = 26 vs. 43 months for patients with high LIF expression and low LIF expression gastric cancer, respectively). To study the potential function of LIF in the downregulation of gastric cancer, we monitored the behavior using proliferation, cell cycle, and flow cytometry analysis. Overexpression of LIF inhibited the gastric cancer cell cycle in the G1 phase. In our experiment, overexpression of LIF by lentivirus upregulated P21 and downregulated cyclin D1. Recombinant human LIF also downregulated P21 and cyclin D1 at various times. A further in vivo tumor formation study in nude mice indicated that overexpression of LIF in gastric cancer significantly delayed the progress of tumor formation. These findings indicate that LIF may serve as a negative regulator of gastric cancer.