OBJECTIVE: To establish the STO cell lines expressing green fluorescent protein (GFP) and mouse leukemia inhibitory factor (LIF) , and try to culture the mouse embryonic stem cells (mESCs) by using the established STO-GFP-mLIF cells as the feeder layer. METHODS: The lentiviral particles containing GFP and mLIF and puromycin-resistance gene were constructed and transduced into STO cell lines. The cell lines stably expressing GFP and mLIF genes were screened out. The expression level of the inserted exogenous LIF gene was tested by Western blot and ELISA. The STO-GFP-mLIF cells were treated with different concentrations of mitomycin C (5, 10, 15, 20 µg/ml) for different time (1.5, 2.5, 3, 3.5 hours) to prepare feeder layers and the cell proliferation level on feeder layer was observed. Mouse embryonic stem cells were cultured on mitomycin C-treated feeder layer and the growth of cell colonies was observed. RESULTS: The expression level of LIF protein in STO-GFP-mLIF cells was up-regulated, as compared with STO cells (P＜0.05). It was confirmed that the optimal concentration and time for inhibiting the proliferetion of STO-GFP-mLIF cells by mitomycin C were 10 µg/ml and 3 hours respectively. The observation also found that the embryonic stem cells could develop into typic "birdnest" shaped stem cell colony on mitomycin C-treated feeder layer. CONCLUSION The stable STO cell lines effectively expressing green fluorescent protein and mouse leukemia inhibitory factor have been established successfully, which can maintain the undifferentiated state of mouse embryonic stem cells.
Animals ; Cell Differentiation ; Cell Line ; Cell Separation ; Embryonic Stem Cells ; Feeder Cells ; Green Fluorescent Proteins ; Leukemia Inhibitory Factor ; Mice

OBJECTIVETo investigate the expression and clinical significances of HGFA, Matriptase, HAI-1 and HAI-2 in patients with acute myeloid leukemia (AML).

METHODSThe bone marrow samples from 91 AML patients, 41 AML patients in complete remission, and 32 normal controls were collected. Real time fluorescence quantitative RT-PCR (qRT-PCR) was used to detect the mRNA expressions levels of HGFA, Matriptase, HAI-1, HAI-2 . The expressions of these genes were compared among AML untreated group, the complete remission group and the healthy control group. The correlation of their expression with clinical characteristics was analyzed.

RESULTSThe level of HGFA in the AML untreated group was higher than that in the healthy control group（P<0.05）, while the HAI-2 mRNA level was lower than that in the healthy control group（P<0.05）. The mRNA levels of HAI-1 and Matriptase were not changed significantly in all groups. The HAI-2 mRNA expression level was significantly lower in the high white blood cell group (P<0.05).

CONCLUSIONThe abnormal activation of HGF/c-Met signaling system in AML may result from the increase of HGFA expression and the decrease of HAI-2 expression of the upstream regulatory factors.

Hepatocyte Growth Factor ; Humans ; Leukemia, Myeloid, Acute ; Membrane Glycoproteins ; Proteinase Inhibitory Proteins, Secretory ; Serine Endopeptidases

OBJECTIVETo study the effects of LIF combined with bFGF on the proliferation, stemness and senescence of hUC-MSC.

METHODSExperiments were divided into 4 groups: control group, in which the cells were treated with complete medium (α-MEM containing 10% FBS); group LIF, in which the cells were treated with complete medium containing 10 ng/ml LIF; group bFGF, in which the cells were treated with complete medium containing 10 ng/ml bFGF; combination group, in which the cells were treated with complete medium containing 10 ng/ml LIF and 10 ng/ml bFGF. The growth curves of hUC-MSC at passage 4 in different groups were assayed by cell counting kit 8. Cellular morphologic changes were observed under inverted phase contrast microscope; hUC-MSC senescence in different groups was detected by β-galactosidase staining. The expression of PCNA, P16, P21, P53, OCT4 and NANOG genes was detected by RT-PCR.

RESULTSThe cell growth curves of each group were similar to the S-shape; the cell proliferation rate from high to low as follows: that in the combination group > group bFGF > group LIF > control group. Senescence and declining of proliferation were observed at hUC-MSC very early in control group; the cells in group LIF maintained good cellular morphology at early stage, but cell proliferation was slow and late senescence was observed; a few cells in group bFGF presented signs of senescence, but with quick proliferation; the cells in combination group grew quickly and maintained cellular morphology of hUC-MSC for long time. The LIF and bFGF up-regulated the expression of PCNA, OCT4 and NANOG, while they down-regulated the expression of P16, P21, P53, and their combinative effects were more significant.

CONCLUSIONLIF combined with bFGF not only can promote the proliferation and maintenance of stemness of hUC-MSC, but also can delay the senescence of hUC-MSC.

Cell Cycle ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Fibroblast Growth Factor 2 ; pharmacology ; Genes, Homeobox ; Humans ; Leukemia Inhibitory Factor ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Octamer Transcription Factor-3 ; metabolism ; Organic Chemicals ; Proliferating Cell Nuclear Antigen ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; Umbilical Cord ; cytology

OBJECTIVETo investigate effects of retinol on the expressions of epidermal growth factor (EGF), stem cell factor (SCF), colony-stimulating factor 1 (CSF1) and leukemia inhibitory factor (LIF) in cultured human umbilical-derived mesenchymal stem cells (UCMSCs).

METHODSHuman UCMSCs were isolated from human umbilical cord and identified for immunophenotypes. The cells were then cultured in DMEM/F12 media supplemented with 12% fetal bovine serum (FBS), 12% FBS+1 µmol/L retinol, 15% knockout serum replacement (KSR) and 15% KSR+ 1 µmol/L retinol. The expressions of the cytokines EGF, SCF, CSF1 and LIF in the cells were detected using RT-PCR and ELISA.

RESULTSThe isolated cells exhibited characteristic immunophenotypes of human UCMSCs and expressed EGF, CSF1 and SCF at both mRNA and protein levels but not LIF protein. Retinol (1 µmol/L) significantly promoted the expressions of SCF and CSF1 at both mRNA and protein levels but did not result in changes of EGF and LIF expressions in human UCMSCs.

CONCLUSIONRetinol at the concentration of 1 µmol/L can promote expression of SCF and CSF1 in human UCMSCs in vitro.

Cell Differentiation ; Cells, Cultured ; EGF Family of Proteins ; metabolism ; Humans ; Immunophenotyping ; Leukemia Inhibitory Factor ; metabolism ; Macrophage Colony-Stimulating Factor ; metabolism ; Mesenchymal Stromal Cells ; drug effects ; metabolism ; Stem Cell Factor ; metabolism ; Umbilical Cord ; cytology ; Vitamin A ; pharmacology

OBJECTIVETo screen the differentially expressed miRNAs and their target genes in adipogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs) to better understand the mechanism for regulating the balance between osteoblast and adipocyte differentiation.

METHODSCultured hMSCs were induced for adipogenic differentiation, and at 0, 7, 14, and 21 days of induction, the cells were examined for miRNA and mRNA expression profiles using miRNA chip and transcriptome sequencing (RNA-seq) techniques. Correlation analysis was carried out for the miRNAs and mRNAs of potential interest. The databases including TargetScan, PicTar and miRanda were used to predict the target genes of the differentially expressed miRNA.

RESULTSThe expression of miR-140-5p was down-regulated and leukemia inhibitory factor receptor (LIFR) expression increased progressively during adipogenic differentiation of hMSCs, showing a negative correlation between them. Target gene prediction using the 3 databases identified LIFR as the target gene of miR-140-5p.

CONCLUSIONmiRNA-140-5p may play an important role by regulating its target gene LIFR during adipogenic differentiation of hMSCs.

Adipocytes ; cytology ; Adipogenesis ; Cell Differentiation ; Cells, Cultured ; Down-Regulation ; Humans ; Leukemia Inhibitory Factor Receptor alpha Subunit ; metabolism ; Mesenchymal Stromal Cells ; cytology ; MicroRNAs ; genetics ; Oligonucleotide Array Sequence Analysis ; Osteoblasts ; cytology ; RNA, Messenger ; Transcriptome

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.
Animals ; Blastocyst ; Cattle* ; Dexamethasone* ; Embryo Transfer* ; Embryonic Structures* ; Fertilization in Vitro ; Humans* ; Leukemia Inhibitory Factor* ; Leukemia* ; Pregnancy Rate* ; Pregnancy* ; Prostaglandins F

OBJECTIVETo investigate the effect of macrophages on embryo implantation by observing the distribution of macrophages in mouse uterine tissues during the peri-implantation period.

METHODSUterine tissues were collected from pregnant (n=30) and pseudopregnant mice (n=30) during the peri-implantation period. The distributions of macrophages, iNOS and leukemia inhibitory factor (LIF) were determined by immunohistochemistry and the correlations of macrophages with iNOS and LIF were analyzed.

RESULTSMacrophages were located mainly in the endometrium before D4.5 in the pregnant rats with D0.5 defined as the morning when a vaginal plug was observed. After D4.5, the macrophages was significantly reduced in number (P<0.05) in the endometrium and gradually migrated to the perimetrium. In the psudopregnant mice, macrophages were located mainly in the endometrium. Before D4.5, iNOS-positive cells were detected mainly in the endometrium and the myometrium in the pregnant rats and became significantly reduced on D4.5 (P<0.05); in the pseudopregnant mice, the positive cells were mostly detected in the endometrium. Significant differences were found in the distribution of the macrophages and LIF between the implantation and non-implantation sites (P=0.013). LIF was mostly located in the endometrium in the pregnant mice but scarcely detected in the pseudopregnant mice.

CONCLUSIONMacrophages are located mainly in the endometrium and the implantation site where iNOS and LIF are expressed, suggesting the important role of macrophages in the determination of implantation.

Animals ; Blood Cell Count ; Embryo Implantation ; Endometrium ; cytology ; Female ; Immunohistochemistry ; Leukemia Inhibitory Factor ; metabolism ; Macrophages ; cytology ; Mice ; Nitric Oxide Synthase Type II ; metabolism ; Pregnancy ; Uterus ; cytology

PURPOSE: To investigate the effect of macrophage migration inhibitory factor (MIF) on corneal sensitivity after laser in situ keratomileusis (LASIK) surgery. METHODS: New Zealand white rabbits were used in this study. A hinged corneal flap (160-microm thick) was created with a microkeratome, and -3.0 diopter excimer laser ablation was performed. Expressions of MIF mRNA in the corneal epithelial cells and surrounding inflammatory cells were analyzed using reverse transcription polymerase chain reaction at 48 hours after LASIK. After LASIK surgery, the rabbits were topically given either 1) a balanced salt solution (BSS), 2) MIF (100 ng/mL) alone, or 3) a combination of nerve growth factor (NGF, 100 ug/mL), neurotrophine-3 (NT-3, 100 ng/mL), interleukin-6 (IL-6, 5 ng/mL), and leukemia inhibitory factor (LIF, 5 ng/mL) four times a day for three days. Preoperative and postoperative corneal sensitivity at two weeks and at 10 weeks were assessed using the Cochet-Bonnet esthesiometer. RESULTS: Expression of MIF mRNA was 2.5-fold upregulated in the corneal epithelium and 1.5-fold upregulated in the surrounding inflammatory cells as compared with the control eyes. Preoperative baseline corneal sensitivity was 40.56 +/- 2.36 mm. At two weeks after LASIK, corneal sensitivity was 9.17 +/- 5.57 mm in the BSS treated group, 21.92 +/- 2.44 mm in the MIF treated group, and 22.42 +/- 1.59 mm in the neuronal growth factors-treated group (MIF vs. BSS, p < 0.0001; neuronal growth factors vs. BSS, p < 0.0001; MIF vs. neuronal growth factors, p = 0.815). At 10 weeks after LASIK, corneal sensitivity was 15.00 +/- 9.65, 35.00 +/- 5.48, and 29.58 +/- 4.31 mm respectively (MIF vs. BSS, p = 0.0001; neuronal growth factors vs. BSS, p = 0.002; MIF vs. neuronal growth factors, p = 0.192). Treatment with MIF alone could achieve as much of an effect on recovery of corneal sensation as treatment with combination of NGF, NT-3, IL-6, and LIF. CONCLUSIONS: Topically administered MIF plays a significant role in the early recovery of corneal sensitivity after LASIK in the experimental animal model.
Animals ; Epithelium, Corneal/*drug effects/innervation/physiology ; Female ; Humans ; Interleukin-6/pharmacology ; Keratomileusis, Laser In Situ/*methods ; Leukemia Inhibitory Factor/pharmacology ; Macrophage Migration-Inhibitory Factors/genetics/*pharmacology ; Models, Animal ; Nerve Growth Factor/pharmacology ; Nerve Regeneration/*drug effects/physiology ; Neurotrophin 3/pharmacology ; RNA, Messenger/metabolism ; Rabbits ; Recovery of Function/*drug effects/physiology ; Sensation/*drug effects/physiology

OBJECTIVETo study the changes of endogenous leukemia inhibitory factor (LIF) in neonatal rats with periventricular leukomalacia (PVL).

METHODSA PVL model of 3-day-old Wistar rats was prepared by left carotid artery ligation followed by 6% oxygen for 4 hours. The rats were sacrificed at 1, 3, 7, 14 and 28 days of hypoxia ischemia (HI), and the brain tissues were sampled. Real-Time PCR and Western blot methods were applied to analyze the expression of LIF mRNA and protein. Double staining immunofluorescence was used to detect the co-expression of LIF and GFAP.

RESULTSAt 1, 3 and 7 days of HI, LIF protein level in the PVL group was higher than in the control group (P<0.01). In the PVL group, the LIF protein level on the third day after HI reached a peak and was higher than the other time points (P<0.01). The change of LIF mRNA expression showed the same tendency with LIF protein. The double staining immunofluorescence showed a co-expression of LIF and GFAP.

CONCLUSIONSLIF mRNA and LIF protein expression in astrocytes show a trend of initial increase followed by steady decline in neonatal rats with PVL, suggesting that endogenous LIF may participate in the repair of PVL.

Animals ; Animals, Newborn ; Disease Models, Animal ; Female ; Glial Fibrillary Acidic Protein ; analysis ; Leukemia Inhibitory Factor ; analysis ; genetics ; physiology ; Leukomalacia, Periventricular ; metabolism ; pathology ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar

BACKGROUND: Because of the heterogeneity of human mesenchymal stem cells (MSCs), methods for cell expansion in culture and the effects on gene expression are critical factors that need to be standardized for preparing MSCs. We investigated gene expression patterns of MSCs with different seeding densities and culture times. METHODS: Bone marrow-derived MSCs were plated at densities from 200 cells/cm2 to 5,000 cells/cm2, and the gene expression patterns were evaluated over time using a reverse-transcription polymerase chain reaction assay. RESULTS: The mRNA levels of factors that play a critical role in cell migration and tissue regeneration, such as podocalyxin-like protein (PODXL), alpha4-integrin, alpha6-integrin, and leukemia inhibitory factor (LIF), were higher in MSCs plated at 200 cells/cm2 than in MSCs plated at 5,000 cells/cm2. The mRNA levels of these factors gradually increased for 10 days and then decreased by day 15 in culture. MSCs seeded at 200 cells/cm2 that were cultured for 10 days expressed high levels of Oct-4 and Nanog. Indoleamine 2,3-dioxygenase, cyclooxygenase-1, and hepatocyte growth factor expression were upregulated in the presence of the proinflammatory cytokine interferon-gamma in these cells. CONCLUSION: We found differences in the gene expression patterns of MSCs under different culture conditions. MSCs from 10-day cultures seeded at a low density were efficiently expanded, expressed PODXL, alpha6-integrin, alpha4-integrin, and LIF, and maintained properties like stemness and immunomodulation. Therefore, ex vivo expansion of MSCs maintained for an adequate culture time after plating at low cell density can provide an effective regenerative medicinal strategy for cell therapies using MSCs.
Cell Count ; Cell Movement ; Cyclooxygenase 1 ; Gene Expression ; Hepatocyte Growth Factor ; Humans ; Immunomodulation ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; Interferon-gamma ; Leukemia Inhibitory Factor ; Mesenchymal Stromal Cells ; Polymerase Chain Reaction ; Population Characteristics ; Regeneration ; RNA, Messenger ; Seeds ; Tissue Therapy