The aim of this study was to investigate the feasibility of the human cord blood adult stem cells (ASCs) to differentiate into hepatocytes in vitro induced by combined stimulation with hepatocyte growth factor (HGF), stem cell factor (SCF) and leukemia inhibitory factor (LIF). The adult stem cells were obtained through density gradient centrifugation and magnetic activated cell sorting (MACS). The adult stem cells were cultured in DMEM with HGF (10 ng/ml)+SCF (10 ng/ml)+LIF (10 ng/ml) in induced group I. In induced group II the enriched cells were cultured in DMEM with SCF (10 ng/ml)+LIF (10 ng/ml) and the undifferentiated cells acted as the control group without the factors. The morphology of cells was observed by the inverted phase contrast microscopy; the expression of albumin (Alb), human hepatocyte cytokeratin (CK18) and alpha-fetoprotein (AFP) were detected by immunofluorescence, immunohistochemistry and RT-PCR assay in the 21-day culture. Alb secreted by hepatocytes in the medium was determined by radioimmunoassay (RIA) at day 7, 14, 21, 23 and 25. The results showed that the shapes of ASCs changed and their sizes and number increased in the course of culture in group I. After being induced for three weeks, the cells turned round and resembled hepatocyte-like cells. The mRNA for Alb could be detected by RT-PCR in the differentiated adult stem cells in group I, and the mRNA for AFP was poorly detected by RT-PCR at day 21. Alb and CK18 were positive through immunofluorescence and immunohistochemistry at day 21, compared with group II and the control group. In group I, Alb in the medium significantly increased, compared with control group, and reached the highest level at day 21, then decreased at day 23. It is concluded that under some definite inducing conditions, human cord blood adult stem cells can differentiate into hepatocyte-like cells and HGF plays a critical role during the course.
Adult Stem Cells ; cytology ; Cell Differentiation ; physiology ; Cells, Cultured ; Fetal Blood ; cytology ; Hepatocyte Growth Factor ; pharmacology ; Hepatocytes ; cytology ; Humans ; Leukemia Inhibitory Factor ; pharmacology

In order to observe the effect of Bushenantai recipe on the expression of endometrial leukemia-inhibitory factor (LIF) in mice with embryonic implantation dysfunction (EID), 120 Kunming mice post coition were randomized into three groups: normal control group, model group and traditional Chinese medicine group (TCM group) (n=40 in each group). Uterus was collected on the pregnancy day (Pd) 4, 5, 6 after an intravenous injection of Evan's blue. The endometrium was dyed by Evan's blue and the mean points of response were observed on Pd 5. The expression of LIF mRNA and protein was detected by RT-PCR and immunohistochemistry respectively and analyzed statistically by image system. The results showed that the number of implantation sites in model group was remarkably less than in normal control group and TCM group. There was no significant difference between normal control group and TCM group. The expression of LIF mRNA and protein in model group was delayed. Bushenantai recipe could increase the expression of LIF mRNA and protein in endometria of mice with EID. It was suggested that Bushenantai recipe could improve embryo implantation of mice with EID by promoting the endometrial LIF expression and endometrial decidualization.
Blastocyst/cytology ; Embryo Implantation ; Endometrium/*metabolism ; Gene Expression ; Gene Expression Regulation, Developmental ; Leukemia Inhibitory Factor/*biosynthesis ; Leukemia Inhibitory Factor/*genetics ; Medicine, Chinese Traditional ; Models, Biological ; Plant Extracts/pharmacology ; RNA, Messenger/metabolism ; Time Factors

OBJECTIVETo search for the optimal approach for hepatocyte-directed differentiation of hepatic progenitor cells and investigate the molecular mechanism of the hepatic differentiation.

METHODSHepatic progenitor cells were infected with recombinant adenovirus which containing human LIF, BMP2 or BMP9 gene. The maturation and differentiation of progenitor cells were examined by PAS staining and ICG uptake methods at 4, 7 and 10 days post infection. The production of Albumin (Alb) was measured by luciferase activity at day 4, 7, 10 and 14.

RESULTSPAS staining assay revealed that BMP2 and BMP9 enhanced glycogen storage in hepatic progenitor cells most obviously at day 7. The percentages of positive cells were 30% and 45% respectively at 7 days post-infection. Meanwhile, 40% and 30% cells were positive by ICG uptake assay after BMP2 and BMP9 induction. Luciferase activity indicated that BMP9 induced ALB-Luc activity most significantly at day 7. However, less inductive activity was found in LIF-treated group.

CONCLUSIONThese results indicated tuat hepatic progenitor cells were differentiated into hepatocyte-like cells by BMPs and LIF induction.

Adenoviridae ; Bone Morphogenetic Proteins ; pharmacology ; Cell Differentiation ; Cells, Cultured ; Hepatocytes ; cytology ; metabolism ; virology ; Humans ; Leukemia Inhibitory Factor ; pharmacology ; Stem Cells ; cytology ; metabolism ; virology

OBJECTIVETo study the effects of growth factors on the survival and proliferation of human spermatogonial stem cells (SSCs) in vitro.

METHODSSSCs were treated with the growth factors SCF, LIF and bFGF added to the culture, each at the concentrations of 0, 5, 10 and 20 microg/L and repeated three times. The survival time and proliferation rate of the cells were determined every 8-12 hours and their morphological features observed with the light microscope and electron microscope.

RESULTSThe survival time and proliferation rate of the SSCs were significantly increased in the treated groups as compared with the control (P < 0.05).

CONCLUSIONThe growth factors SCF, LIF and bFGF can promote the survival and proliferation of SSCs in vitro.

Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Fibroblast Growth Factor 2 ; pharmacology ; Humans ; Intercellular Signaling Peptides and Proteins ; pharmacology ; Leukemia Inhibitory Factor ; pharmacology ; Male ; Spermatogonia ; cytology ; drug effects ; Stem Cell Factor ; pharmacology ; Stem Cells ; cytology ; drug effects

OBJECTIVETo study the effects and underlying mechanisms of Zhuyun Recipe (ZR) on the endometrial receptivity in ovarian stimulation (OS) and blastocyst implantation dysfunction (BID) mice.

METHODSTotally 200 normal female Kunming mice were randomly divided into 6 groups, i. e., the control group (Group A), the OS group (Group B), the OS + ZR group (Group C), the BID group (Group D), the BID + ZR group (Group E), and the ZR group (Group F). The pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG) were intraperitoneally injected to mice in Group B. Mifepristone was subcutaneously injected to mice in Group D at 9:00 am on the 4th gestation day. Corresponding medications were given to mice in Group C, E, and F at 1.5 mL/100 g by gastrogavage at 8:00 am from the first to the 4th gestation day. Eight uterus samples were collected at 9:00 pm on the 4th gestation day and fixed. The expression levels of leukemia inhibitory factor (LIF) and integrin beta3 were detected using immunohistochemical assay. The pregnant mice were sacrificed at 9:30 pm on the 8th gestation day, and their uterus were taken out. The number of blastocysts was counted.

RESULTSCompared with Group A, the pregnant rate was 6.67% (1/15 cases) in Group B and 18.75% (3/16 cases) in Group D, the mean OD value of LIF was 0. 18 +/- 0.02 in Group B and 0.23 +/- 0.02 in Group D, and the mean OD value of integrin beta3 was 0.20 +/- 0.05 in Group B and 0.19 +/- 0. 02 in Group D, showing statistical difference (P < 0.01). The pregnant rate was 54.55% (12/22 cases) in Group C and 65. 22% (15/23 cases) in Group E, the mean OD value of LIF was 0.37 +/- 0. 09 in Group C and 0.39 +/- 0.02 in Group E, and the mean OD value of integrin beta3 was 0.34 +/- 0.04 in Group C and 0.38 +/- 0.08 in Group E, showing statistical difference when compared with those of Group B and Group D respectively (P < 0.05).

CONCLUSIONSOS and BID had negative effects on the endometrial receptivity and hindered the blastocyst implantation. ZR could improve the uterine receptivity and elevate the pregnant rate by up-regulating the expressions of endometrial LIF and integrin beta3.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Embryo Implantation ; drug effects ; Endometrium ; drug effects ; physiology ; Female ; Integrin beta3 ; metabolism ; Leukemia Inhibitory Factor ; metabolism ; Mice ; Mice, Inbred Strains ; Ovulation Induction ; Pregnancy

Undifferentiated embryonic stem (ES) cells can be maintained in vitro if cultured in the presence of the cytokine leukaemia inhibitory factor (LIF). ES cells can also differentiate in vitro. A particularly efficient method for inducing ES cell differentiation is to culture ES cells as aggregates in the absence of LIF. Under these conditions they form structures known as embryoid bodies (EBs). However the current protocols for EB formation are still diverse. In order to facilitate further study, we carefully controlled the culture conditions for EB formation, and here we report an efficient protocol by which uniformly differentiated EBs were obtained, monitored by measuring the differentiation of beating cardiomyocytes. Furthermore, by using this protocol we observed in long-term cultured plating EBs (> 60 days) there still exist cell colony with pluripotency. This observation raised a potential possibility that ES cells may keep pluripotent in a niche provided by differentiated cells.
Animals ; Cell Differentiation ; physiology ; Cells, Cultured ; Culture Media ; Embryonic Development ; physiology ; Embryonic Stem Cells ; cytology ; Leukemia Inhibitory Factor ; pharmacology ; Mice ; Stem Cell Niche ; physiology ; Time Factors

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 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 establish the method for isolation, culture, and differentiation induction of nestin-positive cells in fetal rat hepatic cells.

METHODSHepatic cells were obtained from fetal rats by means of mechanical separation and hanging-drop culture, and after two days of primary culture, the medium was changed for further cell culture in the presence of 20% fetal bovine serum (containing glucose 25 ml/L, mycillin 100 U/ml, pH 7.6), 10 mmol/L nicotinamide, 1 mg/L insulin, affix N2, basic fibroblast growth factor, stem cell factor, epidermal growth factor and leukemia inhibitory factor.

RESULT AND CONCLUSIONNestin-positive cells were obtained from fetal rat liver, which can differentiate into islet beta cells after culture and expansion in vitro.

Animals ; Cell Differentiation ; drug effects ; physiology ; Cell Separation ; methods ; Cell Transdifferentiation ; drug effects ; physiology ; Cells, Cultured ; Epidermal Growth Factor ; pharmacology ; Female ; Fetus ; Fibroblast Growth Factor 2 ; pharmacology ; Hepatocytes ; cytology ; metabolism ; Insulin ; pharmacology ; Intermediate Filament Proteins ; biosynthesis ; Islets of Langerhans ; cytology ; drug effects ; Leukemia Inhibitory Factor ; pharmacology ; Liver ; Male ; Nerve Tissue Proteins ; biosynthesis ; Nestin ; Rats ; Rats, Wistar ; Stem Cell Factor ; pharmacology

OBJECTIVETo evaluate the influence of superovulation by GnRHa protocol and pregnant mare's serum gonadotropin (PMSG) alone on the expression of estrogen receptor (ER), progesterone receptor (PR) and leukemia inhibitory factor (LIF) mRNA on endometrium.

METHODSForty-five female ICR mice were randomly allocated into 3 groups:(1) GnRHa+PMSG group: alarelin was give first for desensitizing the pituitary, then superovulation with PMSG; (2) PMSG group: mice were injected with PMSG only; (3) Natural cycle group: mice were given with same volume of saline. Endometrium samples were taken at 48 hours after given hCG or ovulation (control group). ER and PR in glandular cells were detected with SP immunohistochemistry semiquantitatively. Expression of LIF mRNA on endometrium was detected with reverse transcriptase-polymerase chain reaction (RT-PCR) analysis.

RESULTThe positive rate(%) and expression intense (AU) of ER and PR on glandular epithelium cells were significantly lower in GnRHa+PMSG group and PMSG group than those in natural cycle group (all P <0.01). The expression of LIF mRNA was significantly lower in GnRHa+PMSG group and PMSG group than that in natural cycle group (all P <0.01); but the expressions of ER, PR and LIF in GnRHa+PMSG group were higher than those in PMSG group.

CONCLUSIONThe protocol with GnRHa down regulates the expressions of ER, PR and the LIF mRNA on the mice of secretive phase endometrium, suggesting it may have an adverse effect on the endometrial receptivity in mice, but it may still be better than PMSG alone.

Animals ; Clinical Protocols ; Endometrium ; metabolism ; Female ; Gonadotropin-Releasing Hormone ; analogs & derivatives ; pharmacology ; Gonadotropins ; pharmacology ; Leukemia Inhibitory Factor ; metabolism ; Mice ; Mice, Inbred ICR ; Ovulation Induction ; methods ; RNA, Messenger ; metabolism ; Random Allocation ; Receptors, Estrogen ; metabolism ; Receptors, Progesterone ; metabolism ; Superovulation ; metabolism