Recombinant Human Leukemia Inhibitory Factor Research Articles

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.

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.

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.

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.

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.

Leukemia inhibitory factor promotes extracellular matrix synthesis in degenerative nucleus pulposus cells via MAPK-ERK1/2 signaling pathway

Extracellular matrix (ECM) anabolism and catabolism imbalance is key feature of chondrocyte and intervertebral disc nucleus pulposus (NP) cell degeneration. The role of LIF as a multifunctional cytokine in the ECM metabolism of chondrocytes is controversial, but no relevant research in the ECM metabolism of NP cells. This study aimed to explore the biofunction and related mechanisms of LIF in the degenerative NP cells. We obtained an increase in the expression of LIF in the human degenerated NP specimens. The addition of recombinant human leukemia inhibitory factor (rhLIF) to the degenerated NP cells cultured in vitro was found to stimulate the synthesis of ECM, and rhLIF could activate the ERK1/2 signaling pathway. However, coculture with PD98059, a signal inhibitor of ERK1/2, blocked the effect of rhLIF on the synthesis of ECM. To furtherly clarify the role of LIF, we carried out animal experiments and found that rhLIF treatment could successfully delay the degree of degeneration of the intervertebral disc in a rabbit model; but with the addition of PD98059, the function of rhLIF for degeneration protection disappeared. In summary, this study demonstrates that LIF plays a role in promoting ECM synthesis in the degenerated NP cells as a protective role in intervertebral disc degeneration (IDD), which is related to the activation of ERK1/2 signaling pathway.

Neuroprotective effect of the recombinant human leukemia inhibitory factor in mice with an experimental cuprizone model of multiple sclerosis: possible mechanisms

Neuroprotective effect of the recombinant human leukemia inhibitory factor in mice with an experimental cuprizone model of multiple sclerosis: possible mechanisms

Prokaryotic Expression and Purification of Recombinant Human Leukemia Inhibitory Factor; Analysis of the Ability to Maintain Pluripotency in Embryonic Stem Cells

Background: Leukemia Inhibitory Factor (LIF) is largely used in stem cell researches for the maintenance of Embryonic Stem Cells (ESCs) in a pluripotent state. However, the relatively high costs of LIF is a potential limit of such researches. Objectives: The aim of this study was prokaryotic expression and purification of the recombinant hexahistidine-tagged human LIF (His6-hLIF) fusion protein and assessment of its ability to maintain a pluripotent or undifferentiated state of ESCs. Methods: Encoding the DNA sequence of mature hLIF was codon optimized for expression in Escherichia coli and chemically synthesized and cloned in the expression vector pET- 28a (+). Immobilized Metal Affinity Chromatography (IMAC) was performed to purify the recombinant His6-hLIF. Then, His6-hLIF was tested for its ability to maintain mESC by comparison with commercial LIF as a control. Results: The yield for the recombinant His6-hLIF was assessed to be approximately 1.7 mg from one liter of culture. There were no statistically significant differences in expression of two pluripotency gene markers, oct-4 and Nanog, between mESCs treated with His6-hLIF and those with commercial hLIF (P = 0.09 and P = 0.13, respectively). Besides, morphological characteristics (round cellular morphology) were similar between them. Conclusions: Collectively, the findings showed that the ability of the recombinant His6-hLIF protein in maintaining pluripotent state of ESCs was comparable to commercial hLIF, providing evidence that the presence of the N-terminal hexahistidin tag does not influence biological activities of hLIF.

Protective Effects of Leukemia Inhibitory Factor on Retinal Vasculature and Cells in Streptozotocin-induced Diabetic Mice.

Background:Leukemia inhibitory factor (LIF) has been reported to possess various pharmacological effects, including displaying vascular and neuroprotective properties, during retinal disease. The aim of this study was to investigate the vascular and structural changes in the retina of diabetic mice and to explore whether LIF prevents experimental diabetes-induced retinal injury in the early stages.Methods:Diabetes was induced in C57Bl/6J mice with streptozotocin (STZ) injections. Successful diabetic animal models were randomly separated into two groups: the diabetic group (n = 15) and the LIF-treated group (n = 15). Normal C57BL/6 mice served as the normal control group (n = 14). Recombinant human LIF was intravitreally injected 8 weeks after the diabetic model was successfully established. Retinas were collected and evaluated using histological and immunohistochemical techniques, and flat-mounted retinas and Western blotting were performed at 18 weeks after the induction of diabetes and 2 days after the intravitreal injection of LIF. The analysis of variance test were used.Results:Histological analysis showed that there were fewer retinal ganglion cells (RGCs) and the inner nuclear layer (INL) became thinner in the diabetic model group (RGC 21.8 ± 4.0 and INL 120.2 ± 4.6 μm) compared with the normal control group (RGC 29.0 ± 6.7, t = −3.02, P = 0.007; INL 150.7 ± 10.6 μm, t = −8.88, P < 0.001, respectively). After LIF treatment, the number of RGCs (26.9 ± 5.3) was significantly increased (t = 3.39, P = 0.030) and the INL (134.5 ± 14.2 μm) was thicker compared to the diabetic group (t = 2.75, P = 0.013). In the anti-Brn-3a-labeled retinas, the number of RGCs in the LIF-treated group (3926.0 ± 143.9) was obviously increased compared to the diabetic group (3507.7 ± 286.1, t = 2.38, P = 0.030), while no significance was found between the LIF-treated group and the control group (4188.3 ± 114.7, t = −2.47, P = 0.069). Flat-mounted retinas demonstrated that a disorganized, dense distribution of the vessel was prominent in the diabetic model group. Vessel distribution in the LIF-treated mouse group was typical and the thickness was uniform. The levels of phosphosignal transducer and activator of transcription 3 activation were obviously higher in the LIF-injected retinas than those in the diabetic control group (t = 3.85, P = 0.019) and the normal control (t = −3.20, P = 0.019).Conclusion:The present study provides evidence that LIF treatment protects the integrity of the vasculature and prevents retinal injury in the early stages of diabetic retinopathy in STZ-induced diabetic models.

Effect of exogenous leukemia inhibitory factor on reparative regeneration of the peripheral nerve in mice

The role of trophic and growth factors in the regeneration of the damaged peripheral nerve is known. The leukemia inhibitory factor (LIF) exhibits properties not only of the polyfunctional cytokine, but also the neurotrophic factor. Regeneration of the peripheral nerve is impaired during oxidative stress in the area of injury.The purpose of the work was to investigate the effect of recombinant human LIF (rhLIF) on the efficacy of reparative regeneration of the sciatic nerve.Materials and methods. Adult male FVB/N mice were divided into the following groups: sham-operated; trauma (neurotomy) of the right sciatic nerve; nerve injury and subcutaneous injection of rhLIF at a dose of 1 μg per animal daily from the third day after the nerve injury. The study was conducted 4 weeks after the injury. At morphometry, the density of nerve fibers in distal nerve segments after impregnation with nitric oxide silver was evaluated. The motor function was evaluated in the “open field” test by the number of crossed squares, and in footprint test by the distance between the extreme toes of the right foot. In the area of muscle injury, the content of malondialdehyde, superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase was estimated.Results. The total number of nerve fibers in the distal segment of the nerve of mice with trauma and injection of rhLIF is greater (p &lt; 0.05) than in mice with only nerve injury and does not differ from sham-operated animals. Horizontal motor activity in mice with nerve injury is lower (p &lt;0.05) than in sham-operated and cytokine injected mice. The distance between the extreme toes of the sham-operated mice is greater (p &lt; 0.05) than in mice with nerve injury, as well as injuries and injections of rhLIF, but after the injection of cytokine, the rates are higher (p &lt; 0.05) than in the group of mice without it. After the injury, the content of malondialdehyde and catalase activity increases in muscle tissue, while the activity of glutathione reductase decreases (p &lt; 0.05). Under the influence of rhLIF, the parameters change to the values of sham-operated mice, and, in addition, the activity of the glutathione peroxidase is significantly increased.Conclusions. Injection of rhLIF to mice with a sciatic nerve injury promotes a more complete restoration of the structural organization of the damaged nerve and improves the motor function of the injured limb. At the same time, the balance between the factors of oxidative stress and antioxidant system is improved in the muscle tissue in the area of the nerve injury.

Expression, purification and characterization of active untagged recombinant human leukemia inhibitory factor from E.coli

Leukemia inhibitory factor (LIF), a member of the IL-6 cytokine family, is considered to be a pleiotropic cytokine and functions in both cell proliferation and differentiation. It is widely used in the culture of mouse embryonic stem cells and is implicated in the implantation of mouse model and possibly in humans. Great efforts have been made on the efficient generation of LIF to meet the requirement of this cytokine in biomedical research. However, because of the low expression level in the eukaryotic system and poor purification yields, recombinant human LIF has usually been expressed either as inclusion body or as fusion protein in E. coli (Escherichia coli). Here we introduce a simple method to express hLIF in a soluble form in E. coli and a subsequent purification method. The expression of hLIF was induced at a low temperature (16 °C) and most of the expressed hLIF was observed to be in a soluble form. Then by using three steps of chromatography, which could be easily scaled-up for industrial purposes, active untagged hLIF was purified with similar bioactivity compared to that of the commercial product. The endotoxin level of purified hLIF protein in our method was determined to be lower than 1EU/μg, which was also comparable to the commercial products. Furthermore, as hLIF was expressed in a soluble form, there was no need to develop the denaturation and renaturation methods. The yield of hLIF protein was evaluated to be approximately 0.7 mg hLIF from 1 g wet weight of E.coli in our method.

Acquisition and Expansion of Adult Rat Bone Marrow Multipotent Mesenchymal Stromal Cells

Abstract This study was initiated in order to test a mini-invasive method of mesenchymal stem/progenitor cells (MS/PCs) isolation from a rat bone marrow (BM), and subsequently their expansion, differentiation, and evaluation of their immunophenotypic characteristics; and later their preservation as donor cells in an optimal condition for potential autotransplantation. The study group comprised of 6 adult male Sprague-Dawley (S-D) rats, weighing 480—690 g. The rats were anaesthetised by isoflurane with room air in a Plexiglas box and maintained by inhalation of a mixture of isoflurane and O2. Their femurs were surgically exposed and their diaphyses double-trephined. Then BM cells were flushed out by saline with heparin and aspirated into a syringe with a solution of DMEM (Dulbecco’s modified eagle’s medium) and heparin. The mononuclear cells from the BM were isolated by centrifugation and expanded in a standard culture medium supplemented with ES-FBS (es-cell-qualified foetal bovine serum), L-glutamine and rh LIF (recombinant human leukemia inhibitory factor). Following 14 days of passaging cultures, the cells were split into 2 equal parts. The first culture continued with the original medium. The second culture received additional supplementation with a human FGFβ (fibroblast growth factor beta) and EGF (epidermal growth factor). The populations of these cells were analysed by light-microscopy, then the mean fluorescence intensities (MFIs) of CD90 and Nestin were evaluated by a tricolour flow cytometry using monoclonal antibodies. The type of general anaesthesia used proved to be appropriate for the surgical phase of the experiments. All rats survived the harvesting of the BM without complications. The total number of mononuclear cells was 1.5—4.0 × 106 per sample and the proportion of CD90/Nestin expressing cells was &lt; 1 %. Following 14 days of expansion, the cells became larger, adherent, with fibrillary morphology; the proportion of cells expressing CD90/Nestin increased to almost 25 %, i. e. they earned basic phenotypic characteristics of MSCs. Throughout the further cultivation a gradual decrease of the CD90/Nestin expression occurred. This suggested that the suitability of rat bone marrow derived MS/PCs for replacement therapy would probably be the highest between days 12—15 of cultivation and then would diminish.

Simplified Method to Produce Human Bioactive Leukemia Inhibitory Factor in Escherichia coli

Background: Human leukemia inhibitory factor (hLIF) is a poly functional cytokine with numerous regulatory effects on different cells. Main application of hLIF is maintaining pluripotency of embryonic stem cells. hLIF indicated effective work in implantation rate of fertilized eggs and multiple sclerosis (MS) treatment. Low production of hLIF in eukaryotic cells and prokaryotic host’s problems for human protein production convinced us to develop a simple way to reach high amount of this widely used clinical and research factor. Objectives: In this study we want to purify recombinant human leukemia inhibitory factor in single simple method. Materials and Methods: This is an experimental study, gene expression: human LIF gene was codon optimized for expression in Escherichia coli and attached his-tag tail to make it extractable. After construction and transformation of vector to E. coli, isopropyl β-D-1-thiogalactopyranoside (IPTG) used for induction. Single step immobilized metal affinity chromatography (IMAC) used for purification confirmed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and western blotting. Bioactivity of the hLIF were tested by MTT assay with TF-1 cells and CISH gene stimulation in monocyte and TF-1 by real-time PCR. Induction by 0.4 mM of IPTG in 25°C for 3 hours indicated best result for soluble expression. SPSS indicated P ˂ 0.05 that is significant for our work. Results: Cloning, expression, and extraction of bio active rhLIF was successfully achieved according MTT assay and real time PCR after treatment of TF-1 and monocyte cell lines. Conclusions: We developed an effective single step purification method to produce bioactive recombinant hLIF in E. coli. For the first time we used CISH gene stimulating for bioactivity test for qualifying of recombinant hLIF for application.

Improvement of pregnancy rate by intrauterine administration of dexamethasone and recombinant human leukemia inhibitory factor at the time of embryo transfer in cattle.

Bovine embryos (day 5) were cultured to day 10 with or without 100 ng/mL PGF2α in medium supplemented with control; 100 nM Dex; 1,000 U/mL recombinant human leukemia inhibitory factor (rhLIF); or Dex+rhLIF. Although the rates to development to the blastocyst were not significantly different among groups, the hatching rate after additional culture with Dex +/or rhLIF was significantly higher in all supplemented groups than the control (p < 0.05). In the presence of PGF2α, the hatching rate was significantly restored in all supplemented groups relative to the group treated with only PGF2α and the control (p < 0.05). Embryo transfer (ET) was performed with blastocysts (day 7). PGF2α levels of control recipient cows were significantly higher in the circulatory blood samples collected 60 min after ET than in samples collected 60 min before ET (p < 0.005), and were decreased in cows injected with loading medium supplemented with Dex+rhLIF (p < 0.005). Pregnancy rate was significantly higher in the ET group that received supplemented embryo-loading medium than in the non-supplemented control (p < 0.05). The intrauterine administration of Dex and rhLIF at ET prevented increased PGF2α in circulatory blood and resulted in enhanced pregnancy rate.

Human leukemia inhibitory factor produced by the ExpressTec method from rice (Oryza sativa L.) is active in human neural stem cells and mouse induced pluripotent stem cells

Stem cell-based therapy has the potential to treat an array of human diseases. However, to study the therapeutic potential and safety of these cells, a scalable cell culture medium is needed that is free of human or bovine-derived serum proteins. Thus, cost-effective recombinant serum proteins and cytokines are needed to produce such mediums. One such cytokine, leukemia inhibitory factor (LIF), has been shown to be a critical paracrine factor that maintains stem cell pluripotency in murine embryonic stem cells and human naïve stem cells while simultaneously inhibiting differentiation. We recently produced recombinant human LIF (rhLIF) in a rice-based protein expression system known as ExpressTec.12 We described expression of rice-derived rhLIF and demonstrated its biological equivalency to E. coli-derived rhLIF in traditional and embryonic mouse stem cell systems. Here we describe the expression yield of rice-derived rhLIF and the scale up production capacity. We provide further evidence of the efficacy of rice-derived rhLIF in additional stem cell systems including human neural stem cells and mouse induced pluripotent stem (iPS) cells. The expression level, biological activity, and potential for production at commercial scale of rice-derived rhLIF provides a proof-of-principal for ExpressTec-derived proteins to produce regulatory-friendly, high performance, and dependable stem cell media.

Application of recombinant human leukemia inhibitory factor (LIF) produced in rice (Oryza sativa L.) for maintenance of mouse embryonic stem cells

Embryonic and induced pluripotent stem cells have the ability to differentiate into any somatic cell type, and thus have potential to treat a number of diseases that are currently incurable. Application of these cells for clinical or industrial uses would require an increase in production to yield adequate numbers of viable cells. However, the relatively high costs of cytokines and growth factors required for maintenance of stem cells in the undifferentiated state have the potential to limit translational research. Leukemia inhibitory factor (LIF), a member of the IL-6 cytokine family, is a key regulator in the maintenance of naïve states for both human and mouse stem cells. In this study, we describe a new recombinant human LIF (rhLIF) using a plant-based (rice) expression system. We found that rice-derived rhLIF possessed the same specific activity as commercial Escherichia coli-derived LIF and was capable of supporting mouse embryonic stem cell proliferation in the undifferentiated state as evidenced from pluripotency marker level analysis. Retention of the pluripotent state was found to be indistinguishable between rice-derived rhLIF and other recombinant LIF proteins currently on the market.

Recombinant leukemia inhibitory factor suppresses human medullary thyroid carcinoma cell line xenografts in mice

Medullary thyroid carcinoma (MTC) is a neoplasm of the endocrine system, which originates from parafollicular C-cells of the thyroid gland. For MTC therapy, the Food and Drug Administration recently approved vandetanib and cabozantinib, multi-kinase inhibitors targeting RET and other tyrosine kinase receptors of vascular endothelial growth factor, epidermal growth factor, or hepatocyte growth factor. Nevertheless, not all patients with the progressive MTC respond to these drugs, requiring the development of additional therapeutic modalities that have distinct activity. Previously, we reported that expression of activated Ras or Raf in the human MTC cell lines, TT and MZ-CRC-1, can induce growth arrest and RET downregulation via a leukemia inhibitory factor (LIF)-mediated autocrine/paracrine loop. In this study, we aimed to evaluate bacterially-produced recombinant human LIF for its efficacy to suppress human MTC xenografts in mice. Here, we report that, consistent with its effects in vitro, locally or systemically administered recombinant LIF effectively suppressed growth of TT and MZ-CRC-1 xenografts in mice. Further, as predicted from its effects in TT and MZ-CRC-1 cell cultures in vitro, recombinant LIF activated the JAK/STAT pathway and downregulated RET and E2F1 expression in tumors in mice. These results suggest that LIF is a potent cytostatic agent for MTC cells, which regulates unique mechanisms that are not targeted by currently available therapeutic agents.