OBJECTIVETo investigate the effect of extracellular signal-regulated kinase (ERK) signaling pathway on the endothelial differentiation of periodontal ligament stem cells (PDLSC).

METHODSHuman PDLSC was cultured in the medium with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) to induce endothelial differentiation. Endothelial inducing cells was incubated with U0126, a specific p-ERK1/2 inhibitor. PDLSC from one person were randomly divided into four groups: control group, endothelial induced group, endothelial induced+DMSO group and endothelial induced+U0126 group. The protein expression of the p-EKR1/2 was analyzed by Western blotting at 0, 1, 3, 6 and 12 hours during endonthelial induction. The mRNA expressions of CD31, VE-cadherin, and VEGF were detected by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) after a 7-day induction. The proportion of CD31(+) to VE-cadherin(+) cells was identified by flow cytometry, and the ability of capillary-like tubes formation was detected by Matrigel assay after a 14-day induction. The measurement data were statistically analyzed.

RESULTSPhosphorylated ERK1/2 protein level in PDLSC was increased to 1.24±0.12 and 1.03±0.24 at 1 h and 3 h respectively, during the endothelial induction (P<0.01). The mRNA expressions of CD31 and VEGF in induced+U0126 group were decreased to 0.09±0.18 and 0.49±0.17, which were both significantly different with those in induced group (P<0.05). The proportion of CD31(+) to VE-cadherin(+) cells of induced+U0126 group were decreased to 5.22±0.85 and 3.56±0.87, which were both significantly different with those in induced group (P<0.05). In Matrigel assay, the branching points, tube number and tube length were decreased to 7.0±2.7, 33.5±6.4, and (15 951.0±758.1) pixels, which were all significantly different with those in induced group (P<0.05).

CONCLUSIONSThe endothelial differentiation of PDLSC is positively regulated by ERK signaling pathway. Inhibition of ERK1/2 phosphorylation could suppress endothelial differentiation of PDLSC.

Antigens, CD ; genetics ; metabolism ; Butadienes ; pharmacology ; Cadherins ; genetics ; metabolism ; Cell Differentiation ; Endothelial Cells ; cytology ; physiology ; Enzyme Inhibitors ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; physiology ; Fibroblast Growth Factor 2 ; pharmacology ; Humans ; Mitogen-Activated Protein Kinase 3 ; antagonists & inhibitors ; metabolism ; Nitriles ; pharmacology ; Periodontal Ligament ; cytology ; metabolism ; Phosphorylation ; Platelet Endothelial Cell Adhesion Molecule-1 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Random Allocation ; Signal Transduction ; Stem Cells ; cytology ; physiology ; Time Factors ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; pharmacology

OBJECTIVETo explore the relation between oocyte maturation and growth differentiation factor-9 (GDF-9) gene expression.

METHODSOvariectomy was performed in 50 Kunming female mice of 10 days old, and the preantral follicles were isolated from the ovaries and cultured in medium drops for 12 days. Oocytes and somatic cells were mechanically isolated. The oocytes cultured in vitro for 2, 4, 6, 8, 10, and 12 days constituted the in vitro cultured group and the oocytes obtained from female mice of 12, 14, 16, 18, 20, and 22 days old served as the in vivo group. Semi-quantitative RT-PCR and agar gel electrophoresis were performed to quantify GDF-9 gene expression in each oocyte.

RESULTSFollicle survival, antrum formation and maturation rate was 89.5%, 51.8% and 56.6% in the in vitro cultured follicles, respectively. GDF-9 gene expression on days 2, 4, 6, 8, 10, and 12 in in vitro cultured oocytes was 0.83-/+0.08, 0.52-/+0.09, 0.45-/+0.13, 0.49-/+0.09, 0.49-/+0.09, and 0.68-/+0.08, respectively; GDF-9 gene expression in in vivo grown oocytes of 12, 14, 16, 18, 20, and 22 days were 0.64-/+0.35, 0.48-/+0.10, 0.52-/+0.10, 0.66-/+0.08, 0.72-/+0.09, and 0.91-/+0.11, respectively. Between days 8 and 12, GDF-9 gene expression in in vitro cultured oocyte was significantly lower than that in in vivo grown oocytes (P<0.05).

CONCLUSIONMII oocytes can be obtained from in vitro culture of the preantral follicles. GDF-9 gene expression in the oocytes varies with their growth stages. Between days 8 and 12 of in vitro culture, GDF-9 gene expression in the cultured oocytes is different from that in in vivo grown oocytes.

Animals ; Animals, Newborn ; Bone Morphogenetic Protein 15 ; Cell Survival ; Cells, Cultured ; Electrophoresis, Agar Gel ; Female ; Gene Expression ; Growth Differentiation Factor 9 ; Intercellular Signaling Peptides and Proteins ; genetics ; Mice ; Oocytes ; cytology ; growth & development ; metabolism ; Ovarian Follicle ; cytology ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors

OBJECTIVE: This study was to examine the in vitro neural cell differentiation patterns of human embryonic stem (hES) cells following treatment of various neurotrophic factors [basic fibroblast growth factor (bFGF), retinoic acid (RA), brain derived neurotrophic factor (BDNF) and transforming growth factor (TGF)-alpha], particulary in dopaminergic neuron formation. METHODS: The hES cells were induced to differentiate by bFGF and RA. Group I) In bFGF induction method, embryoid bodies (EBs, for 4 days) derived from hES were plated onto gelatin dish, selected for 8 days in ITSFn medium and expanded at the presence of bFGF (10 ng/ml) for another 6 days followed by a final differentiation in N2 medium for 7, 14 and 21 days. Group II) For RA induction, EBs were exposed of RA (10-6 M) for 4 days and allowed to differentiate in N2 medium for 7, 14 and 21 days. Group III) To examine the effects of additional neurotrophic factors, bFGF or RA induced cells were exposed to either BDNF (10 ng/ml) or TGF-alpha (10 ng/ml) during the 21 days of final differentiation. Neuron differentiation and dopamine secretion were examined by indirect immunocytochemistry and HPLC, respectively. RESULTS: The bFGF or RA treated hES cells were resulted in similar neural cell differentiation patterns at the terminal differentiation stage, specifically, 75% neurons and 11% glial cells. Additionally, treatment of hES cells with BDNF or TGF-alpha during the terminal differentiation stage led to significantly increased tyrosine hydroxylase (TH) expression of a dopaminergic neuron marker, compared to control (p<0.05). In contrast, no effect was observed on the rate of mature neuron (NF-200) or glutamic acid decarboxylase-positive neurons. Immunocytochemistry and HPLC analyses revealed the higher levels of TH expression (20.3%) and dopamine secretion (265.5+/-62.8 pmol/mg) in bFGF and TGF-alpha sequentially treated hES cells than those in RA or BDNF treated hES cells. CONCLUSION: These results indicate that the generation of dopamine secretory neurons from in vitro differentiated hES cells can be improved by TGF-alpha addition in the bFGF induction protocol.
Brain-Derived Neurotrophic Factor ; Cell Differentiation ; Chromatography, High Pressure Liquid ; Dopamine* ; Dopaminergic Neurons ; Embryoid Bodies ; Embryonic Stem Cells* ; Fibroblast Growth Factor 2 ; Fibroblast Growth Factors ; Gelatin ; Glutamic Acid ; Humans* ; Immunohistochemistry ; Nerve Growth Factors* ; Neuroglia ; Neurons* ; Transforming Growth Factor alpha ; Transforming Growth Factors ; Tretinoin ; Tyrosine 3-Monooxygenase

BACKGROUNDNeural stem cells (NSCs) are a self-renewing and multipotent population of the central nervous system (CNS), which are active during development and maintain homeostasis and tissue integrity throughout life. Microglias are an immune cell population resident in the CNS, which have crucial physiological functions in the developing and adult CNS. This study aimed to investigate that whether microglia co-cultured with NSCs could promote astrogliogenesis from NSCs.

METHODSMicroglia and NSCs were co-cultured in 24-well insert plates. NSCs were plated in the bottom of the well and microglia in the insert. Fluorescent staining, Western blotting and RT-PCR were used to determine the effect of microglia on NSCs differentiation.

RESULTSCo-culture of microglia and NSCs promoted astrogliogenesis from NSCs. Several key genes, such as Notch 1, Notch 2, Notch 3, Hes 5, and NRSF were downregulated, while the critical genes Id1 and Id2 were upregulated. BMP2 and FGF2 were upregulated.

CONCLUSIONMicroglias act as a regulator of NSCs astrogliogenesis.

Animals ; Astrocytes ; cytology ; metabolism ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; Blotting, Western ; Bone Morphogenetic Protein 2 ; genetics ; Cell Differentiation ; genetics ; physiology ; Cells, Cultured ; Coculture Techniques ; methods ; Fibroblast Growth Factor 2 ; genetics ; Inhibitor of Differentiation Protein 1 ; genetics ; Inhibitor of Differentiation Protein 2 ; genetics ; Microglia ; cytology ; metabolism ; Microscopy, Fluorescence ; Neural Stem Cells ; cytology ; metabolism ; Rats ; Repressor Proteins ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVEParkinson's disease (PD), a neurodegenerative disorder, has been reported to be associated with brain neuroinflammation in its pathogenesis. Herein, changes in peripheral immune system were determined to better understand PD pathogenesis and provide possible target for treatment of PD through improvement of immune disorder.

METHODS1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was intraperitoneally injected into mice to prepare PD model. Expression levels of pro-inflammatory and anti-inflammatory cytokines and transcription factors of CD4+ T lymphocyte subsets in spleen and mesenteric lymph nodes and concentrations of the cytokines in serum were examined on day 7 after MPTP injection. Percentages of CD4+ T lymphocyte subsets were measured by flow cytometry.

RESULTSMPTP induced PD-like changes such as motor and behavioral deficits and nigrostriatal impairment. Expression levels of the pro-inflammatory cytokines including interferon (IFN)-γ, interleukin (IL)-2, IL-17 and IL-22, in spleen and mesenteric lymph nodes were upregulated and their concentrations in serum were elevated in PD progression. But, the concentrations of the anti-inflammatory cytokines including IL-4, IL-10 and transforming growth factor (TGF)-β were not altered in the two lymphoid tissues or serum of PD mice. In addition, expression of T-box in T cells (T-bet), the specific transcription factor of helper T (Th) 1 cells, was downregulated, but expression of transcription factor forkhead box p3 (Foxp3), the transcription factor of regulatory T (Treg) cells, was upregulated. In support of the results, the numbers of IFN-γ-producing CD4+ cells (Th1 cells) were reduced but CD4+CD25+ cells (Treg cells) were elevated in both the lymphoid tissues of PD mice.

CONCLUSIONPD has a dysfunction of peripheral immune system. It manifests enhancement of proinflammatory response and CD4+ T cell differentiation bias towards Treg cells away from Th1 cells.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; Animals ; CD4-Positive T-Lymphocytes ; pathology ; Cell Differentiation ; Cytokines ; blood ; Disease Models, Animal ; Flow Cytometry ; Forkhead Transcription Factors ; metabolism ; Interferon-gamma ; blood ; Interleukin-10 ; blood ; Interleukin-17 ; blood ; Interleukin-2 ; blood ; Interleukin-4 ; blood ; Interleukins ; blood ; Lymph Nodes ; cytology ; Lymphocyte Activation ; Mice ; Parkinson Disease ; immunology ; physiopathology ; Spleen ; cytology ; T-Box Domain Proteins ; metabolism ; T-Lymphocytes, Regulatory ; Th1 Cells ; Transforming Growth Factor beta ; blood

Immunosuppressive regulatory T lymphocytes (Treg) expressing the transcription factor Foxp3 play a vital role in the maintenance of tolerance of the immune-system to self and innocuous non-self. Most Treg that are critical for the maintenance of tolerance to self, develop as an independent T-cell lineage from common T cell precursors in the thymus. In this organ, their differentiation requires signals from the T cell receptor for antigen, from co-stimulatory molecules, as well as from cytokine-receptors. Here we focus on the cytokines implicated in thymic development of Treg, with a particular emphasis on the roles of interleukin-2 (IL-2) and IL-15. The more recently appreciated involvement of TGF-β in thymic Treg development is also briefly discussed. Finally, we discuss how cytokine-dependence of Treg development allows for temporal, quantitative, and potentially qualitative modulation of this process.
Animals ; Cell Differentiation ; genetics ; Cytokines ; immunology ; Forkhead Transcription Factors ; genetics ; immunology ; Gene Expression Regulation ; Immune Tolerance ; genetics ; Interleukin-15 ; genetics ; immunology ; Interleukin-2 ; genetics ; immunology ; Mice ; Receptors, Antigen, T-Cell ; genetics ; immunology ; T-Lymphocytes, Regulatory ; immunology ; Transforming Growth Factor beta ; genetics ; immunology

Embryonic stem cells are pluripotent and their differentiation in vitro can serve as an experimental model to explore the molecular mechanisms of early embryonic development. To investigate the effect of stromal cell conditioned medium combined with cytokines (sccm + cys) on the differentiation from human embryonic stem cells to hematopoietic cells and endothelial cells, the mouse fibroblast feeder cells to make human embryonic stem cells grown into embryonic bodies (EBs) were initially deleted. After culture for 3 days, EB cells were trypsinized into single cells and induced for 8 days by sccm + cys. Then, the differentiated cells were cultured in the semisolid medium containing 0.9% methylcellulose and cytokines to study the colony forming and self-renewal ability of cells. Immunocytochemical staining was used to check the surface markers of the colony cells. During the induction, mRNA expression of flk-1, BMI-1, scl, and Zeta-globin genes was tested by RT-PCR. Surface markers, such as flk-1, CD34 were tested by the flow cytometry. The results demonstrated that: (1) cell clusters containing 20-30 cells were formed after culture for 8 - 14 days in the semisolid medium, replanting these cells resulted in similar cell cluster forming. In addition, CD45 positive in big cell colonies were also found in the semisolid medium; (2) attached cell colonies appeared after culture for 8 days in the semisolid medium and VIII factor, UEA and KDR could be detected as negative by immunocytochemical staining; (3) on the 4(th) day of induction, mRNAs of flk-1, BMI-1, scl and Zeta-globin were all expressed. On the 8(th) day of induction, all of the above genes except Zeta-globin were expressed, while ES cell and EB cells which served as controls did not express scl and Zeta-globin genes; (4) on the 8(th) day of induction, the proportions of flk-1(+) cells and CD34(+) cells among all the inducing population were 9.8% and 16.8%, respectively, while the corresponding positive populations were 0.36% and 1.16% in spontaneously differentiated 11(th) day's EB, and 0.04% and 0.16%, respectively, in ES cells. If is concluded that embryonic stem cells can differentiate into hematopoietic cells and endothelial cells in combinant culture system of this study.
Cell Differentiation ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; Endothelial Cells ; cytology ; Flow Cytometry ; Globins ; genetics ; metabolism ; Hematopoietic Stem Cells ; cytology ; Humans ; Immunohistochemistry ; Nuclear Proteins ; genetics ; metabolism ; Polycomb Repressive Complex 1 ; Proto-Oncogene Proteins ; genetics ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; metabolism

OBJECTIVE: This study was to investigate the generation of the functional neuron derived from human embryonic stem(hES,MB03) cells on in vitro neural cell differentiation system. METHODS: For neural progenitor cell formation derived from hES cells, we produced embryoid bodies (EB: for 5 days, without mitogen) from hES cells and then neurospheres (for 7~10 days, 20 ng/ml of bFGF added N2 medium) from EB. And then finally for the differentiation into mature neuron, neural progenitor cells were cultured in i) N2 medium only (without bFGF),ii) N2 supplemented with 20 ng/ml platelet derived growth factor-bb (PDGF-bb) or iii) N2 supplemented with 5 ng/ml brain derived neurotrophic factor (BDNF) for 2 weeks. Identification of neural cell differentiation was carried out by immunocytochemistry using betaIII-tubulin (1:100) and GFAP (1:500). Also, generation of functional neuron was identified using anti-glutamate (Sigma, 1:1000), anti-GABA (Sigma, 1:1000), anti-serotonin (Sigma, 1:1000) and anti-tyrosine hydroxylase (Sigma, 1:1000). RESULTS: In vitro neural cell differentiation, neurotrophic factors (PDGF and BDNF) treated cell groups were high expressed MAP-2 and GFAP than non-treated cell group. The highest expression pattern of MAP-2 and betaIII-tubulin was indicated in BDNF treated group. Also, in the presence of PDGF-bb or BDNF, mostof the neural cells derived from hES cells were differentiated into gultamate and GABA neuron in vitro. Furthermore, we confirmed that there were a few serotonin and tyrosine hydroxylase positive neuron in the same culture environment. CONCLUSION: This results suggested that the generation of functional neuron derived from hES cells was increased by addition of neurotrophic factors such as PDGF-bb or BDNF in b-FGF induced neural cell differentiation system and especially gultamate and GABA neurons were mainly produced in the system.
Blood Platelets ; Brain-Derived Neurotrophic Factor* ; Cell Differentiation* ; Embryoid Bodies ; Embryonic Stem Cells* ; gamma-Aminobutyric Acid ; Humans* ; Immunohistochemistry ; Nerve Growth Factors ; Neurons* ; Serotonin ; Stem Cells ; Tyrosine 3-Monooxygenase

The clinical models for studying ovary-determining genes may be premature ovarian failure (POF). POF is a condition causing amenorrhea, hypoestrogenism, and elevated gonadotropins in women under 40 years old. FSH receptor, LH receptor, inhibin, GDF-9 (growth differentiation factor-9), BMP-15 (bone morphogenetic protein-15), DIAPH2 (diaphanous gene) and XPNPEP2 (X-prolyl aminopeptidase) genes were proposed as a possible candidate gene, but until recently, only mutations in FSH receptor, LH receptor and inhibin genes have been identified in POF patients. Therefore mutation screening of another POF gene necessary to reveal the principal causative genes of POF. OBJECTIVE: The present study was performed to analyze the mutation of GDF-9 gene in Korean patient with POF and to investigate whether mutation of these gene is a likely main cause of POF. METHODS: Eighty-six women with POF were studied and thirty-six normal women were enrolled as control. Mutation screening of these genes were performed by denaturing HPLC and were confirmed by automatic sequencing. RESULTS: Three different mutations of GDF-9 gene were identified in Korean women with POF; Arg3Cys mutation in one patient, Leu40Val mutation in one patient, Asp57Tyr mutation in 10 patients and 5 normal controls. Arg3Cys mutation and Leu40Val mutation were likely cause of disease. Frequencies of Arg3Cys mutation and Leu40Val mutation were 1.2%, respectively. Asp57Tyr mutation was common polymorphism in Korean women. All mutations was a novel mutation found in the present study. CONCLUSION: POF was resulted by mutations of GDF-9 gene, but mutations of GDF-9 gene are not likely main causes of POF because of low frequency of mutations.
Adult ; Amenorrhea ; Bone Morphogenetic Protein 15 ; Chromatography, High Pressure Liquid ; Female ; Gonadotropins ; Growth Differentiation Factor 9 ; Humans ; Inhibins ; Mass Screening ; Primary Ovarian Insufficiency* ; Receptors, FSH ; Receptors, LH

OBJECTIVETo investigate the effects of total saponins of panax ginseng (TSPG) on proliferation and differentiation of human embryonic neural stem cell (NSC) into dopaminergic neuron.

METHODIsolation, cultivation and identification of human embryonic NSC from cerebral cortex of 7-12 week abortus. By using flow cytometry and MTT assay, the effects of various concentration of TSPG and TSPG cooperating with cytokines( EGF, bFGF) in NSC culture media for 3 days on proliferation of human embryonic NSC has studied. By employing immunocytochemistry assay of the expression of tyrosine hydroxylase (TH), the effect of different dilution of TSPG and TSPG cooperating with IL-1 on induced differentiation of human embryonic NSC into dopaminergic neuron has researched.

RESULTTSPG can significantly promote the proliferation of NSC. When TSPG cooperating with EGF and bFGF, the proliferation of NSC is much stronger than that of only using FGF and bFGF. TSPG also induces NSC to differentiate into dopaminergic neuron, especially when TSPG is cooperating with IL-1.

CONCLUSIONTSPG can not only obviously accelerate the proliferation of NSC, but also significantly induce differentiation of NSC into dopaminergic neuron.

Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dopamine ; metabolism ; Drug Synergism ; Embryonic Stem Cells ; cytology ; drug effects ; metabolism ; Epidermal Growth Factor ; pharmacology ; Fibroblast Growth Factor 2 ; pharmacology ; Humans ; Immunohistochemistry ; Interleukin-1 ; pharmacology ; Neurons ; cytology ; drug effects ; metabolism ; Panax ; chemistry ; Plants, Medicinal ; chemistry ; Saponins ; isolation & purification ; pharmacology ; Tyrosine 3-Monooxygenase ; metabolism