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Sample records for 3-methylcholanthrene induces differential

  1. Regulation of nonmuscle myosin II during 3-methylcholanthrene induced dedifferentiation of C2C12 myotubes

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    Dey, Sumit K.; Saha, Shekhar; Das, Provas; Das, Mahua R.; Jana, Siddhartha S., E-mail: bcssj@iacs.res.in

    2014-08-01

    3-Methylcholanthrene (3MC) induces tumor formation at the site of injection in the hind leg of mice within 110 days. Recent reports reveal that the transformation of normal muscle cells to atypical cells is one of the causes for tumor formation, however the molecular mechanism behind this process is not well understood. Here, we show in an in vitro study that 3MC induces fragmentation of multinucleate myotubes into viable mononucleates. These mononucleates form colonies when they are seeded into soft agar, indicative of cellular transformation. Immunoblot analysis reveals that phosphorylation of myosin regulatory light chain (RLC{sub 20}) is 5.6±0.5 fold reduced in 3MC treated myotubes in comparison to vehicle treated myotubes during the fragmentation of myotubes. In contrast, levels of myogenic factors such as MyoD, Myogenin and cell cycle regulators such as Cyclin D, Cyclin E1 remain unchanged as assessed by real-time PCR array and reverse transcriptase PCR analysis, respectively. Interestingly, addition of the myosin light chain kinase inhibitor, ML-7, enhances the fragmentation, whereas phosphatase inhibitor perturbs the 3MC induced fragmentation of myotubes. These results suggest that decrease in RLC{sub 20} phosphorylation may be associated with the fragmentation step of dedifferentiation. - Highlights: • 3-Methylcholanthrene induces fragmentation of C2C12-myotubes. • Dedifferentiation can be divided into two steps – fragmentation and proliferation. • Fragmentation is associated with rearrangement of nonmuscle myosin II. • Genes associated with differentiation and proliferation are not altered during fragmentation. • Phosphorylation of myosin regulatory light chain is reduced during fragmentation.

  2. Xenotropic type C virus expression in murine thymomas induced by radiation or 3-methylcholanthrene

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    Mayer, A. (New York Univ. Medical Center, NY); Duran-Reynals, M.L.

    1981-10-01

    Thymic lymphoma incidence and thymic expression of MuLV with xenotropic infectivity was monitored in AKR, RF, and reciprocal F/sub 1/ mice of the AKR X RF cross after treatment with either ..gamma.. radiation or the chemical carcinogen 3-methylcholanthrene (MCA). These two inbred strains and the F/sub 1/ hybrids developed similary high incidences of thymoma, and lymphomatous cells from AKR mice and (ARK) X RF..integral..)F/sub 1/ mice were observed to be expressing MuLV with xenotropic host range. However, lymphoma cells from RF mice and (RF) X AKR..integral..)F/sub 1/ mice did not shed xenotropic MuLV. Thymic xenotropic virus expression was therefore not correlated with a high incidence of radiation or chemically induced thymoma, but rather appeared to be a phenotype genetically transmitted by AKR mice to F/sub 1/ mice of the AKR X RF cross as a dominant trait in induced thymomas. In addition, a maternal effect on thymic xenotropic virus expression in induced thymomas was observed by the comparison of reciprocal F/sub 1/ hybrids in this cross.

  3. Prevention of 3-methylcholanthrene-induced fibrosarcomas in rats pre-inoculated with endogenous rat retrovirus.

    OpenAIRE

    Fish, D C; Demarais, J T; Djurickovic, D B; Huebner, R J

    1981-01-01

    Weanling Fischer 344 rats received a single intraperitoneal injection of a 1000-fold concentrated preparation of endogenous nontransforming rat retrovirus. Ten days later, the rats were each given a single subcutaneous injection of 3-methylcholanthrene. The rats inoculated with the endogenous rat retrovirus were significantly protected against the development of cancer, whereas uninoculated rats and rats given one of several murine retroviruses or baboon retrovirus were not protected.

  4. CD8+ T cells are crucial for the ability of congenic normal mice to reject highly immunogenic sarcomas induced in nude mice with 3-methylcholanthrene

    DEFF Research Database (Denmark)

    Boesen, M; Svane, I M; Engel, A M;

    2000-01-01

    An attempt was made to identify the selection pressures put upon a growing tumour by CD8+ T cells. To this end tumours induced with 3-methylcholanthrene in T cell-deficient nude mice and in congenic T cell-competent nu/+ mice were transplanted to nu/+ recipients. The rejection rate of the sarcomas...

  5. Basal and 3-methylcholanthrene-induced expression of cytochrome P450 1A, 1B and 1C genes in the Brazilian guppy, Poecilia vivipara.

    Science.gov (United States)

    Dorrington, Tarquin; Zanette, Juliano; Zacchi, Flávia L; Stegeman, John J; Bainy, Afonso C D

    2012-11-15

    In fish there are four cytochrome P450 (CYP1) subfamilies: CYP1A, CYP1B, CYP1C, and CYP1D. Here we cloned Poecilia vivipara CYP1A, with an inferred amino acid sequence 91% identical to CYP1A from the killifish Fundulus heteroclitus, another member of the Cypriniformes, and an important model in ecotoxicology. In addition, we examined the expression of CYP1A, CYP1B1, and CYP1C1 by qPCR in liver, gill, and intestine of adult P. vivipara injected with 3-methylcholanthrene (3-MC) or held in clean water (control group) for 24h. All three tissues examined showed basal expression of the three CYP1 genes. CYP1A was most strongly expressed in the liver, while CYP1B1, and CYP1C1 were most strongly expressed in the gill and intestine respectively. 3-MC induced CYP1A, CYP1B1, and CYP1C1 significantly (20-120-fold) in the three organs, consistent with the regulation of CYP1A, CYP1B1 and CYP1C1 via the aryl hydrocarbon receptor. Validation of CYP1 gene biomarkers in fish collected from a contaminated urban mangrove environment was confirmed with significant induction of CYP1A and CYP1C1 in gills (10-15-fold) and CYP1B1 in liver (23-fold), relative to fish from a control site. The responsiveness of these CYP1 genes indicates P. vivipara is suitable as a model for environmental toxicology studies and environmental assessment in Brazil.

  6. A synergistic effect of oestradiol and prolactin influencing the incidence of 3-methylcholanthrene induced cerivical carcinomas in mice.

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    Forsberg, J G; Breistein, L S

    1976-09-01

    Castrated NMRI mice were laparotomized and a thread impregnated with beeswax-methylcholanthrene was inserted into the uterine cervix. Beginning on the day of operation and for a further 5 days the animals were injected with oestradiol, prolactin, oestradiol-prolactin, oestradiol-prolactin-progesterone, or the solvents for the hormones only. One group of animals were injected with oestradiol-prolactin for 6 days and later with progesterone every third day until death. The animals were killed one or 4 weeks after the operation. Among the one-week animals the number of cervices presenting epithelial downgrowths ("buds") into the stroma was higher after treatment with a combination of oestradiol and prolactin than after treatment with each hormone separately or among the controls. Four weeks after operation, the incidence of squamous cervical carcinomas was seen to be significantly higher among animals injected with both oestradiol and prolactin than in controls or in those injected with oestradiol or prolactin alone. Progesterone had no definite effect on the oestradiol-prolactin induced incidence. The mechanism behind the synergistic effect of prolactin and oestradiol is discussed.

  7. 3-methylcholanthrene induces differential recruitment of aryl hydrocarbon receptor to human promoters

    DEFF Research Database (Denmark)

    Pansoy, Andrea; Ahmed, Shaimaa; Valen, Eivind;

    2010-01-01

    The aryl hydrocarbon receptor (AHR) is a ligand-activated protein that mediates the toxic actions of polycyclic aromatic and halogenated compounds. Identifying genes directly regulated by AHR is important in understanding the pathways regulated by this receptor. Here we used chromatin immunopreci......The aryl hydrocarbon receptor (AHR) is a ligand-activated protein that mediates the toxic actions of polycyclic aromatic and halogenated compounds. Identifying genes directly regulated by AHR is important in understanding the pathways regulated by this receptor. Here we used chromatin...

  8. Metabolism of 3-methylcholanthrene by rat liver microsomes: a reinvestigation

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    Stoming, T.A. (Medical Coll., Augusta, GA); Bornstein, W.; Bresnick, E.

    1977-01-01

    Metabolites of 3-methylcholanthrene (3-MC) formed by rat liver microsomes were analyzed by high pressure liquid chromatography. The metabolic profile is significantly different from previous studies using thin layer chromatography. The major metabolites include 1- and 2-hydroxy-3-MC. Use of the high pressure liquid chromatographic system allows for the separation of at least seven new metabolites. The amounts of three of these new metabolites are substantially decreased when the potent epoxide hydrase inhibitor 3,3,3-trichloropropene oxide is added to the incubation system. These results then suggest the formation of epoxides of 3-methylcholanthrene other than the K-region oxide.

  9. Induction of hepatic cytochrome P-450 activity in wild cotton rats (Sigmodon hispidus) by phenobarbital and 3-methylcholanthrene

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    Elangbam, C.S.; Qualls, C.W.,Jr.; Bauduy, M. (Oklahoma State Univ., Stillwater (USA))

    1989-05-01

    Wild cotton rats (Sigmodon hispidus) are ubiquitous throughout the Southeast quadrant of the United States, easy to capture, have a generation interval of less than one year and a limited range of movement (less than one hectare). This species may prove to be an excellent model for monitoring environmental contamination. Traditionally, cytochrome P-450 inducing agents are grouped into two classes. One, represented by phenobarbital, induces P-450b and P-450e; the other, represented by 3-methylcholanthrene, induces P-450c and P-450d isoenzymes. The types and amounts of cytochrome P-450 vary among species, organs, health status, sex, and stress of the animal. If the levels of cytochrome P-450 of wild cotton rats are to be used in monitoring environmental pollution, it is necessary to characterize the inducibility and concentration of cytochrome P-450 in this species. This study was designed to determine the concentration and inducibility of cytochrome P-450 in the livers of cotton rats after intraperitoneal (ip) administration of phenobarbital and 3-methylcholanthrene.

  10. Autoimmunity and tumor resistance. 3-Methylcholanthrene tumorigenesis in New Zealand Black mice.

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    Morton, J I; Siegel, B V; Moore, R D

    1978-11-01

    Skin tumors induced by the subcutaneous injection of 3-methylcholanthrene (3-MC) in New Zealand Black (NZB) mice had a delayed development and lower frequency compared with BALB/c and C57BL mice. In the SJL/J strain, the incidence of tumors was lower than in the NZB, but with the same delayed development. Most of the tumors in the BALB/c, C57BL, and SJL/J strains were sarcomas; more than one third of the tumors in the NZB mice were squamous cell carcinomas. The greatest frequency and most rapid development of tumors in the NZB, as a function of age at the time of injection of 3-MC, occurred at 4 months. Young (3.5 and 7 weeks) and 12-month-old tumor incidence in the 4-month-old NZB was decreased by treatment with antithymocyte serum (ATS). Five hundred rad whole-body x-irradiation accelerated the onset of tumors but did not increase the final incidence. 3-MC injection and the presence of skin tumors had no influence on the development of glomerulonephritis or hematopoietic neoplasms in the NZB mice. Coombs' positive anemia was not influenced by 3-MC injection, but there was an earlier and increased incidence of positive Coombs' tests in tumor-bearing animals. Liver aryl hydrocarbon hydroxylase (AHH) specific activity was low in the young NZB, increased gradually with age, and was higher in the female mice.

  11. Expression of CYP2A3 mRNA and its regulation by 3-methylcholanthrene, pyrazole, and ß-ionone in rat tissues

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    A.B. Robottom-Ferreira

    2003-07-01

    Full Text Available Cytochrome P450 (CYP 2A enzymes are involved in the metabolism of numerous drugs and hormones and activate different carcinogens. Human CYP2A6, mouse CYP2A5 and rat CYP2A3 are orthologous enzymes that present high similarity in their amino acid sequence and share substrate specificities. However, different from the human and mouse enzyme, CYP2A3 is not expressed in the rat liver. There are limited data about expression of CYP2A3 in extrahepatic tissues and its regulation by typical CYP inducers. Therefore, the objective of the present study was to analyze CYP2A3 mRNA expression in different rat tissues by RT-PCR, and to study the influence of 3-methylcholanthrene, pyrazole and ß-ionone treatment on its expression. Male Wistar rats were divided into four groups of 5 rats each, and were treated ip for 4 days with 3-methylcholanthrene (25 mg/kg body weight, pyrazole (150 mg/kg body weight, ß-ionone (1 g/kg body weight, or vehicle. Total RNA was extracted from tissues and CYP2A3 mRNA levels were analyzed by semiquantitative RT-PCR. CYP2A3 mRNA was constitutively expressed in the esophagus, lung and nasal epithelium, but not along the intestine, liver, or kidney. CYP2A3 mRNA levels were increased in the esophagus by treatment with 3-methylcholanthrene and pyrazole (17- and 7-fold, respectively, in lung by pyrazole and ß-ionone (3- and 4-fold, respectively, although not statistically significant, in the distal part of the intestine and kidney by 3-methylcholanthrene and pyrazole, and in the proximal part of the intestine by pyrazole. CYP2A3 mRNA was not induced in nasal epithelium, liver or in the middle part of the intestine. These data show that, in the rat, CYP2A3 is constitutively expressed in several extrahepatic tissues and its regulation occurs through a complex mechanism that is essentially tissue specific.

  12. Modifications of benzene myelotoxicity and metabolism by phenobarbital, SKF-252A and 3-methylcholanthrene

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    Gill, D.P.; Kempen, R.R.; Nash, J.B.; Ellis, S.

    1979-11-05

    It has recently been suggested that the primary myelotoxic species generated from benzene is not produced directly from the parent compound, but from phenol or an even later metabolite. Several compounds that alter the activities of microsomal oxidative and conjugating enzymes were studied for their effects on benzene's myelotoxicity and metabolism. Phenobarbital (PB) protected animals from leucopnia and increased both to total amount of phenol as well as the amount of unconjugated phenol excreted in the urine. SKF-525A had no effect on the leucopenia, whereas it reduced the conversion of benzene to phenol without changing the excretion of unconjugated phenol. 3-Methylcholanthrene also did not prevent the leucopenia, but it did increase the conversion of benzene to phenol and the amount of unconjugated phenol excreted during the first days of the experiment. These data indicate that the early phases of benzene's metabolism may be modulated by the drug pretreatments employed, but myelotoxicity was abated only by PB. We conclude that the marrow effect of benzene is due to a metabolic product other than phenol and, furthermore that the formation of this toxic principle is not strictly dependent on the rate of phenol production.

  13. Influences of 3-methylcholanthrene, phenobarbital and dexamethasone on xenobiotic metabolizing-related cytochrome P450 enzymes and steroidogenesis in human fetal adrenal cortical cells

    Institute of Scientific and Technical Information of China (English)

    Hui WANG; Min HUANG; Ren-xiu PENG; Jiang LE

    2006-01-01

    Aim: To explore the influence and possible mechanism of xenobiotics on adrenal steroidogenesis during fetal development. Methods: Primary human fetal adrenal cortical cells were prepared, cultured and treated with 3-methylcholanthrene, phenobarbital and dexamethasone. The activities of 7-ethoxyresorufin 0-dealkylase, benzphetamine, aminopyrine and erythromycin N-demethylases were measured by enzyme assays. At the same time, quantitative analysis of steroid hormones cortisol, aldosterone, testosterone and progesterone were carried out in cultural medium by radioimmunoassays. Results: The activities of benzphetamine and aminopyrine Ar-demethylase were increased in the cultural fetal adrenal cells treated with phenobarbital (0.25-1 mmol/L) for 24 h. Dexamethasone (25-100 μmol/L) also increased the activity of erythromycin W-demethylase. The activity of 7-ethoxyresorufin 0-dealkylase was undetected in the cells treated without and with 3-methylcholanthrene (0.5-2 μmol/L). Meanwhile, the contents of medium cortisol, aldosterone and progesterone were decreased after treatment with 3-methylcholanthrene. Cortisol, aldosterone and progesterone concentrations were also slightly decreased with phenobarbital. Dexamethasone enhanced the productions of cortisol and progesterone remarkably. The trend of testosterone concentration was uncertain after 3-methylcholanthrene, phenobarbital or dexamethasone treatment. Conclusion: 3-Methylcholanthrene, phenobarbital or dexamethasone could interfere with the synthesis of cortisol, aldosterone and progesterone in primary human fetal adrenal cortical cells, which likely act through xenobiotic metabolizing-related cytochrome P450 isoform activation.

  14. Highly selective bioactivation of 1- and 2-hydroxy-3-methylcholanthrene to mutagens by individual human and other mammalian sulphotransferases expressed in Salmonella typhimurium.

    Science.gov (United States)

    Meinl, Walter; Tsoi, Carrie; Swedmark, Stellan; Tibbs, Zachary E; Falany, Charles N; Glatt, Hansruedi

    2013-09-01

    The benzylic alcohols 1- and 2-hydroxy-3-methylcholanthrene (OH-MC) are major primary metabolites of the carcinogen 3-methylcholanthrene (MC). We investigated them for mutagenicity in TA1538-derived Salmonella typhimurium strains expressing mammalian sulphotransferases (SULTs). 1-OH-MC was efficiently activated by human (h) SULT1B1 (2400 revertants/nmol), weakly activated by hSULT1C3 and hSULT2A1 (2-9 revertants/nmol), but not activated by the other hSULTs studied (1A2, 1A3, 1C2 and 1E1). Mouse, rat and dog SULT1B1 activated 1-OH-MC (8-100 revertants/nmol) with much lower efficiency than their human orthologue. The other isomer, 2-OH-MC, was activated to a potent mutagen by hSULT1A1 (4000-5400 revertants/nmol), weakly activated by hSULT1A2 or hSULT2A1 (1-12 revertants/nmol), but not activated by the other hSULTs. In contrast to their human orthologue, mouse, rat and dog SULT1A1 did not appreciably activate 2-OH-MC (mutagens extremely difficult, in particular as the critical form may even differ for positional isomers, such as 1- and 2-OH-MC. Moreover, the species-dependent differences will complicate the verification of in vitro results in animal studies.

  15. Biphenyl metabolism by rat liver microsomes. Regioselective effects of inducers, inhibitors, and solvents

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    Haugen, D.A.

    1981-01-01

    The effects of the inducers phenobarbital and 3-methylcholanthrene, the inhibitors 7,8-benzoflavone and 1-benzyl-imidazole, and the solvents methanol, acetone, and dimethyl sulfoxide on the 2-, 3-, and 4-hydroxylation of biphenyl and the O-de-ethylation of 7-ethoxycoumarin by rat liver microsomes were examined. Phenobarbital pretreatment primarily induced 2- and 3-hydroxylation, the latter most dramatically. 3-Methylcholanthrene pretreatment induced 2- and 3-hydroxylation to similar extents. The inhibitors and solvents had regioselective effects on biphenyl metabolism that were characteristic of the uninduced, phenobarbital-induced, and 3-methylcholanthrene-induced microsomes. The presence of multiple forms of cytochrome P-450 in uninduced microsomes is indicated by the regioselective effects of the solvents and the inhibitors. The 3-methylcholanthrene-dependent increases in 2- and 3-hydroxylation appear due to induction of a single form of cytochrome P-450, as indicated by similar dose-response relationships and similar changes in sensitivitty to the inhibitors. The phenobarbital-dependent increases in 2- and 3-hydroxylation appear due to the induction of two forms of cytochrome P-450, as indicated by different changes in sensitivity to the effects of dimethyl sulfoxide and 7,8-benzoflavone. The results indicate that examination of the regioselectivity of biphenyl metabolism is a useful approach for characterizing microsomal mono-oxygenases, and they suggest that the approach may also be useful in the characterization of purified mono-oxygenase systems. (JMT)

  16. 3-Methylcholanthrene, an AhR agonist, caused cell-cycle arrest by histone deacetylation through a RhoA-dependent recruitment of HDAC1 and pRb2 to E2F1 complex.

    Directory of Open Access Journals (Sweden)

    Chih-Cheng Chang

    Full Text Available We previously showed that treating vascular endothelial cells with 3-methylcholanthrene (3MC caused cell-cycle arrest in the Go/G1 phase; this resulted from the induction of p21 and p27 and a decreased level and activity of the cyclin-dependent kinase, Cdk2. We further investigated the molecular mechanisms that modulate cell-cycle regulatory proteins through the aryl-hydrocarbon receptor (AhR/Ras homolog gene family, member A (RhoA dependent epigenetic modification of histone. AhR/RhoA activation mediated by 3MC was essential for the upregulation of retinoblastoma 2 (pRb2 and histone deacetylase 1 (HDAC1, whereas their nuclear translocation was primarily modulated by RhoA activation. The combination of increased phosphatase and tensin homolog (PTEN activity and decreased phosphatidylinositide 3-kinase (PI3K activation by 3MC led to the inactivation of the Ras-cRaf pathway, which contributed to pRb2 hypophosphorylation. Increased HDAC1/pRb2 recruitment to the E2F1 complex decreased E2F1-transactivational activity and H3/H4 deacetylation, resulting in the downregulation of cell-cycle regulatory proteins (Cdk2/4 and Cyclin D3/E. Co-immunoprecipitation and electrophoretic mobility shift assay (EMSA results showed that simvastatin prevented the 3MC-increased binding activities of E2F1 proteins in their promoter regions. Additionally, RhoA inhibitors (statins reversed the effect of 3MC in inhibiting DNA synthesis by decreasing the nuclear translocation of pRb2/HDAC1, leading to a recovery of the levels of cell-cycle regulatory proteins. In summary, 3MC decreased cell proliferation by the epigenetic modification of histone through an AhR/RhoA-dependent mechanism that can be rescued by statins.

  17. Sox9 potentiates BMP2-induced chondrogenic differentiation and inhibits BMP2-induced osteogenic differentiation.

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    Liao, Junyi; Hu, Ning; Zhou, Nian; Lin, Liangbo; Zhao, Chen; Yi, Shixiong; Fan, Tingxu; Bao, Wei; Liang, Xi; Chen, Hong; Xu, Wei; Chen, Cheng; Cheng, Qiang; Zeng, Yongming; Si, Weike; Yang, Zhong; Huang, Wei

    2014-01-01

    Bone morphogenetic protein 2 (BMP2) is one of the key chondrogenic growth factors involved in the cartilage regeneration. However, it also exhibits osteogenic abilities and triggers endochondral ossification. Effective chondrogenesis and inhibition of BMP2-induced osteogenesis and endochondral ossification can be achieved by directing the mesenchymal stem cells (MSCs) towards chondrocyte lineage with chodrogenic factors, such as Sox9. Here we investigated the effects of Sox9 on BMP2-induced chondrogenic and osteogenic differentiation of MSCs. We found exogenous overexpression of Sox9 enhanced the BMP2-induced chondrogenic differentiation of MSCs in vitro. Also, it inhibited early and late osteogenic differentiation of MSCs in vitro. Subcutaneous stem cell implantation demonstrated Sox9 potentiated BMP2-induced cartilage formation and inhibited endochondral ossification. Mouse limb cultures indicated that BMP2 and Sox9 acted synergistically to stimulate chondrocytes proliferation, and Sox9 inhibited BMP2-induced chondrocytes hypertrophy and ossification. This study strongly suggests that Sox9 potentiates BMP2-induced MSCs chondrogenic differentiation and cartilage formation, and inhibits BMP2-induced MSCs osteogenic differentiation and endochondral ossification. Thus, exogenous overexpression of Sox9 in BMP2-induced mesenchymal stem cells differentiation may be a new strategy for cartilage tissue engineering.

  18. Epilepsy-induced motility of differentiated neurons.

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    Chai, Xuejun; Münzner, Gert; Zhao, Shanting; Tinnes, Stefanie; Kowalski, Janina; Häussler, Ute; Young, Christina; Haas, Carola A; Frotscher, Michael

    2014-08-01

    Neuronal ectopia, such as granule cell dispersion (GCD) in temporal lobe epilepsy (TLE), has been assumed to result from a migration defect during development. Indeed, recent studies reported that aberrant migration of neonatal-generated dentate granule cells (GCs) increased the risk to develop epilepsy later in life. On the contrary, in the present study, we show that fully differentiated GCs become motile following the induction of epileptiform activity, resulting in GCD. Hippocampal slice cultures from transgenic mice expressing green fluorescent protein in differentiated, but not in newly generated GCs, were incubated with the glutamate receptor agonist kainate (KA), which induced GC burst activity and GCD. Using real-time microscopy, we observed that KA-exposed, differentiated GCs translocated their cell bodies and changed their dendritic organization. As found in human TLE, KA application was associated with decreased expression of the extracellular matrix protein Reelin, particularly in hilar interneurons. Together these findings suggest that KA-induced motility of differentiated GCs contributes to the development of GCD and establish slice cultures as a model to study neuronal changes induced by epileptiform activity.

  19. Impact-induced differentiation in icy bodies

    CERN Document Server

    Tonks, W Brian; Melosh, H Jay

    2016-01-01

    By the time icy objects grow to about the mass of Europa and silicate bodies grow to approximately lunar mass, a large high-speed impact can generate an intact melt region that allows the dense material to gravitationally segregate, forming a large density anomaly. If this anomaly generates sufficient differential stress, it rapidly segregates to the object's center, triggering whole body differentiation. We used an impact melting model based on the Hugoniot equations, the linear shock-particle velocity relationship, and the empirical relationship between shock pressure and distance coupled with a Monte Carlo simulation of the late accretion process to determine conditions under which large impacts trigger differentiation in icy bodies. In a gas-free environment, impacts of projectiles in the satellite's accretion zone have a small probability of triggering differentiation in surviving proto-satellites as small as Triton. The probability increases to 100% by the time surviving proto-satellites grow to the mas...

  20. On Volatility Induced Stationarity for Stochastic Differential Equations

    DEFF Research Database (Denmark)

    Albin, J.M.P.; Astrup Jensen, Bjarne; Muszta, Anders;

    2006-01-01

    This article deals with stochastic differential equations with volatility induced stationarity. We study of theoretical properties of such equations, as well as numerical aspects, together with a detailed study of three examples.......This article deals with stochastic differential equations with volatility induced stationarity. We study of theoretical properties of such equations, as well as numerical aspects, together with a detailed study of three examples....

  1. BMP2 induces chondrogenic differentiation, osteogenic differentiation and endochondral ossification in stem cells.

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    Zhou, Nian; Li, Qi; Lin, Xin; Hu, Ning; Liao, Jun-Yi; Lin, Liang-Bo; Zhao, Chen; Hu, Zhen-Ming; Liang, Xi; Xu, Wei; Chen, Hong; Huang, Wei

    2016-10-01

    Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-β (TGF-β) super-family, is one of the main chondrogenic growth factors involved in cartilage regeneration. BMP2 is known to induce chondrogenic differentiation in various types of stem cells in vitro. However, BMP2 also induces osteogenic differentiation and endochondral ossification in mesenchymal stem cells (MSCs). Although information regarding BMP2-induced chondrogenic and osteogenic differentiation within the same system might be essential for cartilage tissue engineering, few studies concerning these issues have been conducted. In this study, BMP2 was identified as a regulator of chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. BMP2 was used to regulate chondrogenic and osteogenic differentiation in stem cells within the same culture system in vitro and in vivo. Any changes in the differentiation markers were assessed. BMP2 was found to induce chondrogenesis and osteogenesis in vitro via the expression of Sox9, Runx2 and its downstream markers. According to the results of the subcutaneous stem cell implantation studies, BMP2 not only induced cartilage formation but also promoted endochondral ossification during ectopic bone/cartilage formation. In fetal limb cultures, BMP2 promoted chondrocyte hypertrophy and endochondral ossification. Our data reveal that BMP2 can spontaneously induce chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. Thus, BMP2 can be used in cartilage tissue engineering to regulate cartilage formation but has to be properly regulated for cartilage tissue engineering in order to retain the cartilage phenotype.

  2. Dexamethasone Suppresses Oxysterol-Induced Differentiation of Monocytic Cells

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    Yonghae Son

    2016-01-01

    Full Text Available Oxysterol like 27-hydroxycholesterol (27OHChol has been reported to induce differentiation of monocytic cells into a mature dendritic cell phenotype. We examined whether dexamethasone (Dx affects 27OHChol-induced differentiation using THP-1 cells. Treatment of monocytic cells with Dx resulted in almost complete inhibition of transcription and surface expression of CD80, CD83, and CD88 induced by 27OHChol. Elevated surface levels of MHC class I and II molecules induced by 27OHChol were reduced to basal levels by treatment with Dx. A decreased endocytosis ability caused by 27OHChol was recovered by Dx. We also examined effects of Dx on expression of CD molecules involved in atherosclerosis. Increased levels of surface protein and transcription of CD105, CD137, and CD166 by treatment with 27OHChol were significantly inhibited by cotreatment with Dx. These results indicate that Dx inhibits 27OHChol-induced differentiation of monocytic cells into a mature dendritic cell phenotype and expression of CD molecules whose levels are associated with atherosclerosis. In addition, we examined phosphorylation of AKT induced by 27OHChol and effect of Dx, where cotreatment with Dx inhibited the phosphorylation of AKT. The current study reports that Dx regulates oxysterol-mediated dendritic cell differentiation of monocytic cells.

  3. NEW DESIGNED HMBA AGENTS AS INDUCERS OF ERYTHROLEUKEMIA CELL DIFFERENTIATION

    Institute of Scientific and Technical Information of China (English)

    王华力; 张世馥; 周建平; 章静波

    2002-01-01

    Objective.Searching for more potent and less toxic HMBA related agents. Methods.Human erythroleukemia cell K562,murine erythroleukemia cell (MEL) and its sub line MEL DS19 were used as target cells to select a cell line which is the most sensitive to HMBA,then analyzed the activity of inducing differentiation of two new designed HMBA derivatives:HMBPA [hexamethylenebi (3 pyridin) amide] and Co HDTA (ethylenediaminetetra acetic acid cobalt) using cell biology,cytochemical and molecular biology techniques. Results.We found that the MEL DS19 cells were most sensitive to HMBA (benzidine positive,B+ ~76% ).Co HDTA can inhibit the growth of MEL DS19,but induces differentiation just in a small population (B+ 2% ~4.5% ).Between 0.02~5μ mol/L,HMBPA induces 3% ~8% cells committed to differentiation with little inhibition of cell proliferation.1μ mol/L HMBPA and 2mmol/L HMBA together,can obviously increase the percentage of differentiated cell (B+ ~72% ),inhibit DNA synthesis and accelerate β globin transcription. Conclusion.The new HMBA derivatives may provide potential cancer differentiation inducers.

  4. Induced Differentiation of Adipose-derived Stromal Cells into Myoblasts

    Institute of Scientific and Technical Information of China (English)

    吴桂珠; 郑秀; 江忠清; 王金华; 宋岩峰

    2010-01-01

    This study aimed to induce the differentiation of isolated and purified adipose-derived stromal cells(ADSCs) into myoblasts,which may provide a new strategy for tissue engineering in patients with stress urinary incontinence(SUI).ADSCs,isolated and cultured ex vivo,were identified by flow cytometry and induced to differentiate into myoblasts in the presence of an induction solution consisting of DMEM supplemented with 5-azacytidine(5-aza),5% FBS,and 5% horse serum.Cellular morphology was observed under an i...

  5. Platelet-Released Growth Factors Induce Differentiation of Primary Keratinocytes

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    Andreas Bayer

    2017-01-01

    Full Text Available Autologous thrombocyte concentrate lysates, for example, platelet-released growth factors, (PRGFs or their clinically related formulations (e.g., Vivostat PRF® came recently into the physicians’ focus as they revealed promising effects in regenerative and reparative medicine such as the support of healing of chronic wounds. To elucidate the underlying mechanisms, we analyzed the influence of PRGF and Vivostat PRF on human keratinocyte differentiation in vitro and on epidermal differentiation status of skin wounds in vivo. Therefore, we investigated the expression of early (keratin 1 and keratin 10 and late (transglutaminase-1 and involucrin differentiation markers. PRGF treatment of primary human keratinocytes decreased keratin 1 and keratin 10 gene expression but induced involucrin and transglutaminase-1 gene expression in an epidermal growth factor receptor- (EGFR- dependent manner. In concordance with these results, microscopic analyses revealed that PRGF-treated human keratinocytes displayed morphological features typical of keratinocytes undergoing terminal differentiation. In vivo treatment of artificial human wounds with Vivostat PRF revealed a significant induction of involucrin and transglutaminase-1 gene expression. Together, our results indicate that PRGF and Vivostat PRF induce terminal differentiation of primary human keratinocytes. This potential mechanism may contribute to the observed beneficial effects in the treatment of hard-to-heal wounds with autologous thrombocyte concentrate lysates in vivo.

  6. Globular adiponectin induces differentiation and fusion of skeletal muscle cells

    Institute of Scientific and Technical Information of China (English)

    Tania Fiaschi; Domenico Cirelli; Giuseppina Comito; Stefania Gelmini; Giampietro Ramponi; Maria Serio; Paola Chiarugi

    2009-01-01

    The growing interest in skeletal muscle regeneration is associated with the opening of new therapeutic strategies for muscle injury after trauma, as well as several muscular degenerative pathologies, including dystrophies, muscu-lar atrophy, and cachexia. Studies focused on the ability of extracellular factors to promote myogenesis are therefore highly promising. We now report that an adipocyte-derived factor, globular adiponectin (gAd), is able to induce mus-cle gene expression and cell differentiation, gAd, besides its well-known ability to regulate several metabolic func-tions in muscle, including glucose uptake and consumption and fatty acid catabolism, is able to block cell cycle entry of myoblasts, to induce the expression of specific skeletal muscle markers such as myosin heavy chain or eaveolin-3, as well as to provoke cell fusion into multinucleated syneytia and, finally, muscle fibre formation, gAd exerts its pro-differentiative activity through redox-dependent activation of p38, Akt and 5'-AMP-activated protein kinase path-ways. Interestingly, differentiating myoblasts are autocrine for adiponectiu, and the mimicking of pro-inflammatory settings or exposure to oxidative stress strongly increases the production of the hormone from differentiating cells. These data suggest a novel function of adiponectin, directly coordinating the myogenic differentiation program and serving an autocrine function during skeletal myogenesis.

  7. Did 26Al and impact-induced heating differentiate Mercury?

    Science.gov (United States)

    Bhatia, G. K.; Sahijpal, S.

    2017-02-01

    Numerical models dealing with the planetary scale differentiation of Mercury are presented with the short-lived nuclide, 26Al, as the major heat source along with the impact-induced heating during the accretion of planets. These two heat sources are considered to have caused differentiation of Mars, a planet with size comparable to Mercury. The chronological records and the thermal modeling of Mars indicate an early differentiation during the initial 1 million years (Ma) of the formation of the solar system. We theorize that in case Mercury also accreted over an identical time scale, the two heat sources could have differentiated the planets. Although unlike Mars there is no chronological record of Mercury's differentiation, the proposed mechanism is worth investigation. We demonstrate distinct viable scenarios for a wide range of planetary compositions that could have produced the internal structure of Mercury as deduced by the MESSENGER mission, with a metallic iron (Fe-Ni-FeS) core of radius 2000 km and a silicate mantle thickness of 400 km. The initial compositions were derived from the enstatite and CB (Bencubbin) chondrites that were formed in the reducing environments of the early solar system. We have also considered distinct planetary accretion scenarios to understand their influence on thermal processing. The majority of our models would require impact-induced mantle stripping of Mercury by hit and run mechanism with a protoplanet subsequent to its differentiation in order to produce the right size of mantle. However, this can be avoided if we increase the Fe-Ni-FeS contents to 71% by weight. Finally, the models presented here can be used to understand the differentiation of Mercury-like exoplanets and the planetary embryos of Venus and Earth.

  8. Hypercholesterolemia Induces Differentiation of Regulatory T Cells in the Liver.

    Science.gov (United States)

    Mailer, Reiner K W; Gisterå, Anton; Polyzos, Konstantinos A; Ketelhuth, Daniel F J; Hansson, Göran K

    2017-05-26

    The liver is the central organ that responds to dietary cholesterol intake and facilitates the release and clearance of lipoprotein particles. Persistent hypercholesterolemia leads to immune responses against lipoprotein particles that drive atherosclerosis. However, the effect of hypercholesterolemia on hepatic T-cell differentiation remains unknown. To investigate hepatic T-cell subsets upon hypercholesterolemia. We observed that hypercholesterolemia elevated the intrahepatic regulatory T (Treg) cell population and increased the expression of transforming growth factor-β1 in the liver. Adoptive transfer experiments revealed that intrahepatically differentiated Treg cells relocated to the inflamed aorta in atherosclerosis-prone low-density lipoprotein receptor deficient (Ldlr(-/-)) mice. Moreover, hypercholesterolemia induced the differentiation of intrahepatic, but not intrasplenic, Th17 cells in wild-type mice, whereas the disrupted liver homeostasis in hypercholesterolemic Ldlr(-/-) mice led to intrahepatic Th1 cell differentiation and CD11b(+)CD11c(+) leukocyte accumulation. Our results elucidate a new mechanism that controls intrahepatic T-cell differentiation during atherosclerosis development and indicates that intrahepatically differentiated T cells contribute to the CD4(+) T-cell pool in the atherosclerotic aorta. © 2017 American Heart Association, Inc.

  9. Sambucus williamsii induced embryonic stem cells differentiated into neurons.

    Science.gov (United States)

    Liu, Shih-Ping; Hsu, Chien-Yu; Fu, Ru-Huei; Huang, Yu-Chuen; Chen, Shih-Yin; Lin, Shinn-Zong; Shyu, Woei-Cherng

    2015-01-01

    The pluripotent stem cells, including embryonic stem cells (ESCs), are capable of self-renewal and differentiation into any cell type, thus making them the focus of many clinical application studies. However, the efficiency of ESCs differentiated into neurons needs to improve. In this study, we tried to increase efficiently to a neural fate in the presence of various transitional Chinese medicines through a three-step differentiation strategy. From extracts of 10 transitional Chinese medicine candidates, we determined that Sambucus williamsii (SW) extract triggers the up-regulation of Nestin and Tuj1 (neuron cells markers) gene expression levels. After determining the different concentrations of SW extract, the number of neurons in the 200 μg/ml SW extract group was higher than the control, 50, 100, and 400 μg/ml SW extract groups. In addition, the number of neurons in the 200 μg/ml SW extract group was higher and higher after each time passage (three times). We also detected the Oct4, Sox2 (stem cells markers), Tuj1, and Nestin genes expression levels by RT-PCR. In the differentiated process, Oct4 and Sox2 genes decreased while the Tuj1 and Nestin genes expression levels increased. In summary, we demonstrated that SW could induce pluripotent stem cells differentiated into neurons. Thus, SW might become a powerful material for neurons-differentiating strategies.

  10. Demethylation of the pesticide methoxychlor in liver and intestine from untreated, methoxychlor-treated, and 3-methylcholanthrene-treated channel catfish (Ictalurus punctatus): evidence for roles of CYP1 and CYP3A family isozymes.

    Science.gov (United States)

    Stuchal, Leah D; Kleinow, Kevin M; Stegeman, John J; James, Margaret O

    2006-06-01

    Exposure to the organochlorine pesticide methoxychlor (MXC) is associated with endocrine disruption in several species through biotransformation to mono-desmethyl-MXC (OH-MXC) and bis-desmethyl-MXC (HPTE), which interact with estrogen receptors. The biotransformation of [14C]methoxychlor was examined in channel catfish (Ictalurus punctatus), a freshwater species found in the southern United States. Hepatic microsomes formed OH-MXC and HPTE, assessed by comigration with authentic standards. The Km for OH-MXC formation by control liver microsomes was 3.8 +/- 1.3 microM (mean +/- S.D., n = 4), and Vmax was 131 +/- 53 pmol/min/mg protein. These values were similar to those of catfish pretreated with 2 mg/kg methoxychlor i.p. for 6 days (Km 3.3 +/- 0.8 microM and Vmax 99 +/- 17 pmol/min/mg) but less (p Methoxychlor pretreatment significantly reduced intestinal metabolite formation from 32 +/- 4 to 15 +/- 6 pmol/min/mg (mean +/- S.D., n = 4), whereas 3-MC treatment significantly increased OH-MXC production to 72 +/- 22 pmol/min/mg. Ketoconazole, clotrimazole, and alpha-naphthoflavone all decreased the production of OH-MXC in liver microsomes, whereas alpha-naphthoflavone stimulated HPTE formation, suggesting that CYP1 and CYP3 family isozymes demethylated methoxychlor. The results suggest that the formation of estrogenic metabolites from methoxychlor would be more rapid in catfish coexposed to CYP1 inducers.

  11. Radiation-Induced Differentiation in Human Lung Fibroblast

    Energy Technology Data Exchange (ETDEWEB)

    Park, Sa-Rah; Ahn, Ji-Yeon; Han, Young-Soo; Shim, Jie-Young; Yun, Yeon-Sook; Song, Jie-Young [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2007-10-15

    One of the most common tumors in many countries is lung cancer and patients with lung cancer may take radiotherapy. Although radiotherapy may have its own advantages, it can also induce serious problems such as acute radiation pneumonitis and pulmonary fibrosis. Pulmonary fibrosis is characterized by excessive production of {alpha}-SMA and accumulation of extracellular matrix (ECM) such as collagen and fibronectin. There has been a great amount of research about fibrosis but the exact mechanism causing the reaction is not elucidated especially in radiation-induced fibrosis. Until now it has been known that several factors such as transforming growth factor (TGF-{beta}), tumor necrosis factor (TNF), interleukin (IL)-1, IL-6, platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) are related to fibrosis. Among them TGF-{beta} with Smad signaling is known to be the main stream and other signaling molecules such as MAPK, ERK and JNK (3) also participates in the process. In addition to those above factors, it is thought that more diverse and complicate mechanisms may involve in the radiationinduced fibrosis. Therefore, to investigate the underlying mechanisms in radiation induced fibrosis, first of all, we confirmed whether radiation induces trans differentiation in human normal lung fibroblasts. Here, we suggest that not only TGF-{beta} but also radiation can induce trans differentiation in human lung fibroblast WI-38 and IMR-90.

  12. Insulin Cannot Induce Adipogenic Differentiation in Primary Cardiac Cultures

    Science.gov (United States)

    Parameswaran, Sreejit; Sharma, Rajendra K.

    2016-01-01

    Cardiac tissue contains a heterogeneous population of cardiomyocytes and nonmyocyte population especially fibroblasts. Fibroblast differentiation into adipogenic lineage is important for fat accumulation around the heart which is important in cardiac pathology. The differentiation in fibroblast has been observed both spontaneously and due to increased insulin stimulation. The present study aims to observe the effect of insulin in adipogenic differentiation of cardiac cells present in primary murine cardiomyocyte cultures. Oil Red O (ORO) staining has been used for observing the lipid accumulations formed due to adipogenic differentiation in murine cardiomyocyte cultures. The accumulated lipids were quantified by ORO assay and normalized using protein estimation. The lipid accumulation in cardiac cultures did not increase in presence of insulin. However, addition of other growth factors like insulin-like growth factor 1 and epidermal growth factor promoted adipogenic differentiation even in the presence of insulin and other inhibitory molecules such as vitamins. Lipid accumulation also increased in cells grown in media without insulin after an initial exposure to insulin-containing growth media. The current study adds to the existing knowledge that the insulin by itself cannot induce adipogenic induction in the cardiac cultures. The data have significance in the understanding of cardiovascular health especially in diabetic patients. PMID:27574386

  13. Cyclosporin A induces cardiac differentiation but inhibits hemato-endothelial differentiation of P19 cells.

    Directory of Open Access Journals (Sweden)

    Seung-Cheol Choi

    Full Text Available Little is known about the mechanisms underlying the effects of Cyclosporin A (CsA on the fate of stem cells, including cardiomyogenic differentiation. Therefore, we investigated the effects and the molecular mechanisms behind the actions of CsA on cell lineage determination of P19 cells. CsA induced cardiomyocyte-specific differentiation of P19 cells, with the highest efficiency at a concentration of 0.32 μM during embryoid body (EB formation via activation of the Wnt signaling pathway molecules, Wnt3a, Wnt5a, and Wnt8a, and the cardiac mesoderm markers, Mixl1, Mesp1, and Mesp2. Interestingly, cotreatment of P19 cells with CsA plus dimethyl sulfoxide (DMSO during EB formation significantly increases cardiac differentiation. In contrast, mRNA expression levels of hematopoietic and endothelial lineage markers, including Flk1 and Er71, were severely reduced in CsA-treated P19 cells. Furthermore, expression of Flk1 protein and the percentage of Flk1+ cells were severely reduced in 0.32 μM CsA-treated P19 cells compared to control cells. CsA significantly modulated mRNA expression levels of the cell cycle molecules, p53 and Cyclins D1, D2, and E2 in P19 cells during EB formation. Moreover, CsA significantly increased cell death and reduced cell number in P19 cells during EB formation. These results demonstrate that CsA induces cardiac differentiation but inhibits hemato-endothelial differentiation via activation of the Wnt signaling pathway, followed by modulation of cell lineage-determining genes in P19 cells during EB formation.

  14. Strain-induced negative differential resistance in ultrasmall carbon nanotube

    Science.gov (United States)

    Fang, Hui; Zhang, Fei-Peng; Ruan, Xing-Xiang; Huang, Can-Sheng; Jiang, Zhi-Nian; Peng, Jin-Yun; Wang, Ru-Zhi

    2017-08-01

    The transport properties in ultrasmall single-wall carbon nanotubes (SWCNTs) under tensile strain have been theoretically investigated. The regular negative differential resistance (NDR) induced by the strain undergoes a process from enhancement to weakening in the zigzag (3,0) SWCNT. The NDR achieves maximum with applying 4% tensile strain. Compared to the case of (3,0) SWCNT, that NDR cannot be manipulated by applying strain clearly in (4,0) and (5,0) ultrasmall SWCNTs with tensile strain lower than 10%. It proposes this strain-induced NDR effect to demonstrate the possibility of finding potential applications in SWCNT-based NDR nanodevices such as in memory devices, oscillators and fast switching devices.

  15. Mineralized gelatin methacrylate-based matrices induce osteogenic differentiation of human induced pluripotent stem cells

    Science.gov (United States)

    Kang, Heemin; Shih, Yu-Ru V.; Hwang, Yongsung; Wen, Cai; Rao, Vikram; Seo, Timothy; Varghese, Shyni

    2014-01-01

    Human induced pluripotent stem cells (hiPSCs) are a promising cell source with pluripotency and self-renewal properties. Design of simple and robust biomaterials with an innate ability to induce lineage-specificity of hiPSCs is desirable to realize their applications in regenerative medicine. In this study, we investigated the potential of biomaterials containing calcium phosphate minerals to induce osteogenic differentiation of hiPSCs. hiPSCs cultured using mineralized gelatin methacrylate-based matrices underwent osteogenic differentiation ex vivo, both in two- dimensional (2-D) and three-dimensional (3-D) cultures, in growth medium devoid of any osteogenic-inducing chemical components or growth factors. Our findings that osteogenic differentiation of hiPSCs can be achieved through biomaterial-based cues alone present new avenues for personalized regenerative medicine. Such biomaterials that could not only act as structural scaffolds, but could also provide tissue-specific functions such as directing stem cell differentiation commitment, have great potential in bone tissue engineering. PMID:25153779

  16. DIFFERENTIATION AND MALIGNANT SUPPRESSION INDUCED BY MOUSE ERYTHROID DIFFERENTIATION AND DENUCLEATION FACTOR ON MOUSE ERYTHROLEUKEMIA CELLS

    Institute of Scientific and Technical Information of China (English)

    韩代书; 赵青; 葛晔华; 周建平; 马静; 陈克铨; 薛社普

    2002-01-01

    Objective. To investigate the roles of mouse erythroid differentiation and denueleation factor (MEDDF), a novel factor cloned in our laboratory recently, in erythroid terminal differentiation.Methods. Mouse erythroleukemia (MEL) cells were transfected with eukaryotic expression plasmid pcD-NA-MEDDF. Then we investigated the changes on characteristics of cell growth by analyzing cells growth rate,mitotic index and colony-forming rate in semi-solid medium. The expressions of c-myc and β-globin genes were analysed by semi-quantitative RT-PCR.Results. MEL ceils transfected with pcDNA-MEDDF showed significant lower growth rate, mitotic index,and colony-forming rate in semi-solid medium ( P<0.01 ). The percentage of benzidine-positive cells was 32.8% after transfection. The expression of β-globin in cells transfected with pcDNA-MEDDF was 3.43 times higher than that of control (MEL transfected with blank vector, pcDNA3. 1 ), and the expression of c-myc decreased by 66.3%.Conclusions. MEDDF can induce differentiation of MEL cell and suppress its malignancy.

  17. Decreased Intracellular pH Induced by Cariporide Differentially Contributes to Human Umbilical Cord-Derived Mesenchymal Stem Cells Differentiation

    Directory of Open Access Journals (Sweden)

    Wei Gao

    2014-01-01

    Full Text Available Background/Aims: Na+/H+ exchanger 1 (NHE1 is an important regulator of intracellular pH (pHi. High pHi is required for cell proliferation and differentiation. Our previous study has proven that the pHi of mesenchymal stem cells is higher than that of normal differentiated cells and similar to tumor cells. NHE1 is highly expressed in both mesenchymal stem cells and tumor cells. Targeted inhibition of NHE1 could induce differentiation of K562 leukemia cells. In the present paper we explored whether inhibition of NHE1 could induce differentiation of mesenchymal stem cells. Methods: MSCs were obtained from human umbilical cord and both the surface phenotype and functional characteristics were analyzed. Selective NHE1 inhibitor cariporide was used to treat human umbilical cord-derived mesenchymal stem cells (hUC-MSCs. The pHi and the differentiation of hUC-MSCs were compared upon cariporide treatment. The putative signaling pathway involved was also explored. Results: The pHi of hUC-MSCs was decreased upon cariporide treatment. Cariporide up-regulated the osteogenic differentiation of hUC-MSCs while the adipogenic differentiation was not affected. For osteogenic differentiation, β-catenin expression was up-regulated upon cariporide treatment. Conclusion: Decreased pHi induced by cariporide differentially contributes to hUC-MSCs differentiation.

  18. Bitter taste stimuli induce differential neural codes in mouse brain.

    Directory of Open Access Journals (Sweden)

    David M Wilson

    Full Text Available A growing literature suggests taste stimuli commonly classified as "bitter" induce heterogeneous neural and perceptual responses. Here, the central processing of bitter stimuli was studied in mice with genetically controlled bitter taste profiles. Using these mice removed genetic heterogeneity as a factor influencing gustatory neural codes for bitter stimuli. Electrophysiological activity (spikes was recorded from single neurons in the nucleus tractus solitarius during oral delivery of taste solutions (26 total, including concentration series of the bitter tastants quinine, denatonium benzoate, cycloheximide, and sucrose octaacetate (SOA, presented to the whole mouth for 5 s. Seventy-nine neurons were sampled; in many cases multiple cells (2 to 5 were recorded from a mouse. Results showed bitter stimuli induced variable gustatory activity. For example, although some neurons responded robustly to quinine and cycloheximide, others displayed concentration-dependent activity (p<0.05 to quinine but not cycloheximide. Differential activity to bitter stimuli was observed across multiple neurons recorded from one animal in several mice. Across all cells, quinine and denatonium induced correlated spatial responses that differed (p<0.05 from those to cycloheximide and SOA. Modeling spatiotemporal neural ensemble activity revealed responses to quinine/denatonium and cycloheximide/SOA diverged during only an early, at least 1 s wide period of the taste response. Our findings highlight how temporal features of sensory processing contribute differences among bitter taste codes and build on data suggesting heterogeneity among "bitter" stimuli, data that challenge a strict monoguesia model for the bitter quality.

  19. Statins induce differentiation and cell death in neurons and astroglia.

    Science.gov (United States)

    März, Pia; Otten, Uwe; Miserez, André R

    2007-01-01

    Statins are potent inhibitors of the hydroxy-methyl-glutaryl-coenzyme A reductase, the rate limiting enzyme for cholesterol biosynthesis. Experimental and clinical studies with statins suggest that they have beneficial effects on neurodegenerative disorders. Thus, it was of interest to characterize the direct effects of statins on CNS neurons and glial cells. We have treated defined cultures of neurons and astrocytes of newborn rats with two lipophilic statins, atorvastatin and simvastatin, and analyzed their effects on morphology and survival. Treatment of astrocytes with statins induced a time- and dose-dependent stellation, followed by apoptosis. Similarly, statins elicited programmed cell death of cerebellar granule neurons but with a higher sensitivity. Analysis of different signaling cascades revealed that statins fail to influence classical pathways such as Akt or MAP kinases, known to be activated in CNS cells. In addition, astrocyte stellation triggered by statins resembled dibutryl-cyclic AMP (db-cAMP) induced morphological differentiation. However, in contrast to db-cAMP, statins induced upregulation of low-density lipoprotein receptors, without affecting GFAP expression, indicating separate underlying mechanisms. Analysis of the cholesterol biosynthetic pathway revealed that lack of mevalonate and of its downstream metabolites, mainly geranylgeranyl-pyrophosphate (GGPP), is responsible for the statin-induced apoptosis of neurons and astrocytes. Moreover, astrocytic stellation triggered by statins was inhibited by mevalonate and GGPP. Interestingly, neuronal cell death was significantly reduced in astrocyte/neuron co-cultures treated with statins. We postulate that under these conditions signals provided by astrocytes, e.g., isoprenoids play a key role in neuronal survival.

  20. An effective inducer of dopaminergic neuron-like differentiation

    Institute of Scientific and Technical Information of China (English)

    Wenyu Fu; Cui Lv; Wenxin Zhuang; Dandan Chen; E Lv; Fengjie Li; Xiaocui Wang

    2013-01-01

    Rat bone marrow-derived mesenchymal stem cells were cultured and passaged in vitro. After induction with basic fibroblast growth factor for 24 hours, passage 3 bone marrow-derived mesenchymal stem cells were additionally induced into dopaminergic neurons using three different combinations with basic fibroblast growth factor as follows: 20% Xiangdan injection; all-trans retinoic acid + glial-derived neurotrophic factor; or sonic hedgehog + fibroblast growth factor 8. Results suggest that the bone marrow-derived mesenchymal stem cells showed typical neuronal morphological characteristics after induction. In particular, after treatment with sonic hedgehog + fibroblast growth factor 8, the expressions of nestin, neuron-specific enolase, microtubuleassociated protein 2, tyrosine hydroxylase and vesicular monoamine transporter-2 in cells were significantly increased. Moreover, the levels of catecholamines in the culture supernatant were significantly increased. These findings indicate that Xiangdan injection, all-trans retinoic acid + glial-derived neurotrophic factor, and sonic hedgehog + fibroblast growth factor 8 can all induce dopaminergic neuronal differentiation from bone marrow-derived mesenchymal stem cells. In particular, the efficiency of sonic hedgehog + fibroblast growth factor 8 was highest.

  1. Hematopoietic cell differentiation from embryonic and induced pluripotent stem cells

    Science.gov (United States)

    2013-01-01

    Pluripotent stem cells, both embryonic stem cells and induced pluripotent stem cells, are undifferentiated cells that can self-renew and potentially differentiate into all hematopoietic lineages, such as hematopoietic stem cells (HSCs), hematopoietic progenitor cells and mature hematopoietic cells in the presence of a suitable culture system. Establishment of pluripotent stem cells provides a comprehensive model to study early hematopoietic development and has emerged as a powerful research tool to explore regenerative medicine. Nowadays, HSC transplantation and hematopoietic cell transfusion have successfully cured some patients, especially in malignant hematological diseases. Owing to a shortage of donors and a limited number of the cells, hematopoietic cell induction from pluripotent stem cells has been regarded as an alternative source of HSCs and mature hematopoietic cells for intended therapeutic purposes. Pluripotent stem cells are therefore extensively utilized to facilitate better understanding in hematopoietic development by recapitulating embryonic development in vivo, in which efficient strategies can be easily designed and deployed for the generation of hematopoietic lineages in vitro. We hereby review the current progress of hematopoietic cell induction from embryonic stem/induced pluripotent stem cells. PMID:23796405

  2. Derivation, characterization and retinal differentiation of induced pluripotent stem cells

    Indian Academy of Sciences (India)

    Subba Rao Mekala; Vasundhara Vauhini; Usha Nagarajan; Savitri Maddileti; Subhash Gaddipati; Indumathi Mariappan

    2013-03-01

    Millions of people world over suffer visual disability due to retinal dystrophies which can be age-related or a genetic disorder resulting in gradual degeneration of the retinal pigmented epithelial (RPE) cells and photoreceptors. Therefore, cell replacement therapy offers a great promise in treating such diseases. Since the adult retina does not harbour any stem cells, alternative stem cell sources like the embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer a great promise for generating different cell types of the retina. Here, we report the derivation of four iPSC lines from mouse embryonic fibroblasts (MEFs) using a cocktail of recombinant retroviruses carrying the genes for Oct4, Sox2, Klf4 and cMyc. The iPS clone MEF-4F3 was further characterized for stemness marker expression and stable reprogramming by immunocytochemistry, FACS and RT-PCR analysis. Methylation analysis of the nanog promoter confirmed the reprogrammed epigenetic state. Pluripotency was confirmed by embryoid body (EB) formation and lineage-specific marker expression. Also, upon retinal differentiation, patches of pigmented cells with typical cobble-stone phenotype similar to RPE cells are generated within 6 weeks and they expressed ZO-1 (tight junction protein), RPE65 and bestrophin (mature RPE markers) and showed phagocytic activity by the uptake of fluorescent latex beads.

  3. Angiotensin II induces differential insulin action in rat skeletal muscle.

    Science.gov (United States)

    Surapongchai, Juthamard; Prasannarong, Mujalin; Bupha-Intr, Tepmanas; Saengsirisuwan, Vitoon

    2017-03-01

    Angiotensin II (ANGII) is reportedly involved in the development of skeletal muscle insulin resistance. The present investigation evaluated the effects of two ANGII doses on the phenotypic characteristics of insulin resistance syndrome and insulin action and signaling in rat skeletal muscle. Male Sprague-Dawley rats were infused with either saline (SHAM) or ANGII at a commonly used pressor dose (100 ng/kg/min; ANGII-100) or a higher pressor dose (500 ng/kg/min; ANGII-500) via osmotic minipumps for 14 days. We demonstrated that ANGII-100-infused rats exhibited the phenotypic features of non-obese insulin resistance syndrome, including hypertension, impaired glucose tolerance and insulin resistance of glucose uptake in the soleus muscle, whereas ANGII-500-treated rats exhibited diabetes-like symptoms, such as post-prandial hyperglycemia, impaired insulin secretion and hypertriglyceridemia. At the cellular level, insulin-stimulated glucose uptake in the soleus muscle of the ANGII-100 group was 33% lower (P study demonstrates for the first time that chronic infusion with these two pressor doses of ANGII induced differential metabolic responses at both the systemic and skeletal muscle levels.

  4. Monocyte cell surface glycosaminoglycans positively modulate IL-4-induced differentiation toward dendritic cells.

    NARCIS (Netherlands)

    Dekker, E. den; Grefte, S.; Huijs, T.; Dam, G.B. ten; Versteeg, E.M.M.; Berk, L.C.J. van den; Bladergroen, B.A.; Kuppevelt, A.H.M.S.M. van; Figdor, C.G.; Torensma, R.

    2008-01-01

    IL-4 induces the differentiation of monocytes toward dendritic cells (DCs). The activity of many cytokines is modulated by glycosaminoglycans (GAGs). In this study, we explored the effect of GAGs on the IL-4-induced differentiation of monocytes toward DCs. IL-4 dose-dependently up-regulated the expr

  5. Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin

    OpenAIRE

    2015-01-01

    Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34+ hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex...

  6. Identification of Pathways Mediating Growth Differentiation Factor5-Induced Tenogenic Differentiation in Human Bone Marrow Stromal Cells.

    Directory of Open Access Journals (Sweden)

    Sik-Loo Tan

    Full Text Available To date, the molecular signalling mechanisms which regulate growth factors-induced MSCs tenogenic differentiation remain largely unknown. Therefore, a study to determine the global gene expression profile of tenogenic differentiation in human bone marrow stromal cells (hMSCs using growth differentiation factor 5 (GDF5 was conducted. Microarray analyses were conducted on hMSCs cultures supplemented with 100 ng/ml of GDF5 and compared to undifferentiated hMSCs and adult tenocytes. Results of QuantiGene® Plex assay support the use and interpretation of the inferred gene expression profiles and pathways information. From the 27,216 genes assessed, 873 genes (3.21% of the overall human transcriptome were significantly altered during the tenogenic differentiation process (corrected p<0.05. The genes identified as potentially associated with tenogenic differentiation were ARHGAP29, CCL2, integrin alpha 8 and neurofilament medium polypeptides. These genes, were mainly associated with cytoskeleton reorganization (stress fibers formation signaling. Pathway analysis demonstrated the potential molecular pathways involved in tenogenic differentiation were: cytoskeleton reorganization related i.e. keratin filament signaling and activin A signaling; cell adhesion related i.e. chemokine and adhesion signaling; and extracellular matrix related i.e. arachidonic acid production signaling. Further investigation using atomic force microscopy and confocal laser scanning microscopy demonstrated apparent cytoskeleton reorganization in GDF5-induced hMSCs suggesting that cytoskeleton reorganization signaling is an important event involved in tenogenic differentiation. Besides, a reduced nucleostemin expression observed suggested a lower cell proliferation rate in hMSCs undergoing tenogenic differentiation. Understanding and elucidating the tenogenic differentiation signalling pathways are important for future optimization of tenogenic hMSCs for functional tendon cell

  7. PI3K/AKT and ERK regulate retinoic acid-induced neuroblastoma cellular differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Qiao, Jingbo [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Paul, Pritha; Lee, Sora [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Qiao, Lan; Josifi, Erlena; Tiao, Joshua R. [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Chung, Dai H., E-mail: dai.chung@vanderbilt.edu [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Retinoic acid (RA) induces neuroblastoma cells differentiation, which is accompanied by G0/G1 cell cycle arrest. Black-Right-Pointing-Pointer RA resulted in neuroblastoma cell survival and inhibition of DNA fragmentation; this is regulated by PI3K pathway. Black-Right-Pointing-Pointer RA activates PI3K and ERK1/2 pathway; PI3K pathway mediates RA-induced neuroblastoma cell differentiation. Black-Right-Pointing-Pointer Upregulation of p21 is necessary for RA-induced neuroblastoma cell differentiation. -- Abstract: Neuroblastoma, the most common extra-cranial solid tumor in infants and children, is characterized by a high rate of spontaneous remissions in infancy. Retinoic acid (RA) has been known to induce neuroblastoma differentiation; however, the molecular mechanisms and signaling pathways that are responsible for RA-mediated neuroblastoma cell differentiation remain unclear. Here, we sought to determine the cell signaling processes involved in RA-induced cellular differentiation. Upon RA administration, human neuroblastoma cell lines, SK-N-SH and BE(2)-C, demonstrated neurite extensions, which is an indicator of neuronal cell differentiation. Moreover, cell cycle arrest occurred in G1/G0 phase. The protein levels of cyclin-dependent kinase inhibitors, p21 and p27{sup Kip}, which inhibit cell proliferation by blocking cell cycle progression at G1/S phase, increased after RA treatment. Interestingly, RA promoted cell survival during the differentiation process, hence suggesting a potential mechanism for neuroblastoma resistance to RA therapy. Importantly, we found that the PI3K/AKT pathway is required for RA-induced neuroblastoma cell differentiation. Our results elucidated the molecular mechanism of RA-induced neuroblastoma cellular differentiation, which may be important for developing novel therapeutic strategy against poorly differentiated neuroblastoma.

  8. Protein palmitoylation regulates osteoblast differentiation through BMP-induced osterix expression.

    Directory of Open Access Journals (Sweden)

    Wai Fook Leong

    Full Text Available Osteoporosis is one of the most common diseases and can be treated by either anti-resorption drugs, anabolic drugs, or both. To search for anabolic drug targets for osteoporosis therapy, it is crucial to understand the biology of bone forming cells, osteoblasts, in terms of their proliferation, differentiation, and function. Here we found that protein palmitoylation participates in signaling pathways that control osterix expression and osteoblast differentiation. Mouse calvarial osteoblasts express most of the 24 palmitoyl transferases, with some being up-regulated during differentiation. Inhibition of protein palmitoylation, with a substrate-analog inhibitor, diminished osteoblast differentiation and mineralization, but not proliferation or survival. The decrease in differentiation capacity is associated with a reduction in osterix, but not Runx2 or Atf4. Inhibition of palmitoyl transferases had little effect in p53(-/- osteoblasts that show accelerated differentiation due to overexpression of osterix, suggesting that osterix, at least partially, mediated the effect of inhibition of palmitoyl transferases on osteoblast differentiation. BMPs are the major driving force of osteoblast differentiation in the differentiation assays. We found that inhibition of palmitoyl transferases also compromised BMP2-induced osteoblast differentiation through down-regulating osterix induction. However, palmitoyl transferases inhibitor did not inhibit Smad1/5/8 activation. Instead, it compromised the activation of p38 MAPK, which are known positive regulators of osterix expression and differentiation. These results indicate that protein palmitoylation plays an important role in BMP-induced MAPK activation, osterix expression, and osteoblast differentiation.

  9. Sonic Hedgehog activation is implicated in diosgenin-induced megakaryocytic differentiation of human erythroleukemia cells.

    Science.gov (United States)

    Ghezali, Lamia; Liagre, Bertrand; Limami, Youness; Beneytout, Jean-Louis; Leger, David Yannick

    2014-01-01

    Differentiation therapy is a means to treat cancer and is induced by different agents with low toxicity and more specificity than traditional ones. Diosgenin, a plant steroid, is able to induce megakaryocytic differentiation or apoptosis in human HEL erythroleukemia cells in a dose-dependent manner. However, the exact mechanism by which diosgenin induces megakaryocytic differentiation has not been elucidated. In this study, we studied the involvement of Sonic Hedgehog in megakaryocytic differentiation induced by diosgenin in HEL cells. First, we showed that different elements of the Hedgehog pathway are expressed in our model by qRT-PCR. Then, we focused our interest on key elements in the Sonic Hedgehog pathway: Smoothened receptor, GLI transcription factor and the ligand Sonic Hedgehog. We showed that Smoothened and Sonic Hedgehog were overexpressed in disogenin-treated cells and that GLI transcription factors were activated. Then, we showed that SMO inhibition using siSMO or the GLI antagonist GANT-61, blocked megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, we demonstrated that Sonic Hedgehog pathway inhibition led to inhibition of ERK1/2 activation, a major physiological pathway involved in megakaryocytic differentiation. In conclusion, our study reports, for the first time, a crucial role for the Sonic Hedgehog pathway in diosgenin-induced megakaryocytic differentiation in HEL cells.

  10. Sonic Hedgehog activation is implicated in diosgenin-induced megakaryocytic differentiation of human erythroleukemia cells.

    Directory of Open Access Journals (Sweden)

    Lamia Ghezali

    Full Text Available Differentiation therapy is a means to treat cancer and is induced by different agents with low toxicity and more specificity than traditional ones. Diosgenin, a plant steroid, is able to induce megakaryocytic differentiation or apoptosis in human HEL erythroleukemia cells in a dose-dependent manner. However, the exact mechanism by which diosgenin induces megakaryocytic differentiation has not been elucidated. In this study, we studied the involvement of Sonic Hedgehog in megakaryocytic differentiation induced by diosgenin in HEL cells. First, we showed that different elements of the Hedgehog pathway are expressed in our model by qRT-PCR. Then, we focused our interest on key elements in the Sonic Hedgehog pathway: Smoothened receptor, GLI transcription factor and the ligand Sonic Hedgehog. We showed that Smoothened and Sonic Hedgehog were overexpressed in disogenin-treated cells and that GLI transcription factors were activated. Then, we showed that SMO inhibition using siSMO or the GLI antagonist GANT-61, blocked megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, we demonstrated that Sonic Hedgehog pathway inhibition led to inhibition of ERK1/2 activation, a major physiological pathway involved in megakaryocytic differentiation. In conclusion, our study reports, for the first time, a crucial role for the Sonic Hedgehog pathway in diosgenin-induced megakaryocytic differentiation in HEL cells.

  11. Differentiation-inducing and anti-proliferative activities of lupeol on canine melanoma cells.

    Science.gov (United States)

    Ogihara, Kikumi; Naya, Yuko; Okamoto, Yoshiharu; Hata, Keishi

    2014-01-01

    Canine melanoma is the most common oral malignant tumor reported in the field of veterinary medicine. We found that lupeol, a lupine triterpene, inhibited mouse melanoma cell growth in vitro and in vivo by inducing cell differentiation. In the present study, we examined the differentiation-inducing activities of lupeol on 4 canine melanoma cells in vitro and in vivo. The induction of canine melanoma cell differentiation by lupeol was confirmed by evaluating some differentiation markers such as tyrosinase with real-time RT-PCR. Furthermore, we transplanted canine melanoma cells into a severe combined immunodeficiency mouse, and studied the anti-progressive effects of lupeol on tumor tissue. The gene expression of microphthalmia-associated transcription factor, tyrosinase, and tyrosinase-related protein-2, which are markers of pigment cell differentiation, was induced in 4 canine oral malignant melanoma cells by lupeol, and the agent markedly inhibited tumor progression in canine melanoma-bearing mice.

  12. BMP-2 Induced Expression of Alx3 That Is a Positive Regulator of Osteoblast Differentiation.

    Directory of Open Access Journals (Sweden)

    Takashi Matsumoto

    Full Text Available Bone morphogenetic proteins (BMPs regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP-2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation, while it inhibits myogenic differentiation in C2C12 cells. To evaluate genes involved in BMP-2-induced osteoblast differentiation, we performed cDNA microarray analyses to compare BMP-2-treated and -untreated C2C12 cells. We focused on Alx3 (aristaless-like homeobox 3 which was clearly induced during osteoblast differentiation. Alx3, a homeobox gene related to the Drosophilaaristaless gene, has been linked to developmental functions in craniofacial structures and limb development. However, little is known about its direct relationship with bone formation. In the present study, we focused on the mechanisms of Alx3 gene expression and function during osteoblast differentiation induced by BMP-2. In C2C12 cells, BMP-2 induced increase of Alx3 gene expression in both time- and dose-dependent manners through the BMP receptors-mediated SMAD signaling pathway. In addition, silencing of Alx3 by siRNA inhibited osteoblast differentiation induced by BMP-2, as showed by the expressions of alkaline phosphatase (Alp, Osteocalcin, and Osterix, while over-expression of Alx3 enhanced osteoblast differentiation induced by BMP-2. These results indicate that Alx3 expression is enhanced by BMP-2 via the BMP receptors mediated-Smad signaling and that Alx3 is a positive regulator of osteoblast differentiation induced by BMP-2.

  13. Metformin Inhibits Angiotensin II-Induced Differentiation of Cardiac Fibroblasts into Myofibroblasts

    OpenAIRE

    Jian Bai; Na Zhang; Ying Hua; Bingjian Wang; Lin Ling; Albert Ferro; Biao Xu

    2013-01-01

    Differentiation of cardiac fibroblasts into myofibroblasts is a critical event in the progression of cardiac fibrosis that leads to pathological cardiac remodeling. Metformin, an antidiabetic agent, exhibits a number of cardioprotective properties. However, much less is known regarding the effect of metformin on cardiac fibroblast differentiation. Thus, in the present study, we examined the effect of metformin on angiotensin (Ang) II-induced differentiation of cardiac fibroblasts into myofibr...

  14. Regulation of Notch signaling genes during BMP2-induced differentiation of osteoblast precursor cells.

    NARCIS (Netherlands)

    Jong, D.S. de; Steegenga, W.T.; Hendriks, J.M.; Zoelen, E.J.J. van; Olijve, W.; Dechering, K.J.

    2004-01-01

    The bone morphogenetic protein (BMP)-induced Smad signal transduction pathway is an important positive regulator of osteoblast differentiation. BMP and other members of the transforming growth factor-beta (TGF-beta) family have distinct effects on osteoblast differentiation, depending on cell type a

  15. Regulation of Notch signaling genes during BMP2-induced differentiation of osteoblast precursor cells

    NARCIS (Netherlands)

    Jong, de D.S.; Steegenga, W.T.; Hendriks, J.M.A.; Zoelen, van E.J.J.; Olijve, W.; Dechering, K.J.

    2004-01-01

    The bone morphogenetic protein (BMP)-induced Smad signal transduction pathway is an important positive regulator of osteoblast differentiation. BMP and other members of the transforming growth factor-beta (TGF-beta) family have distinct effects on osteoblast differentiation, depending on cell type a

  16. Tinospora cordifolia Induces Differentiation and Senescence Pathways in Neuroblastoma Cells.

    Science.gov (United States)

    Mishra, Rachana; Kaur, Gurcharan

    2015-08-01

    Children diagnosed with neuroblastomas often suffer from severe side as well as late effects of conventional treatments like chemotherapy and radiotherapy. Recent advances in understanding of molecular pathways involved in cellular differentiation and apoptosis have helped in the development of new therapeutic approach based on differentiation-based therapy of malignant tumours. Natural medicines with their holistic therapeutic approach are known to selectively eliminate cancer cells thus provide a better substitute for the conventional treatment modes. The current study was aimed to investigate the anti-cancer potential of aqueous ethanolic extract of Tinospora cordifolia (TCE) using IMR-32 human neuroblastoma cell line as a model system. TCE is highly recommended in Ayurveda for its general body and metal health-promoting properties. TCE treatment was seen to arrest the majority of cells in G0/G1 phase and modulated the expression of DNA clamp sliding protein (PCNA) and cyclin D1. Further, TCE-treated cells showed differentiation as revealed by their morphology and the expression of neuronal cell specific differentiation markers NF200, MAP-2 and NeuN in neuroblastoma cells. The differentiated phenotype was associated with induction of senescence and pro-apoptosis pathways by enhancing expression of senescence marker mortalin and Rel A subunit of nuclear factor kappa beta (NFkB) along with decreased expression of anti-apoptotic marker, Bcl-xl. TCE exhibited anti-metastatic activity and significantly reduced cell migration in the scratched area along with downregulation of neural cell adhesion molecule (NCAM) polysialylation and secretion of matrix metalloproteinases (MMPs). Our data suggest that crude extract or active phytochemicals from this plant may be a potential candidate for differentiation-based therapy of malignant neuroblastoma cells.

  17. Graphene induces spontaneous cardiac differentiation in embryoid bodies

    Science.gov (United States)

    Ahadian, Samad; Zhou, Yuanshu; Yamada, Shukuyo; Estili, Mehdi; Liang, Xiaobin; Nakajima, Ken; Shiku, Hitoshi; Matsue, Tomokazu

    2016-03-01

    Graphene was embedded into the structure of mouse embryoid bodies (EBs) using the hanging drop technique. The inclusion of 0.2 mg per mL graphene in the EBs did not affect the viability of the stem cells. However, the graphene decreased the stem cell proliferation, probably by accelerating cell differentiation. The graphene also enhanced the mechanical properties and electrical conductivity of the EBs. Interestingly, the cardiac differentiation of the EB-graphene was significantly greater than that of the EBs at day 5 of culture, as confirmed by high-throughput gene analysis. Electrical stimulation (voltage, 4 V; frequency, 1 Hz; and duration, 10 ms for 2 continuous days) further enhanced the cardiac differentiation of the EBs, as demonstrated by analyses of the cardiac protein and gene expression and the beating activity of the EBs. Taken together, the results demonstrated that graphene played a major role in directing the cardiac differentiation of EBs, which has potential cell therapy and tissue regeneration applications.Graphene was embedded into the structure of mouse embryoid bodies (EBs) using the hanging drop technique. The inclusion of 0.2 mg per mL graphene in the EBs did not affect the viability of the stem cells. However, the graphene decreased the stem cell proliferation, probably by accelerating cell differentiation. The graphene also enhanced the mechanical properties and electrical conductivity of the EBs. Interestingly, the cardiac differentiation of the EB-graphene was significantly greater than that of the EBs at day 5 of culture, as confirmed by high-throughput gene analysis. Electrical stimulation (voltage, 4 V; frequency, 1 Hz; and duration, 10 ms for 2 continuous days) further enhanced the cardiac differentiation of the EBs, as demonstrated by analyses of the cardiac protein and gene expression and the beating activity of the EBs. Taken together, the results demonstrated that graphene played a major role in directing the cardiac

  18. The Preliminary Experimental Study of Induced Differentiation of Embryonic Stem Cells into Corneal Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    Ling Yu; Jian Ge; Zhichong Wang; Bing Huang; Keming Yu; Chongde Long; Xigu Chen

    2001-01-01

    Purpose:To study preliminarily induced differentiation of embryonic stem cells intocorneal epithelial cells in vitro.Methods: Murine embryonic stem cells were co-cultured with Rabbit limbal cornealepithelial cells in Transwell system to induce differentiation. Mophological andimmunohistochemical examination were implemented.Results: The induced cells from embryonic stem cells have an epithelial appearance.The cells formed a network and were confluent into film gradually after beingco-cultured with rabbit limbal corneal epithelial cells for 24 ~ 96 hours. The cells rangedmosaic structure and localized together with clear rim. Most of the cells showedpolygonal appearance. Transmission electron microscope showed lots of microvilli on thesurface of induced cells and tight junctions between them. These epithelial-like cellsexpressed the corneal epithelial cell specific marker cytokeratin3/cytokeratinl2.Conclusion: The potential mechanism of the differentiation of murine embryonic stemcells into corneal epithelial cells induced by limbal corneal epithelial cell-derivedinducing activity is to be further verified.

  19. Graphene induces spontaneous cardiac differentiation in embryoid bodies.

    Science.gov (United States)

    Ahadian, Samad; Zhou, Yuanshu; Yamada, Shukuyo; Estili, Mehdi; Liang, Xiaobin; Nakajima, Ken; Shiku, Hitoshi; Matsue, Tomokazu

    2016-04-07

    Graphene was embedded into the structure of mouse embryoid bodies (EBs) using the hanging drop technique. The inclusion of 0.2 mg per mL graphene in the EBs did not affect the viability of the stem cells. However, the graphene decreased the stem cell proliferation, probably by accelerating cell differentiation. The graphene also enhanced the mechanical properties and electrical conductivity of the EBs. Interestingly, the cardiac differentiation of the EB-graphene was significantly greater than that of the EBs at day 5 of culture, as confirmed by high-throughput gene analysis. Electrical stimulation (voltage, 4 V; frequency, 1 Hz; and duration, 10 ms for 2 continuous days) further enhanced the cardiac differentiation of the EBs, as demonstrated by analyses of the cardiac protein and gene expression and the beating activity of the EBs. Taken together, the results demonstrated that graphene played a major role in directing the cardiac differentiation of EBs, which has potential cell therapy and tissue regeneration applications.

  20. Effect of inducers and inhibitors of glucuronidation on the biliary excretion and choleretic action of valproic acid in the rat.

    Science.gov (United States)

    Watkins, J B; Klaassen, C D

    1982-02-01

    Valproic acid (VPA) induces an immediate choleresis in the rat which may be attributable to the osmotic properties of VPA-glucuronic acid conjugates in bile. The influence of inducers and inhibitors of glucuronidation of VPA on the biliary excretion and choleretic effect of VPA was studied. Hepatic UDP-glucuronyltransferase activity toward VPA was determined in vitro. Pretreatment with phenobarbital (75 mg/kg/day for 4 days) enhanced VPA glucuronidation; borneol (750 mg/kg) decreased VPA conjugation; 3-methylcholanthrene (20 mg/kg/day for 4 days) and galactosamine (600 mg/kg) had no effect on glucuronidation of VPA in vitro. Hepatic UDP-glucuronic acid content was decreased by borneol and galactosamine administration and was enhanced by phenobarbital and 3-methylcholanthrene pretreatment. The enzyme inducers increased the plasma disappearance of VPA in vivo but did not augment its biliary excretion or choleretic effect. Borneol and galactosamine, which inhibited the conjugation and plasma disappearance of VPA, decreased its biliary excretion and inhibited the VPA-induced increase in bile flow. Thus, the bile flow rate after VPA administration is closely related to the excretion of VPA-glucuronic acid. These data support the conclusion that the choleretic effect of VPA is due to the osmotic activity of VPA conjugates in bile.

  1. Isolation of furocoumarins from bergamot fruits as HL-60 differentiation-inducing compounds.

    Science.gov (United States)

    Kawaii, S; Tomono, Y; Katase, E; Ogawa, K; Yano, M

    1999-10-01

    The HL-60 differentiation-inducing compounds in bergamot fruits were isolated with column chromatography and identified as bergamottin, bergapten, and citropten by (1)H and (13)C NMR. Their HL-60 differentiation-inducing activity was measured by examining nitro blue tetrazolium (NBT) reducing, nonspecific acid esterase (NSE), specific esterase (SE), and phagocytic activities, and bergamottin showed the strongest activity among the coumarins isolated from bergamot fruits. The structure-activity relationship obtained from HL-60 differentiation assay suggests that hydrophobicity of furocoumarins is correlated with their activity.

  2. Use of laser-induced breakdown spectroscopy for the differentiation of pathogens and viruses on substrates.

    Science.gov (United States)

    Multari, Rosalie A; Cremers, David A; Bostian, Melissa L

    2012-03-01

    In this work, the use of laser-induced breakdown spectroscopy (LIBS) to differentiate live pathogens and killed viruses on substrates is investigated. Live pathogens B. anthracis Sterne strain and F. tularensis live vaccine strain were interrogated as lawn and colonies on agar; dilutions on agar; and dilutions on glass slides, and it was found possible to differentiate among all samples. UV killed hantavirusstrains were studied as dilutions on slides and it was also found possible to differentiate among strains. To the best of our knowledge, this is the first study in which LIBS has been used to differentiate virus samples. © 2012 Optical Society of America

  3. Natural Product Vibsanin A Induces Differentiation of Myeloid Leukemia Cells through PKC Activation.

    Science.gov (United States)

    Yu, Zu-Yin; Xiao, He; Wang, Li-Mei; Shen, Xing; Jing, Yu; Wang, Lin; Sun, Wen-Feng; Zhang, Yan-Feng; Cui, Yu; Shan, Ya-Jun; Zhou, Wen-Bing; Xing, Shuang; Xiong, Guo-Lin; Liu, Xiao-Lan; Dong, Bo; Feng, Jian-Nan; Wang, Li-Sheng; Luo, Qing-Liang; Zhao, Qin-Shi; Cong, Yu-Wen

    2016-05-01

    All-trans retinoic acid (ATRA)-based cell differentiation therapy has been successful in treating acute promyelocytic leukemia, a unique subtype of acute myeloid leukemia (AML). However, other subtypes of AML display resistance to ATRA-based treatment. In this study, we screened natural, plant-derived vibsane-type diterpenoids for their ability to induce differentiation of myeloid leukemia cells, discovering that vibsanin A potently induced differentiation of AML cell lines and primary blasts. The differentiation-inducing activity of vibsanin A was mediated through direct interaction with and activation of protein kinase C (PKC). Consistent with these findings, pharmacological blockade of PKC activity suppressed vibsanin A-induced differentiation. Mechanistically, vibsanin A-mediated activation of PKC led to induction of the ERK pathway and decreased c-Myc expression. In mouse xenograft models of AML, vibsanin A administration prolonged host survival and inhibited PKC-mediated inflammatory responses correlated with promotion of skin tumors in mice. Collectively, our results offer a preclinical proof of concept for vibsanin A as a myeloid differentiation-inducing compound, with potential application as an antileukemic agent. Cancer Res; 76(9); 2698-709. ©2016 AACR.

  4. Cardiomyocyte differentiation induced in cardiac progenitor cells by cardiac fibroblast-conditioned medium.

    Science.gov (United States)

    Zhang, Xi; Shen, Man-Ru; Xu, Zhen-Dong; Hu, Zhe; Chen, Chao; Chi, Ya-Li; Kong, Zhen-Dong; Li, Zi-Fu; Li, Xiao-Tong; Guo, Shi-Lei; Xiong, Shao-Hu; Zhang, Chuan-Sen

    2014-05-01

    Our previous study showed that after being treated with 5-azacytidine, Nkx2.5(+) human cardiac progenitor cells (CPCs) derived from embryonic heart tubes could differentiate into cardiomyocytes. Although 5-azacytidine is a classical agent that induces myogenic differentiation in various types of cells, the drug is toxic and unspecific for myogenic differentiation. To investigate the possibility of inducing CPCs to differentiate into cardiomyocytes by a specific and non-toxic method, CPCs of passage 15 and mesenchymal stem cells (MSCs) were treated with cardiac ventricular fibroblast-conditioned medium (CVF-conditioned medium). Following this treatment, the Nkx2.5(+) CPCs underwent cardiomyogenic differentiation. Phase-contrast microscopy showed that the morphology of the treated CPCs gradually changed. Ultrastructural observation confirmed that the cells contained typical sarcomeres. The expression of cardiomyocyte-associated genes, such as alpha-cardiac actin, cardiac troponin T, and beta-myosin heavy chain (MHC), was increased in the CPCs that had undergone cardiomyogenic differentiation compared with untreated cells. In contrast, the MSCs did not exhibit changes in morphology or molecular expression after being treated with CVF-conditioned medium. The results indicated that Nkx2.5(+) CPCs treated with CVF-conditioned medium were capable of differentiating into a cardiac phenotype, whereas treated MSCs did not appear to undergo cardiomyogenic differentiation. Subsequently, following the addition of Dkk1 and the blocking of Wnt signaling pathway, CVF-conditioned medium-induced morphological changes and expression of cardiomyocyte-associated genes of Nkx2.5(+) CPCs were inhibited, which indicates that CVF-conditioned medium-induced cardiomyogenic differentiation of Nkx2.5(+) CPCs is associated with Wnt signaling pathway. In addition, we also found that the activation of Wnt signaling pathway was accompanied by higher expression of GATA-4 and the blocking of the

  5. BMP9-Induced Osteogenetic Differentiation and Bone Formation of Muscle-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Li Xiang

    2012-01-01

    Full Text Available Efficient osteogenetic differentiation and bone formation from muscle-derived stem cells (MDSCs should have potential clinical applications in treating nonunion fracture healing or bone defects. Here, we investigate osteogenetic differentiation ability of MDSCs induced by bone morphogenetic protein 9 (BMP9 in vitro and bone formation ability in rabbit radius defects repairing model. Rabbit's MDSCs were extracted by type I collagenase and trypsin methods, and BMP9 was introduced into MDSCs by infection with recombinant adenovirus. Effects of BMP9-induced osteogenetic differentiation of MDSCs were identified with alkaline phosphatase (ALP activity and expression of later marker. In stem-cell implantation assay, MDSCs have also shown valuable potential bone formation ability induced by BMP9 in rabbit radius defects repairing test. Taken together, our findings suggest that MDSCs are potentiated osteogenetic stem cells which can be induced by BMP9 to treat large segmental bone defects, nonunion fracture, and/or osteoporotic fracture.

  6. Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin.

    Science.gov (United States)

    Dorn, Isabel; Klich, Katharina; Arauzo-Bravo, Marcos J; Radstaak, Martina; Santourlidis, Simeon; Ghanjati, Foued; Radke, Teja F; Psathaki, Olympia E; Hargus, Gunnar; Kramer, Jan; Einhaus, Martin; Kim, Jeong Beom; Kögler, Gesine; Wernet, Peter; Schöler, Hans R; Schlenke, Peter; Zaehres, Holm

    2015-01-01

    Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34(+) hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34(+) hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34(+) hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34(+) cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential.

  7. Involvement of PIKE in icariin induced cardiomyocyte differentiation from murine embryonic stem cells.

    Science.gov (United States)

    Zhou, Limin; Zheng, Bei; Tang, Leilei; Huang, Yujie; Zhu, Danyan

    2014-03-01

    Icariin (ICA) has demonstrated to induce cardiomyocyte differentiation from murine embryonic stem (ES) cells in vitro, however, the mechanisms have not been fully elucidated. In the present study, we investigated whether phosphatidylinositol 3-kinase enhancer (PIKE) was involved in ICA induced cardiomyocyte differentiation of ES cells. Small interfering RNA (siRNA) of PIKE was applied to investigate the role of PIKE in ICA induced cardiomyocyte differentiation. The cardiomyocytes derived from ES cells were verified using immunofluorescence. The expressions of Troponin T, PIKE, phosphatidylinositol 3-kinase (PI3K), and nuclear factor-kappaB (NF-kappaB) were detected by western blot. The change of reactive oxygen species (ROS) generation was estimated using the fluorescent dye 2', 7' - dichlorodihydrofluorescein diacetate. The results showed that PIKE expression increased during cardiomyocyte differentiation. ICA markedly enhanced PIKE and PI3K expression in a time-dependent manner. Knockdown of PIKE by siRNAs blocked the differentiation of ES cells into cardiomyocytes expressing alpha-actinin for cardiac sarcomeric structures. Moreover, reduced ROS generation and NF-kappaB nuclear translocation were responsible for the inhibitory effect of si-PIKE. In conclusion, PIKE was involved in ICA induced cardiomyocyte differentiation, and ROS generation and NF-kappaB nuclear translocation were associated with PIKE activation.

  8. Lineage- and developmental stage-specific mechanomodulation of induced pluripotent stem cell differentiation.

    Science.gov (United States)

    Maldonado, Maricela; Luu, Rebeccah J; Ico, Gerardo; Ospina, Alex; Myung, Danielle; Shih, Hung Ping; Nam, Jin

    2017-09-29

    To maximize the translational utility of human induced pluripotent stem cells (iPSCs), the ability to precisely modulate the differentiation of iPSCs to target phenotypes is critical. Although the effects of the physical cell niche on stem cell differentiation are well documented, current approaches to direct step-wise differentiation of iPSCs have been typically limited to the optimization of soluble factors. In this regard, we investigated how temporally varied substrate stiffness affects the step-wise differentiation of iPSCs towards various lineages/phenotypes. Electrospun nanofibrous substrates with different reduced Young's modulus were utilized to subject cells to different mechanical environments during the differentiation process towards representative phenotypes from each of three germ layer derivatives including motor neuron, pancreatic endoderm, and chondrocyte. Phenotype-specific markers of each lineage/stage were utilized to determine differentiation efficiency by reverse-transcription polymerase chain reaction (RT-PCR) and immunofluorescence imaging for gene and protein expression analysis, respectively. The results presented in this proof-of-concept study are the first to systematically demonstrate the significant role of the temporally varied mechanical microenvironment on the differentiation of stem cells. Our results demonstrate that the process of differentiation from pluripotent cells to functional end-phenotypes is mechanoresponsive in a lineage- and differentiation stage-specific manner. Lineage/developmental stage-dependent optimization of electrospun substrate stiffness provides a unique opportunity to enhance differentiation efficiency of iPSCs for their facilitated therapeutic applications.

  9. Nucleoside drugs induce cellular differentiation by caspase-dependent degradation of stem cell factors.

    Directory of Open Access Journals (Sweden)

    Tanja Musch

    Full Text Available BACKGROUND: Stem cell characteristics are an important feature of human cancer cells and play a major role in the therapy resistance of tumours. Strategies to target cancer stem cells are thus of major importance for cancer therapy. Differentiation therapy by nucleoside drugs represents an attractive approach for the elimination of cancer stem cells. However, even if it is generally assumed that the activity of these drugs is mediated by their ability to modulate epigenetic pathways, their precise mode of action remains to be established. We therefore analysed the potential of three nucleoside analogues to induce differentiation of the embryonic cancer stem cell line NTERA 2 D1 and compared their effect to the natural ligand retinoic acid. METHODOLOGY/PRINCIPAL FINDINGS: All nucleoside analogues analyzed, but not retinoic acid, triggered proteolytic degradation of the Polycomb group protein EZH2. Two of them, 3-Deazaneplanocin A (DZNep and 2'-deoxy-5-azacytidine (decitabine, also induced a decrease in global DNA methylation. Nevertheless, only decitabine and 1beta-arabinofuranosylcytosine (cytarabine effectively triggered neuronal differentiation of NT2 cells. We show that drug-induced differentiation, in contrast to retinoic acid induction, is caused by caspase activation, which mediates depletion of the stem cell factors NANOG and OCT4. Consistent with this observation, protein degradation and differentiation could be counteracted by co-treatment with caspase inhibitors or by depletion of CASPASE-3 and CASPASE-7 through dsRNA interference. In agreement with this, OCT4 was found to be a direct in-vitro-target of CASPASE-7. CONCLUSIONS/SIGNIFICANCE: We show that drug-induced differentiation is not a consequence of pharmacologic epigenetic modulation, but is induced by the degradation of stem-cell-specific proteins by caspases. Our results thus uncover a novel pathway that induces differentiation of embryonic cancer stem cells and is triggered by

  10. Boswellic acid acetate induces differentiation and apoptosis in leukemia cell lines.

    Science.gov (United States)

    Jing, Y; Nakajo, S; Xia, L; Nakaya, K; Fang, Q; Waxman, S; Han, R

    1999-01-01

    Boswellic acid acetate (BC-4), a compound isolated from the herb Boswellia carterii Birdw., can induce differentiation and apoptosis of leukemia cells. Based on cell morphology and NBT reduction, BC-4 induced monocytic differentiation of myeloid leukemia HL-60, U937 and ML-1 cells at a dose under 12.5 microg/ml (24.2 microM). BC-4 was a potent inducer, with 90% of the cells showing morphologic changes and 80-90% of the cells showing NBT reduction. Specific and non-specific esterase were also increased by BC-4. Based on benzidine staining assay, BC-4 failed to induce erythroid leukemia DS-19 and K562 cells differentiation. In contrast to its selective differentiation effect, BC-4 strongly inhibited growth of all cell lines tested. The growth inhibition effect was dose- and time-dependent. In HL-60 cells, 20 microg/ml (38.8 microM) of BC-4 decreased viable cell number by 60% at 24 h, whereas at 3 days there was virtually no viable cells. Morphologic and DNA fragmentation analysis proved that BC-4 induced cell apoptosis. The dual apoptotic and differentiation effects of BC-4 suggest that it may be a powerful agent in the treatment of leukemia.

  11. Forced expression of Hnf4a induces hepatic gene activation through directed differentiation.

    Science.gov (United States)

    Yahoo, Neda; Pournasr, Behshad; Rostamzadeh, Jalal; Fathi, Fardin

    2016-08-05

    Embryonic stem (ES) cells are capable of unlimited self-renewal and have a diverse differentiation potential. These unique features make ES cells as an attractive source for developmental biology studies. Having the mature hepatocyte in the lab with functional activities is valuable in drug discovery studies. Overexpression of hepatocyte lineage-specific transcription factors (TFs) becomes a promising approach in pluripotent cell differentiation toward liver cells. Many studies generate transgenic ES cell lines to examine the effects of specific TFs overexpression in cell differentiation. In the present report, we have addressed whether a suspension or adherent model of differentiation is an appropriate way to study the role of Hnf4a overexpression. We generated ES cells that carried a doxycycline (Dox)-inducible Hnf4a using lentiviral vectors. The transduced cells were subjected to induced Hnf4a overexpression through both spontaneous and directed differentiation methods. Gene expression analysis showed substantially increased expression of hepatic gene markers, particularly Ttr and endogenous Hnf4a, in transduced cells differentiated by the directed approach. These results demonstrated that forced expression of TFs during directed differentiation would be an appropriate way to study relevant gene activation and the effects of overexpression in the context of hepatic differentiation.

  12. Differential effects of pannexins on noise-induced hearing loss.

    Science.gov (United States)

    Abitbol, Julia M; Kelly, John J; Barr, Kevin; Schormans, Ashley L; Laird, Dale W; Allman, Brian L

    2016-12-15

    Hearing loss, including noise-induced hearing loss, is highly prevalent and severely hinders an individual's quality of life, yet many of the mechanisms that cause hearing loss are unknown. The pannexin (Panx) channel proteins, Panx1 and Panx3, are regionally expressed in many cell types along the auditory pathway, and mice lacking Panx1 in specific cells of the inner ear exhibit hearing loss, suggesting a vital role for Panxs in hearing. We proposed that Panx1 and/or Panx3 null mice would exhibit severe hearing loss and increased susceptibility to noise-induced hearing loss. Using the auditory brainstem response, we surprisingly found that Panx1(-/-) and Panx3(-/-) mice did not harbor hearing or cochlear nerve deficits. Furthermore, while Panx1(-/-) mice displayed no protection against loud noise-induced hearing loss, Panx3(-/-) mice exhibited enhanced 16- and 24-kHz hearing recovery 7 days after a loud noise exposure (NE; 12 kHz tone, 115 dB sound pressure level, 1 h). Interestingly, Cx26, Cx30, Cx43, and Panx2 were up-regulated in Panx3(-/-) mice compared with wild-type and/or Panx1(-/-) mice, and assessment of the auditory tract revealed morphological changes in the middle ear bones of Panx3(-/-) mice. It is unclear if these changes alone are sufficient to provide protection against loud noise-induced hearing loss. Contrary to what we expected, these data suggest that Panx1 and Panx3 are not essential for baseline hearing in mice tested, but the therapeutic targeting of Panx3 may prove protective against mid-high-frequency hearing loss caused by loud NE.

  13. Platelet-Activating Factor Induces Th17 Cell Differentiation

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    Anne-Marie Drolet

    2011-01-01

    Full Text Available Th17 cells have been implicated in a number of inflammatory and autoimmune diseases. The phospholipid mediator platelet-activating factor (PAF is found in increased concentrations in inflammatory lesions and has been shown to induce IL-6 production. We investigated whether PAF could affect the development of Th17 cells. Picomolar concentrations of PAF induced IL-23, IL-6, and IL-1β expression in monocyte-derived Langerhans cells (LCs and in keratinocytes. Moreover, when LC were pretreated with PAF and then cocultured with anti-CD3- and anti-CD28-activated T cells, the latter developed a Th17 phenotype, with a significant increase in the expression of the transcriptional regulator RORγt and enhanced expression of IL-17, IL-21, and IL-22. PAF-induced Th17 development was prevented by the PAF receptor antagonist WEB2086 and by neutralizing antibodies to IL-23 and IL-6R. This may constitute a previously unknown stimulus for the development and persistence of inflammatory processes that could be amenable to pharmacologic intervention.

  14. Differentiation of murine embryonic stem and induced pluripotent stem cells to renal lineage in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Morizane, Ryuji [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan); Monkawa, Toshiaki, E-mail: monkawa@sc.itc.keio.ac.jp [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan); Itoh, Hiroshi [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan)

    2009-12-25

    Embryonic stem (ES) cells which have the unlimited proliferative capacity and extensive differentiation potency can be an attractive source for kidney regeneration therapies. Recent breakthroughs in the generation of induced pluripotent stem (iPS) cells have provided with another potential source for the artificially-generated kidney. The purpose of this study is to know how to differentiate mouse ES and iPS cells into renal lineage. We used iPS cells from mouse fibroblasts by transfection of four transcription factors, namely Oct4, Sox2, c-Myc and Klf4. Real-time PCR showed that renal lineage markers were expressed in both ES and iPS cells after the induction of differentiation. It also showed that a tubular specific marker, KSP progressively increased to day 18, although the differentiation of iPS cells was slower than ES cells. The results indicated that renal lineage cells can be differentiated from both murine ES and iPS cells. Several inducing factors were tested whether they influenced on cell differentiation. In ES cells, both of GDNF and BMP7 enhanced the differentiation to metanephric mesenchyme, and Activin enhanced the differentiation of ES cells to tubular cells. Activin also enhanced the differentiation of iPS cells to tubular cells, although the enhancement was lower than in ES cells. ES and iPS cells have a potential to differentiate to renal lineage cells, and they will be an attractive resource of kidney regeneration therapy. This differentiation is enhanced by Activin in both ES and iPS cells.

  15. Apoptosis during β-mercaptoethanol-induced differentiation of adult adipose-derived stromal cells into neurons

    Institute of Scientific and Technical Information of China (English)

    Yanan Cai; Xiaodong Yuan; Ya Ou; Yanhui Lu

    2011-01-01

    β-mercaptoethanol can induce adipose-derived stromal cells to rapidly and efficiently differentiate into neurons in vitro. However, because of the short survival time of the differentiated cells, clinical applications for this technique are limited. As such, we examined apoptosis of neurons differentiated from adipose-derived stromal cells induced with β-mercaptoethanol in vitro using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and transmission electron microscopy. The results revealed that the number of surviving cells decreased and apoptosis rate increased as induction time extended. Taken together, these results suggest that apoptosis occurring in the process of adipose-derived stromal cells differentiating into neurons is the main cause of cell death. However, the mechanism underlying cellular apoptosis should be researched further to develop methods of controlling apoptosis for clinical applications.

  16. Chemical screen identifies FDA-approved drugs and target pathways that induce precocious pancreatic endocrine differentiation.

    Science.gov (United States)

    Rovira, Meritxell; Huang, Wei; Yusuff, Shamila; Shim, Joong Sup; Ferrante, Anthony A; Liu, Jun O; Parsons, Michael J

    2011-11-29

    Pancreatic β-cells are an essential source of insulin and their destruction because of autoimmunity causes type I diabetes. We conducted a chemical screen to identify compounds that would induce the differentiation of insulin-producing β-cells in vivo. To do this screen, we brought together the use of transgenic zebrafish as a model of β-cell differentiation, a unique multiwell plate that allows easy visualization of lateral views of swimming larval fish and a library of clinical drugs. We identified six hits that can induce precocious differentiation of secondary islets in larval zebrafish. Three of these six hits were known drugs with a considerable background of published data on mechanism of action. Using pharmacological approaches, we have identified and characterized two unique pathways in β-cell differentiation in the zebrafish, including down-regulation of GTP production and retinoic acid biosynthesis.

  17. Metformin induces differentiation in acute promyelocytic leukemia by activating the MEK/ERK signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Huai, Lei; Wang, Cuicui; Zhang, Cuiping; Li, Qihui; Chen, Yirui; Jia, Yujiao; Li, Yan; Xing, Haiyan; Tian, Zheng; Rao, Qing; Wang, Min [State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020 (China); Wang, Jianxiang, E-mail: wangjx@ihcams.ac.cn [State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020 (China)

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer Metformin induces differentiation in NB4 and primary APL cells. Black-Right-Pointing-Pointer Metformin induces activation of the MEK/ERK signaling pathway in APL cells. Black-Right-Pointing-Pointer Metformin synergizes with ATRA to trigger maturation of NB4 and primary APL cells. Black-Right-Pointing-Pointer Metformin induces the relocalization and degradation of the PML-RAR{alpha} fusion protein. Black-Right-Pointing-Pointer The study may be applicable for new differentiation therapy in cancer treatment. -- Abstract: Recent studies have shown that metformin, a widely used antidiabetic agent, may reduce the risk of cancer development. In this study, we investigated the antitumoral effect of metformin on both acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL) cells. Metformin induced apoptosis with partial differentiation in an APL cell line, NB4, but only displayed a proapoptotic effect on several non-M3 AML cell lines. Further analysis revealed that a strong synergistic effect existed between metformin and all-trans retinoic acid (ATRA) during APL cell maturation and that metformin induced the hyperphosphorylation of extracellular signal-regulated kinase (ERK) in APL cells. U0126, a specific MEK/ERK activation inhibitor, abrogated metformin-induced differentiation. Finally, we found that metformin induced the degradation of the oncoproteins PML-RAR{alpha} and c-Myc and activated caspase-3. In conclusion, these results suggest that metformin treatment may contribute to the enhancement of ATRA-induced differentiation in APL, which may deepen the understanding of APL maturation and thus provide insight for new therapy strategies.

  18. RhoA/ROCK signaling in chondrogenic differentiation of MSCs induced by TGF-β1

    Institute of Scientific and Technical Information of China (English)

    Liang Xu; Shuhua Yang; Hongtao Tian

    2006-01-01

    Objective: To investigate the role of RhoA/ROCK in the process of chondrogenic differentiation of MSCs in vitro induced by TGF-β1. Methods: MSCs were isolated from rat bone marrow, cultured and passaged. The cultured MSCs at the 3rd passage were induced to differentiate into chondrocytes in induction medium containing TGF-β1, the expressions of RhoA and ROCK1/2 in the induced MSCs cells were detected by Western-blot and RT-PCR. Results: In parallel to chondrogenic marker gene expressions of collagen Ⅱ and aggrecan, ROCK inhibition by Y27632 in these cells caused a significant decrease in mRNA level of collagen Ⅱ. Conclusion: The results suggest that RhoA/ROCK pathway may play an important and complex role in regulation of chondrogenic differentiation and gene expression.

  19. Inducing dopaminergic differentiation of expanded rat mesencephalic neural stem cells by ascorbic acid in vitro

    Institute of Scientific and Technical Information of China (English)

    ZHENG Min; WANG Dongmei; HOU Lingling; LI Haimin; XIE Chao; JIAO Wencang; BAI Cixian; WANG Yaping; PEI Xuetao

    2004-01-01

    Ascorbic acid (AA) induced differentiation of neural stem cells (NSCs) into dopaminergic (DAergic) neurons is reported.NSCs derived from rat mesencephalon were maintained and expanded in a defined medium containing mitogens of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF).Compared with the control, ascorbic acid treatment led to more DAergic neuronal differentiation as indicated by the expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT), which are specific markers of dopamine neurons.AA induction also enhanced expression of Nurr1 and Shh.PD98059, an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, could block AA-induced Nurr1, TH and DAT mRNA expression.The results might suggest a new strategy to provide enough dopaminergic cells for the therapy of Parkinson's disease (PD), and Nurr1 and ERK signaling pathway might participate in the AA-induced DAergic differentiation.

  20. Direct hepatic differentiation of mouse embryonic stem cells induced by valproic acid and cytokines

    Institute of Scientific and Technical Information of China (English)

    Xue-Jun Dong; Guo-Rong Zhang; Qing-Jun Zhou; Ruo-Lang Pan; Ye Chen; Li-Xin Xiang; Jian-Zhong Shao

    2009-01-01

    AIM: To develop a protocol for direct hepatic lineage differentiation from early developmental progenitors to a population of mature hepatocytes. METHODS: Hepatic progenitor cells and then mature hepatocytes from mouse embryonic stem (ES) cells were obtained in a sequential manner, induced by valproic acid (VPA) and cytokines (hepatocyte growth factor, epidermal growth factor and insulin). Morphological changes of the differentiated cells were examined by phase-contrast microscopy and electron microscopy. Reverse transcription polymerase chain reaction and immunocytochemical analyses were used to evaluate the gene expression profiles of the VPA-induced hepatic progenitors and the hepatic progenitor-derived hepatocytes. Glycogen storage, cytochrome P450 activity, transplantation assay, differentiation of bile duct-like structures and tumorigenic analyses were performed for the functional identification of the differentiated cells. Furthermore, FACS and electron microscopy were used for the analyses of cell cycle profile and apoptosis in VPA-induced hepatic differentiated cells. RESULTS: Based on the combination of VPA and cytokines, mouse ES cells differentiated into a uniform and homogeneous cell population of hepatic progenitor cells and then matured into functional hepatocytes. The progenitor population shared several characteristics with ES cells and hepatic stem/progenitor cells, and represented a novel progenitor cell between ES and hepatic oval cells in embryonic development. The differentiated hepatocytes from progenitor cells shared typical characteristics with mature hepatocytes, including the patterns of gene expression, immunological markers, in vitro hepatocyte functions and in vivo capacity to restore acute-damaged liver function. In addition, the differentiation of hepatic progenitor cells from ES cells was accompanied by significant cell cycle arrest and selective survival of differentiating cells towards hepatic lineages. CONCLUSION: Hepatic cells

  1. Synaptic network activity induces neuronal differentiation of adult hippocampal precursor cells through BDNF signaling

    Directory of Open Access Journals (Sweden)

    Harish Babu

    2009-09-01

    Full Text Available Adult hippocampal neurogenesis is regulated by activity. But how do neural precursor cells in the hippocampus respond to surrounding network activity and translate increased neural activity into a developmental program? Here we show that long-term potential (LTP-like synaptic activity within a cellular network of mature hippocampal neurons promotes neuronal differentiation of newly generated cells. In co-cultures of precursor cells with primary hippocampal neurons, LTP-like synaptic plasticity induced by addition of glycine in Mg2+-free media for 5 min, produced synchronous network activity and subsequently increased synaptic strength between neurons. Furthermore, this synchronous network activity led to a significant increase in neuronal differentiation from the co-cultured neural precursor cells. When applied directly to precursor cells, glycine and Mg2+-free solution did not induce neuronal differentiation. Synaptic plasticity-induced neuronal differentiation of precursor cells was observed in the presence of GABAergic neurotransmission blockers but was dependent on NMDA-mediated Ca2+ influx. Most importantly, neuronal differentiation required the release of brain-derived neurotrophic factor (BDNF from the underlying substrate hippocampal neurons as well as TrkB receptor phosphorylation in precursor cells. This suggests that activity-dependent stem cell differentiation within the hippocampal network is mediated via synaptically evoked BDNF signaling.

  2. MicroRNA and DNA methylation alterations mediating retinoic acid induced neuroblastoma cell differentiation.

    Science.gov (United States)

    Stallings, Raymond L; Foley, Niamh H; Bray, Isabella M; Das, Sudipto; Buckley, Patrick G

    2011-10-01

    Many neuroblastoma cell lines can be induced to differentiate into a mature neuronal cell type with retinoic acid and other compounds, providing an important model system for elucidating signalling pathways involved in this highly complex process. Recently, it has become apparent that miRNAs, which act as regulators of gene expression at a post-transcriptional level, are differentially expressed in differentiating cells and play important roles governing many aspects of this process. This includes the down-regulation of DNA methyltransferases that cause the de-methylation and transcriptional activation of numerous protein coding gene sequences. The purpose of this article is to review involvement of miRNAs and DNA methylation alterations in the process of neuroblastoma cell differentiation. A thorough understanding of miRNA and genetic pathways regulating neuroblastoma cell differentiation potentially could lead to targeted therapies for this disease.

  3. Identification of differentially expressed radiation-induced genes in cervix carcinoma cells using suppression subtractive hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jun Sang; Lee, Young Sook; Lee, Jeung Hoon; Lee, Woong Hee; Seo, Eun Young; Cho, Moon June [Chungnam National University, Daejeon (Korea, Republic of)

    2005-03-15

    A number of genes and their products are induced early or late following exposure of cells to ionizing radiation. These radiation-induced genes have various effects of irradiated cells and tissues. Suppression subtractive hybridization (SSH) based on PCR was used to identify the differentially expressed genes by radiation in cervix carcinoma cells. Total RNA and poly (A){sup +} mRNA were isolated from irradiated and non-irradiated HeLa cells. Forward-and reverse-subtracted cDNA libraries were constructed using SSH. Eighty-eight clones of each were used to randomly select differentially expressed genes using reverse Northern blotting (dot blot analysis). Northern blotting was used to verify the screened genes. Of the 176 clones, 10 genes in the forward-subtracted library and 9 genes in the reverse-subtracted library were identified as differentially expressed radiation-induced genes by PCR-select differential screening. Three clones from the forward-subtracted library were confirmed by Northern blotting, and showed increased expression in a dose-dependent manner, including a telomerase catalytic subunit and sodium channel-like protein gene, and an ESTs (expressed sequence tags) gene. We identified differentially expressed radiation-induced genes with low-abundance genes with SSH, but further characterization of theses genes are necessary to clarify the biological functions of them.

  4. Silver nanoparticles impede phorbol myristate acetate-induced monocyte-macrophage differentiation and autophagy

    Science.gov (United States)

    Xu, Yingying; Wang, Liming; Bai, Ru; Zhang, Tianlu; Chen, Chunying

    2015-09-01

    Monocytes/macrophages are important constituents of the innate immune system. Monocyte-macrophage differentiation is not only crucial for innate immune responses, but is also related to some cardiovascular diseases. Silver nanoparticles (AgNPs) are one of the most widely used nanomaterials because of their broad-spectrum antimicrobial properties. However, the effect of AgNPs on the functions of blood monocytes is scarcely reported. Here, we report the impedance effect of AgNPs on THP-1 monocyte differentiation, and that this effect was mediated by autophagy blockade and lysosomal impairment. Firstly, AgNPs inhibit phorbol 12-myristate 13-acetate (PMA)-induced monocyte differentiation by down-regulating both expression of surface marker CD11b and response to lipopolysaccharide (LPS) stimulation. Secondly, autophagy is activated during PMA-induced THP-1 monocyte differentiation, and the autophagy inhibitor chloroquine (CQ) can inhibit this process. Thirdly, AgNPs block the degradation of the autophagy substrate p62 and induce autophagosome accumulation, which demonstrates the blockade of autophagic flux. Fourthly, lysosomal impairments including alkalization and decrease of lysosomal membrane stability were observed in AgNP-treated THP-1 cells. In conclusion, we demonstrate that the impedance of monocyte-macrophage differentiation by AgNPs is mediated by autophagy blockade and lysosomal dysfunction. Our results suggest that crosstalk exists in different biological effects induced by AgNPs.

  5. Differential effects of energy balance on experimentally-induced colitis

    Institute of Scientific and Technical Information of China (English)

    Sarah J McCaskey; Elizabeth A Rondini; Ingeborg M Langohr; Jenifer I Fenton

    2012-01-01

    AIM:To characterize the influence of diet-induced changes in body fat on colitis severity in SMAD3-/-mice.METHODS:SMAD3-/-mice (6-8 wk of age) were randomly assigned to receive a calorie restricted (30%of control; CR),control (CON),or high fat (HF) diet for 20 wk and were gavaged with sterile broth or with Helicobacter hepaticus (H.hepaticus) to induce colitis.Four weeks after infection,mice were sacrificed and the cecum and colons were processed for histological evaluation.RESULTS:Dietary treatment significantly influenced body composition prior to infection (P < 0.05),with CR mice having less (14% ± 2%) and HF-fed mice more body fat (32% ± 7%) compared to controls (22% ±4%).Differences in body composition were associated with alterations in plasma levels of leptin (HF > CON > CR) and adiponectin (CON > HF ≥ CR) (P < 0.05).There were no significant differences in colitis scores between CON and HF-fed mice 4 wk post-infection.Consistent with this,differences in proliferation and inflammation markers (COX-2,iNOS),and infiltrating cell types (CD3+ T lymphocytes,macrophages) were not observed.Unexpectedly,only 40% of CR mice survived infection with H.hepaticus,with mortality observed as early as 1 wk following induction of colitis.CONCLUSION:Increased adiposity does not influence colitis severity in SMAD3-/-mice.Importantly,caloric restriction negatively impacts survival following pathogen challenge,potentially due to an impaired immune response.

  6. Second generation hybrid polar compounds are potent inducers of transformed cell differentiation.

    Science.gov (United States)

    Richon, V M; Webb, Y; Merger, R; Sheppard, T; Jursic, B; Ngo, L; Civoli, F; Breslow, R; Rifkind, R A; Marks, P A

    1996-06-11

    Hybrid polar compounds, of which hexamethylenebisacetamide (HMBA) is the prototype, are potent inducers of differentiation of murine erythroleukemia (MEL) cells and a wide variety of other transformed cells. HMBA has been shown to induce differentiation of neoplastic cells in patients, but is not an adequate therapeutic agent because of dose-limiting toxicity. We report on a group of three potent second generation hybrid polar compounds, diethyl bis-(pentamethylene-N,N-dimethylcarboxamide) malonate (EMBA), suberoylanilide hydroxamic acid (SAHA), and m-carboxycinnamic acid bis-hydroxamide (CBHA) with optimal concentrations for inducing MEL cells of 0.4 mM, 2 microM, and 4 microM, respectively, compared to 5 mM for HMBA. All three agents induce accumulation of underphosphorylated pRB; increased levels of p2l protein, a prolongation of the initial G1 phase of the cell cycle; and accumulation of hemoglobin. However, based upon their effective concentrations, the cross-resistance or sensitivity of an HMBA-resistant MEL cell variant, and differences in c-myb expression during induction, these differentiation-inducing hybrid polar compounds can be grouped into two subsets, HMBA/EMBA and SAHA/CBHA. This classification may prove of value in selecting and planning prospective preclinical and clinical studies toward the treatment of cancer by differentiation therapy.

  7. Activin A induces Langerhans cell differentiation in vitro and in human skin explants.

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    Tiziana Musso

    Full Text Available Langerhans cells (LC represent a well characterized subset of dendritic cells located in the epidermis of skin and mucosae. In vivo, they originate from resident and blood-borne precursors in the presence of keratinocyte-derived TGFbeta. In vitro, LC can be generated from monocytes in the presence of GM-CSF, IL-4 and TGFbeta. However, the signals that induce LC during an inflammatory reaction are not fully investigated. Here we report that Activin A, a TGFbeta family member induced by pro-inflammatory cytokines and involved in skin morphogenesis and wound healing, induces the differentiation of human monocytes into LC in the absence of TGFbeta. Activin A-induced LC are Langerin+, Birbeck granules+, E-cadherin+, CLA+ and CCR6+ and possess typical APC functions. In human skin explants, intradermal injection of Activin A increased the number of CD1a+ and Langerin+ cells in both the epidermis and dermis by promoting the differentiation of resident precursor cells. High levels of Activin A were present in the upper epidermal layers and in the dermis of Lichen Planus biopsies in association with a marked infiltration of CD1a+ and Langerin+ cells. This study reports that Activin A induces the differentiation of circulating CD14+ cells into LC. Since Activin A is abundantly produced during inflammatory conditions which are also characterized by increased numbers of LC, we propose that this cytokine represents a new pathway, alternative to TGFbeta, responsible for LC differentiation during inflammatory/autoimmune conditions.

  8. Induction of oligodendrocyte differentiation from adult human fibroblast-derived induced pluripotent stem cells.

    Science.gov (United States)

    Ogawa, Shin-ichiro; Tokumoto, Yasuhito; Miyake, Jun; Nagamune, Teruyuki

    2011-08-01

    Induced pluripotent stem cells (iPSCs) prepared from somatic cells might become a novel therapeutic tool in regenerative medicine, especially for the central nervous system (CNS). In this study, we attempted to induce O4-positive (O4(+)) oligodendrocytes from adult human fibroblast-derived iPSCs in vitro. We used two adult human iPSC cell lines, 201B7 and 253G1. 201B7 was induced by four-gene transduction (oct4, sox2, klf4, c-myc), and 253G1 was induced by three-gene transduction (oct4, sox2, klf4). We treated these cells with two in vitro oligodendrocyte-directed differentiation protocols that were optimized for human embryonic stem cells. One protocol used platelet-derived growth factor as the major mitogen for oligodendrocyte lineage cells, and the other protocol used epidermal growth factor (EGF) as the mitogen. Although the differentiation efficiency was low (less than 0.01%), we could induce O4(+) oligodendrocytes from 253G1 cells using the EGF-dependent differentiation protocol. This is the first report of the in vitro induction of oligodendrocytes differentiation from human iPSCs.

  9. Modulating the actin cytoskeleton affects mechanically induced signal transduction and differentiation in mesenchymal stem cells.

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    Petra Müller

    Full Text Available Mechanical interactions of mesenchymal stem cells (MSC with the environment play a significant role in controlling the diverse biological functions of these cells. Mechanical forces are transduced by integrins to the actin cytoskeleton that functions as a scaffold to switch mechanical signals into biochemical pathways. To explore the significance of cytoskeletal mechanisms in human MSC we modulated the actin cytoskeleton using the depolymerising drugs cytochalasin D (CytD and latrunculin A (LatA, as well as the stabilizing drug jasplakinolide (Jasp and examined the activation of the signalling molecules ERK and AKT during mechanical loading. All three drugs provoked significant changes in cell morphology and organisation of the cytoskeleton. Application of mechanical forces to β1-integrin receptors using magnetic beads without deformation of the cell shape induced a phosphorylation of ERK and AKT. Of the two drugs that inhibited the cytoskeletal polymerization, LatA completely blocked the activation of ERK and AKT due to mechanical forces, whereas CytD inhibited the activation of AKT but not of ERK. Activation of both signalling molecules by integrin loading was not affected due to cell treatment with the cytoskeleton stabilizing drug Jasp. To correlate the effects of the drugs on mechanically induced activation of AKT and ERK with parameters of MSC differentiation, we studied ALP activity as a marker for osteogenic differentiation and examined the uptake of fat droplets as marker for adipogenic differentiation in the presence of the drugs. All three drugs inhibited ALP activity of MSC in osteogenic differentiation medium. Adipogenic differentiation was enhanced by CytD and Jasp, but not by LatA. The results indicate that modulation of the cytoskeleton using perturbing drugs can differentially modify both mechanically induced signal transduction and MSC differentiation. In addition to activation of the signalling molecules ERK and AKT, other

  10. Metformin inhibits angiotensin II-induced differentiation of cardiac fibroblasts into myofibroblasts.

    Science.gov (United States)

    Bai, Jian; Zhang, Na; Hua, Ying; Wang, Bingjian; Ling, Lin; Ferro, Albert; Xu, Biao

    2013-01-01

    Differentiation of cardiac fibroblasts into myofibroblasts is a critical event in the progression of cardiac fibrosis that leads to pathological cardiac remodeling. Metformin, an antidiabetic agent, exhibits a number of cardioprotective properties. However, much less is known regarding the effect of metformin on cardiac fibroblast differentiation. Thus, in the present study, we examined the effect of metformin on angiotensin (Ang) II-induced differentiation of cardiac fibroblasts into myofibroblasts and its underlying mechanism. Adult rat cardiac fibroblasts were stimulated with Ang II (100 nM) in the presence or absence of metformin (10-200 µM). Ang II stimulation induced the differentiation of cardiac fibroblasts into myofibroblasts, as indicated by increased expression of α-smooth muscle actin (α-SMA) and collagen types I and III, and this effect of Ang II was inhibited by pretreatment of cardiac fibroblasts with metformin. Metformin also decreased Ang II-induced reactive oxygen species (ROS) generation in cardiac fibroblasts via inhibiting the activation of the PKC-NADPH oxidase pathway. Further experiments using PKC inhibitor calphostin C and NADPH oxidase inhibitor apocynin confirmed that inhibition of the PKC-NADPH oxidase pathway markedly attenuated Ang II-induced ROS generation and myofibroblast differentiation. These data indicate that metformin inhibits Ang II-induced myofibroblast differentiation by suppressing ROS generation via the inhibition of the PKC-NADPH oxidase pathway in adult rat cardiac fibroblasts. Our results provide new mechanistic insights regarding the cardioprotective effects of metformin and provide an efficient therapeutic strategy to attenuate cardiac fibrosis.

  11. Metformin inhibits angiotensin II-induced differentiation of cardiac fibroblasts into myofibroblasts.

    Directory of Open Access Journals (Sweden)

    Jian Bai

    Full Text Available Differentiation of cardiac fibroblasts into myofibroblasts is a critical event in the progression of cardiac fibrosis that leads to pathological cardiac remodeling. Metformin, an antidiabetic agent, exhibits a number of cardioprotective properties. However, much less is known regarding the effect of metformin on cardiac fibroblast differentiation. Thus, in the present study, we examined the effect of metformin on angiotensin (Ang II-induced differentiation of cardiac fibroblasts into myofibroblasts and its underlying mechanism. Adult rat cardiac fibroblasts were stimulated with Ang II (100 nM in the presence or absence of metformin (10-200 µM. Ang II stimulation induced the differentiation of cardiac fibroblasts into myofibroblasts, as indicated by increased expression of α-smooth muscle actin (α-SMA and collagen types I and III, and this effect of Ang II was inhibited by pretreatment of cardiac fibroblasts with metformin. Metformin also decreased Ang II-induced reactive oxygen species (ROS generation in cardiac fibroblasts via inhibiting the activation of the PKC-NADPH oxidase pathway. Further experiments using PKC inhibitor calphostin C and NADPH oxidase inhibitor apocynin confirmed that inhibition of the PKC-NADPH oxidase pathway markedly attenuated Ang II-induced ROS generation and myofibroblast differentiation. These data indicate that metformin inhibits Ang II-induced myofibroblast differentiation by suppressing ROS generation via the inhibition of the PKC-NADPH oxidase pathway in adult rat cardiac fibroblasts. Our results provide new mechanistic insights regarding the cardioprotective effects of metformin and provide an efficient therapeutic strategy to attenuate cardiac fibrosis.

  12. Wnt signaling induces epithelial differentiation during cutaneous wound healing

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    Hocking Anne

    2006-01-01

    Full Text Available Abstract Background Cutaneous wound repair in adult mammals does not regenerate the original epithelial architecture and results in altered skin function. We propose that lack of regeneration may be due to the absence of appropriate molecular signals to promote regeneration. In this study, we investigated the regulation of Wnt signaling during cutaneous wound healing and the consequence of activating either the beta-catenin-dependent or beta-catenin-independent Wnt signaling on epidermal architecture during wound repair. Results We determined that the expression of Wnt ligands that typically signal via the beta-catenin-independent pathway is up-regulated in the wound while the beta-catenin-dependent Wnt signaling is activated in the hair follicles adjacent to the wound edge. Ectopic activation of beta-catenin-dependent Wnt signaling with lithium chloride in the wound resulted in epithelial cysts and occasional rudimentary hair follicle structures within the epidermis. In contrast, forced expression of Wnt-5a in the deeper wound induced changes in the interfollicular epithelium mimicking regeneration, including formation of epithelia-lined cysts in the wound dermis, rudimentary hair follicles and sebaceous glands, without formation of tumors. Conclusion These findings suggest that adult interfollicular epithelium is capable of responding to Wnt morphogenic signals necessary for restoring epithelial tissue patterning in the skin during wound repair.

  13. BMP4 and FGF strongly induce differentiation of mouse ES cells into oral ectoderm

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    Hiroshi Ochiai

    2015-09-01

    Full Text Available During embryonic development, oral ectoderm differentiates into the adenohypophysis, dental epithelia, salivary glands, and nasal pit. Few reports exist concerning the induction of oral ectoderm from embryonic stem (ES cells. Generally, any lot differences in fetal bovine serum (FBS and serum replacer may affect the induction of ES cell-differentiation. Using a previously established culture strategy for differentiation, the proportion of cell aggregates containing Pitx1+ oral ectoderm varied widely between 9–36% when several different lots of FBS or serum replacer were used. We therefore tried to enhance the differentiation method. We found that bone morphogenetic protein (BMP 4 and fibroblast growth factor (FGF treatments improved oral ectoderm induction. Such treatment also improved the differentiation of oral ectoderm into the adenohypophysis. Furthermore, increased BMP4 treatment induced dental epithelium and mesenchyme. Such differentiation suggests that the Pitx1+ layer displays similar properties to oral ectoderm, as found in vivo. Differentiation of ES cells into oral ectoderm using different lots of FBS and serum replacer increased 78–90% after treatment with BMP4 and FGF. In summary, we have established a robust strategy for the induction of oral ectoderm differentiation from mouse ES cells.

  14. Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells.

    Science.gov (United States)

    Kajiwara, Masatoshi; Aoi, Takashi; Okita, Keisuke; Takahashi, Ryosuke; Inoue, Haruhisa; Takayama, Naoya; Endo, Hiroshi; Eto, Koji; Toguchida, Junya; Uemoto, Shinji; Yamanaka, Shinya

    2012-07-31

    Hepatocytes generated from human induced pluripotent stem cells (hiPSCs) are unprecedented resources for pharmaceuticals and cell therapy. However, the in vitro directed differentiation of human pluripotent stem cells into mature hepatocytes remains challenging. Little attention has so far been paid to variations among hiPSC lines in terms of their hepatic differentiation. In the current study, we developed an improved hepatic differentiation protocol and compared 28 hiPSC lines originated from various somatic cells and derived using retroviruses, Sendai viruses, or episomal plasmids. This comparison indicated that the origins, but not the derivation methods, may be a major determinant of variation in hepatic differentiation. The hiPSC clones derived from peripheral blood cells consistently showed good differentiation efficiency, whereas many hiPSC clones from adult dermal fibroblasts showed poor differentiation. However, when we compared hiPSCs from peripheral blood and dermal fibroblasts from the same individuals, we found that variations in hepatic differentiation were largely attributable to donor differences, rather than to the types of the original cells. These data underscore the importance of donor differences when comparing the differentiation propensities of hiPSC clones.

  15. Differential Gene Expression in Chemically Induced Mouse Lung Adenomas

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    Ruisheng Yao

    2003-01-01

    Full Text Available Because of similarities in histopathology and tumor progression stages between mouse and human lung adenocarcinomas, the mouse lung tumor model with lung adenomas as the endpoint has been used extensively to evaluate the efficacy of putative lung cancer chemopreventive agents. In this study, a competitive cDNA library screening (CCLS was employed to determine changes in the expression of mRNA in chemically induced lung adenomas compared with paired normal lung tissues. A total of 2555 clones having altered expression in tumors were observed following competitive hybridization between normal lung and lung adenomas after primary screening of over 160,000 clones from a mouse lung cDNA library. Among the 755 clones confirmed by dot blot hybridization, 240 clones were underexpressed, whereas 515 clones were overexpressed in tumors. Sixty-five clones with the most frequently altered expression in six individual tumors were confirmed by semiquantitative RT-PCR. When examining the 58 known genes, 39 clones had increased expression and 19 had decreased expression, whereas the 7 novel genes showed overexpression. A high percentage (>60% of overexpressed or underexpressed genes was observed in at least two or three of the lesions. Reproducibly overexpressed genes included ERK-1, JAK-1, surfactant proteins A, B, and C, NFAT1, α-1 protease inhibitor, helix-loop-helix ubiquitous kinase (CHUK, α-adaptin, α-1 PI2, thioether S-methyltransferase, and CYP2C40. Reproducibly underexpressed genes included paroxanase, ALDH II, CC10, von Ebner salivary gland protein, and α- and β-globin. In addition, CCLS identified several novel genes or genes not previously associated with lung carcinogenesis, including a hypothetical protein (FLJ11240 and a guanine nucleotide exchange factor homologue. This study shows the efficacy of this methodology for identifying genes with altered expression. These genes may prove to be helpful in our understanding of the genetic basis of

  16. Differential activities of glucocorticoid-induced leucine zipper protein isoforms.

    Science.gov (United States)

    Soundararajan, Rama; Wang, Jian; Melters, Daniël; Pearce, David

    2007-12-14

    Glucocorticoid-induced leucine zipper protein (GILZ) is expressed in both epithelial and immune tissues and modulates a variety of cellular functions, including proliferation and epithelial sodium channel (ENaC) activity. A number of reports have described various GILZ activities, focusing on a single isoform with molecular mass of approximately 17 kDa, now termed GILZ1. In GILZ immunoblots using a newly developed antiserum, we detected multiple species in extracts from cultured kidney cells. Mass spectrometric analysis revealed that one of these represented a previously uncharacterized distinct isoform of GILZ, GILZ2. Rapid amplification of cDNA ends was used to clone cDNAs corresponding to four isoforms, which, in addition to GILZ1 and GILZ2, included new isoforms GILZ3 and GILZ4. Heterologous expression of these four GILZ isoforms in cultured cells revealed striking functional differences. Notably, GILZ1 was the only isoform that significantly stimulated ENaC-mediated Na+ current in a kidney collecting duct cell line, although GILZ2 and GILZ3 also stimulated ENaC surface expression in HEK 293 cells. GILZ1 and GILZ3, and to a lesser extent GILZ2, inhibited ERK phosphorylation. Interestingly, GILZ4, which had no effect on either ENaC or ERK, potently suppressed cellular proliferation, as did GILZ1, but not GILZ2 or GILZ3. Finally, rat and mouse tissues all expressed multiple GILZ species but varied in the relative abundance of each. These data suggest that multiple GILZ isoforms are expressed in most cells and tissues and that these play distinct roles in regulating key cellular functions, including proliferation and ion transport. Furthermore, GILZ inhibition of ERK appears to play an essential role in stimulation of cell surface ENaC but not in inhibition of proliferation.

  17. Lignin Induces ES Cells to Differentiate into Neuroectodermal Cells through Mediation of the Wnt Signaling Pathway.

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    Yu Inoue

    Full Text Available Embryonic stem cells (ES cells are characterized by their pluripotency and infinite proliferation potential. Ever since ES cells were first established in 1981, there have been a growing number of studies aimed at clinical applications of ES cells. In recent years, various types of differentiation inducement systems using ES cells have been established. Further studies have been conducted to utilize differentiation inducement systems in the field of regenerative medicine. For cellular treatments using stem cells including ES cells, differentiation induction should be performed in a sufficient manner to obtain the intended cell lineages. Lignin is a high-molecular amorphous material that forms plants together with cellulose and hemicelluloses, in which phenylpropane fundamental units are complexly condensed. Lignin derivatives have been shown to have several bioactive functions. In spite of these findings, few studies have focused on the effects of lignin on stem cells. Our study aimed to develop a novel technology using lignin to effectively induce ES cells to differentiate into neuroectodermal cells including ocular cells and neural cells. Since lignin can be produced at a relatively low cost in large volumes, its utilization is expected for more convenient differentiation induction technologies and in the field of regenerative medicine in the future.

  18. An improved protocol that induces human embryonic stem cells to differentiate into neural cells in vitro.

    Science.gov (United States)

    Zhou, Jun-Mei; Chu, Jian-Xin; Chen, Xue-Jin

    2008-01-01

    Human embryonic stem (ES) cells have the capacity for self-renewal and are able to differentiate into any cell type. However, obtaining high-efficient neural differentiation from human ES cells remains a challenge. This study describes an improved 4-stage protocol to induce a human ES cell line derived from a Chinese population to differentiate into neural cells. At the first stage, embryonic bodies (EBs) were formed in a chemically-defined neural inducing medium rather than in traditional serum or serum-replacement medium. At the second stage, rosette-like structures were formed. At the third stage, the rosette-like structures were manually selected rather than enzymatically digested to form floating neurospheres. At the fourth stage, the neurospheres were further differentiated into neurons. The results show that, at the second stage, the rate of the formation of rosette-like structures from EBs induced by noggin was 88+/-6.32%, higher than that of retinoic acid 55+/-5.27%. Immunocytochemistry staining was used to confirm the neural identity of the cells. These results show a major improvement in obtaining efficient neural differentiation of human ES cells.

  19. Murine bone cell lines as models for spaceflight induced effects on differentiation and gene expression

    Science.gov (United States)

    Lau, P.; Hellweg, C. E.; Baumstark-Khan, C.; Reitz, G.

    Critical health factors for space crews especially on long-term missions are radiation exposure and the absence of gravity DNA double strand breaks DSB are presumed to be the most deleterious DNA lesions after radiation as they disrupt both DNA strands in close proximity Besides radiation risk the absence of gravity influences the complex skeletal apparatus concerning muscle and especially bone remodelling which results from mechanical forces exerting on the body Bone is a dynamic tissue which is life-long remodelled by cells from the osteoblast and osteoclast lineage Any imbalance of this system leads to pathological conditions such as osteoporosis or osteopetrosis Osteoblastic cells play a crucial role in bone matrix synthesis and differentiate either into bone-lining cells or into osteocytes Premature terminal differentiation has been reported to be induced by a number of DNA damaging or cell stress inducing agents including ionising and ultraviolet radiation as well as treatment with mitomycin C In the present study we compare the effects of sequential differentiation by adding osteoinductive substances ss -glycerophosphate and ascorbic acid Radiation-induced premature differentiation was investigated regarding the biosynthesis of specific osteogenic marker molecules and the differentiation dependent expression of marker genes The bone cell model established in our laboratory consists of the osteocyte cell line MLO-Y4 the osteoblast cell line OCT-1 and the subclones 4 and 24 of the osteoblast cell line MC3T3-E1 expressing several

  20. Differentiation induced by physiological and pharmacological stimuli leads to increased antigenicity of human neuroblastoma cells

    Institute of Scientific and Technical Information of China (English)

    Lena-Maria Carlson; Sven P(a)hlman; Anna De Geer; Per Kogner; Jelena Levitskaya

    2008-01-01

    Sympathetic neuronal differentiation is associated with favorable prognosis of neuroblastoma (NB), the most common extra-cranial solid tumor of early childhood. Differentiation agents have proved useful in clinical protocols of NB treatment, but using them as a sole treatment is not sufficient to induce tumor elimination in patients. Therefore, complementary approaches, such as immunotherapy, are warranted. Here we demonstrate that differentiation of NB cell lines and ex vivo isolated tumor cells in response to physiological or pharmacological stimuli is associated with acquisition of increased antigenicity. This manifests as increased expression of surface major histocompatibility class I complexes and ICAM-1 molecules and translates into increased sensitivity of NB cells to lysis by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. The latter is paralleled by enhanced ability of differentiated cells to form immune conjugates and bind increased amounts of granzyme B to the cell surface. We demonstrate, for the first time, that, regardless of the stimulus applied, the differentiation state in NBs is associated with increased tumor antigenicity that enables more efficient elimination of tumor cells by cytotoxic lymphocytes and paves the way for combined application of differentiation-inducing agents and immunotherapy as an auxiliary approach in NB patients.

  1. Cytokine-Regulated GADD45G Induces Differentiation and Lineage Selection in Hematopoietic Stem Cells

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    Frederic B. Thalheimer

    2014-07-01

    Full Text Available The balance of self-renewal and differentiation in long-term repopulating hematopoietic stem cells (LT-HSC must be strictly controlled to maintain blood homeostasis and to prevent leukemogenesis. Hematopoietic cytokines can induce differentiation in LT-HSCs; however, the molecular mechanism orchestrating this delicate balance requires further elucidation. We identified the tumor suppressor GADD45G as an instructor of LT-HSC differentiation under the control of differentiation-promoting cytokine receptor signaling. GADD45G immediately induces and accelerates differentiation in LT-HSCs and overrides the self-renewal program by specifically activating MAP3K4-mediated MAPK p38. Conversely, the absence of GADD45G enhances the self-renewal potential of LT-HSCs. Videomicroscopy-based tracking of single LT-HSCs revealed that, once GADD45G is expressed, the development of LT-HSCs into lineage-committed progeny occurred within 36 hr and uncovered a selective lineage choice with a severe reduction in megakaryocytic-erythroid cells. Here, we report an unrecognized role of GADD45G as a central molecular linker of extrinsic cytokine differentiation and lineage choice control in hematopoiesis.

  2. How-to-Do-It: Cytokinin Induced Cell Division & Differentiation Using Intact Plants.

    Science.gov (United States)

    Bohnsack, Charles W.

    1989-01-01

    Presents a procedure by which cytokinins are used to induce a population of dividing and differentiating cells on the cut surface of the roots of an intact plant. Includes the method used, results, and suggestions for a variety of variables that may be tested. (RT)

  3. Profiling the Changes in Signaling Pathways in Ascorbic Acid/beta-Glycerophosphate-Induced Osteoblastic Differentiation

    NARCIS (Netherlands)

    Chaves Neto, Antonio Hernandes; Queiroz, Karla Cristiana; Milani, Renato; Paredes-Gamero, Edgar Julian; Justo, Giselle Zenker; Peppelenbosch, Maikel P.; Ferreira, Carmen Verissima

    2011-01-01

    Despite numerous reports on the ability of ascorbic acid and beta-glycerophosphate (AA/beta-GP) to induce osteoblast differentiation, little is known about the molecular mechanisms involved in this phenomenon. In this work, we used a peptide array containing specific consensus sequences (potential s

  4. Differential saliva-induced breakdown of starch filled protein gels in relation to sensory perception

    NARCIS (Netherlands)

    Janssen, A.M.; Pijpekamp, van de A.M.; Labiausse, D.

    2009-01-01

    In this study, the differential breakdown of protein gels containing four types of high and low cross-linked starch granules were studied. Susceptibility to saliva-induced breakdown of starch granules and the consequences of these for overall breakdown of the gel matrix were captured using a multipl

  5. Secreted Cyclic Di-GMP Induces Stalk Cell Differentiation in the Eukaryote Dictyostelium discoideum.

    Science.gov (United States)

    Chen, Zhi-hui; Schaap, Pauline

    2016-01-01

    Cyclic di-GMP (c-di-GMP) is currently recognized as the most widely used intracellular signal molecule in prokaryotes, but roles in eukaryotes were only recently discovered. In the social amoeba Dictyostelium discoideum, c-di-GMP, produced by a prokaryote-type diguanylate cyclase, induces the differentiation of stalk cells, thereby enabling the formation of spore-bearing fruiting bodies. In this review, we summarize the currently known mechanisms that control the major life cycle transitions of Dictyostelium and focus particularly on the role of c-di-GMP in stalk formation. Stalk cell differentiation has characteristics of autophagic cell death, a process that also occurs in higher eukaryotes. We discuss the respective roles of c-di-GMP and of another signal molecule, differentiation-inducing factor 1, in autophagic cell death in vitro and in stalk formation in vivo.

  6. The Apoplastic Copper AMINE OXIDASE1 Mediates Jasmonic Acid-Induced Protoxylem Differentiation in Arabidopsis Roots.

    Science.gov (United States)

    Ghuge, Sandip A; Carucci, Andrea; Rodrigues-Pousada, Renato A; Tisi, Alessandra; Franchi, Stefano; Tavladoraki, Paraskevi; Angelini, Riccardo; Cona, Alessandra

    2015-06-01

    Polyamines are involved in key developmental processes and stress responses. Copper amine oxidases oxidize the polyamine putrescine (Put), producing an aldehyde, ammonia, and hydrogen peroxide (H2O2). The Arabidopsis (Arabidopsis thaliana) amine oxidase gene At4g14940 (AtAO1) encodes an apoplastic copper amine oxidase expressed at the early stages of vascular tissue differentiation in roots. Here, its role in root development and xylem differentiation was explored by pharmacological and forward/reverse genetic approaches. Analysis of the AtAO1 expression pattern in roots by a promoter::green fluorescent protein-β-glucuronidase fusion revealed strong gene expression in the protoxylem at the transition, elongation, and maturation zones. Methyl jasmonate (MeJA) induced AtAO1 gene expression in vascular tissues, especially at the transition and elongation zones. Early protoxylem differentiation was observed upon MeJA treatment along with Put level decrease and H2O2 accumulation in wild-type roots, whereas Atao1 loss-of-function mutants were unresponsive to the hormone. The H2O2 scavenger N,N(1)-dimethylthiourea reversed the MeJA-induced early protoxylem differentiation in wild-type seedlings. Likewise, Put, which had no effect on Atao1 mutants, induced early protoxylem differentiation in the wild type, this event being counteracted by N,N(1)-dimethylthiourea treatment. Consistently, AtAO1-overexpressing plants showed lower Put levels and early protoxylem differentiation concurrent with H2O2 accumulation in the root zone where the first protoxylem cells with fully developed secondary wall thickenings are found. These results show that the H2O2 produced via AtAO1-driven Put oxidation plays a role in MeJA signaling leading to early protoxylem differentiation in root. © 2015 American Society of Plant Biologists. All Rights Reserved.

  7. Smurf1 plays a role in EGF inhibition of BMP2-induced osteogenic differentiation

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    Lee, Hye-Lim; Park, Hyun-Jung; Kwon, Arang [Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of); Baek, Kyunghwa [Department of Pharmacology, College of Dentistry and Research Institute of Oral Science, Gangneung-Wonju National University, Gangneung 210-702, Gangwondo (Korea, Republic of); Woo, Kyung Mi; Ryoo, Hyun-Mo; Kim, Gwan-Shik [Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of); Baek, Jeong-Hwa, E-mail: baekjh@snu.ac.kr [Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of)

    2014-05-01

    It has been demonstrated that epidermal growth factor (EGF) plays a role in supporting the proliferation of bone marrow stromal cells in bone but inhibits their osteogenic differentiation. However, the mechanism underlying EGF inhibition of osteoblast differentiation remains unclear. Smurf1 is an E3 ubiquitin ligase that targets Smad1/5 and Runx2, which are critical transcription factors for bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation. In this study, we investigated the effect of EGF on the expression of Smurf1, and the role of Smurf1 in EGF inhibition of osteogenic differentiation using C2C12 cells, a murine myoblast cell line. EGF increased Smurf1 expression, which was blocked by inhibiting the activity of either JNK or ERK. Chromatin immunoprecipitation and Smurf1 promoter assays demonstrated that c-Jun and Runx2 play roles in the EGF induction of Smurf1 transcription. EGF suppressed BMP2-induced expression of osteogenic marker genes, which were rescued by Smurf1 knockdown. EGF downregulated the protein levels of Runx2 and Smad1 in a proteasome-dependent manner. EGF decreased the transcriptional activity of Runx2 and Smurf1, which was partially rescued by Smurf1 silencing. Taken together, these results suggest that EGF increases Smurf1 expression via the activation of JNK and ERK and the subsequent binding of c-Jun and Runx2 to the Smurf1 promoter and that Smurf1 mediates the inhibitory effect of EGF on BMP2-induced osteoblast differentiation. - Highlights: • EGF increases the expression level of Smurf1 in mesenchymal precursor cells. • EGF reduces the protein levels and transcriptional activity of Runx2 and Smad1. • EGF suppresses BMP2-induced osteogenic differentiation, which is rescued by Smurf1 knockdown.

  8. Quantitative glycomics monitoring of induced pluripotent- and embryonic stem cells during neuronal differentiation

    Directory of Open Access Journals (Sweden)

    Michiyo Terashima

    2014-11-01

    Full Text Available Alterations in the structure of cell surface glycoforms occurring during the stages of stem cell differentiation remain unclear. We describe a rapid glycoblotting-based cellular glycomics method for quantitatively evaluating changes in glycoform expression and structure during neuronal differentiation of murine induced pluripotent stem cells (iPSCs and embryonic stem cells (ESCs. Our results show that changes in the expression of cellular N-glycans are comparable during the differentiation of iPSCs and ESCs. The expression of bisect-type N-glycans was significantly up-regulated in neurons that differentiated from both iPSCs and ESCs. From a glycobiological standpoint, iPSCs are an alternative neural cell source in addition to ESCs.

  9. Modulating notochordal differentiation of human induced pluripotent stem cells using natural nucleus pulposus tissue matrix.

    Directory of Open Access Journals (Sweden)

    Yongxing Liu

    Full Text Available Human induced pluripotent stem cells (hiPSCs can differentiate into notochordal cell (NC-like cells when cultured in the presence of natural porcine nucleus pulposus (NP tissue matrix. The method promises massive production of high-quality, functional cells to treat degenerative intervertebral discs (IVDs. Based on our previous work, we further examined the effect of cell-NP matrix contact and culture medium on the differentiation, and further assessed the functional differentiation ability of the generated NC-like. The study showed that direct contact between hiPSCs and NP matrix can promote the differentiation yield, whilst both the contact and non-contact cultures can generate functional NC-like cells. The generated NC-like cells are highly homogenous regarding the expression of notochordal marker genes. A culture medium containing a cocktail of growth factors (FGF, EGF, VEGF and IGF-1 also supported the notochordal differentiation in the presence of NP matrix. The NC-like cells showed excellent functional differentiation ability to generate NP-like tissue which was rich in aggrecan and collagen type II; and particularly, the proteoglycan to collagen content ratio was as high as 12.5-17.5 which represents a phenotype close to NP rather than hyaline cartilage. Collectively, the present study confirmed the effectiveness and flexibility of using natural NP tissue matrix to direct notochordal differentiation of hiPSCs, and the potential of using the generated NC-like cells for treating IVD degeneration.

  10. Lapatinib induces autophagy, apoptosis and megakaryocytic differentiation in chronic myelogenous leukemia K562 cells.

    Directory of Open Access Journals (Sweden)

    Huey-Lan Huang

    Full Text Available Lapatinib is an oral, small-molecule, dual tyrosine kinase inhibitor of epidermal growth factor receptors (EGFR, or ErbB/Her in solid tumors. Little is known about the effect of lapatinib on leukemia. Using human chronic myelogenous leukemia (CML K562 cells as an experimental model, we found that lapatinib simultaneously induced morphological changes resembling apoptosis, autophagy, and megakaryocytic differentiation. Lapatinib-induced apoptosis was accompanied by a decrease in mitochondrial transmembrane potential and was attenuated by the pancaspase inhibitor z-VAD-fmk, indicating a mitochondria-mediated and caspase-dependent pathway. Lapatinib-induced autophagic cell death was verified by LC3-II conversion, and upregulation of Beclin-1. Further, autophagy inhibitor 3-methyladenine as well as autophagy-related proteins Beclin-1 (ATG6, ATG7, and ATG5 shRNA knockdown rescued the cells from lapatinib-induced growth inhibition. A moderate number of lapatinib-treated K562 cells exhibited features of megakaryocytic differentiation. In summary, lapatinib inhibited viability and induced multiple cellular events including apoptosis, autophagic cell death, and megakaryocytic differentiation in human CML K562 cells. This distinct activity of lapatinib against CML cells suggests potential for lapatinib as a therapeutic agent for treatment of CML. Further validation of lapatinib activity in vivo is warranted.

  11. Radotinib Induces Apoptosis of CD11b+ Cells Differentiated from Acute Myeloid Leukemia Cells.

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    Sook-Kyoung Heo

    Full Text Available Radotinib, developed as a BCR/ABL tyrosine kinase inhibitor (TKI, is approved for the second-line treatment of chronic myeloid leukemia (CML in South Korea. However, therapeutic effects of radotinib in acute myeloid leukemia (AML are unknown. In the present study, we demonstrate that radotinib significantly decreases the viability of AML cells in a dose-dependent manner. Kasumi-1 cells were more sensitive to radotinib than NB4, HL60, or THP-1 cell lines. Furthermore, radotinib induced CD11b expression in NB4, THP-1, and Kasumi-1 cells either in presence or absence of all trans-retinoic acid (ATRA. We found that radotinib promoted differentiation and induced CD11b expression in AML cells by downregulating LYN. However, CD11b expression induced by ATRA in HL60 cells was decreased by radotinib through upregulation of LYN. Furthermore, radotinib mainly induced apoptosis of CD11b+ cells in the total population of AML cells. Radotinib also increased apoptosis of CD11b+ HL60 cells when they were differentiated by ATRA/dasatinib treatment. We show that radotinib induced apoptosis via caspase-3 activation and the loss of mitochondrial membrane potential (ΔΨm in CD11b+ cells differentiated from AML cells. Our results suggest that radotinib may be used as a candidate drug in AML or a chemosensitizer for treatment of AML by other therapeutics.

  12. Human amnion epithelial cells can be induced to differentiate into functional insulin-producing cells

    Institute of Scientific and Technical Information of China (English)

    Yanan Hou; Qin Huang; Tianjin Liu; Lihe Guo

    2008-01-01

    Pancreatic islet transplantation has demonstrated that long-term insulin independence may be achieved in patients suffering from diabetes mellitus type 1. However, limited availability of islet tissue means that new sources of insulinproducing cells that are responsive to glucose are required. Here, we show that human amnion epithelial cells (HAEC) can be induced to differentiate into functional insulinproducing cells in vitro. After induction of differentiation, HAEC expressed multiple pancreatic --cell genes, including insulin, pancreas duodenum homeobox-1, paired box gene 6,NK2 transcription factor-related locus 2, Islet 1, glucokinase,and glucose transporter-2, and released C-peptide in a glucose-regulated manner in response to other extracellular stimulations. The transplantation of induced HAEC into streptozotocin-induced diabetic C57 mice reversed hyperglycemia, restored body weight, and maintained euglycemia for 30 d. These findings indicated that HAEC may be a new source for cell replacement therapy in type 1 diabetes.

  13. Differentiation of Neonatal Human-Induced Pluripotent Stem Cells to Prostate Epithelial Cells: A Model to Study Prostate Cancer Development

    Science.gov (United States)

    2014-06-01

    1 AD_________________ Award Number: W81XWH-12-1-0189 TITLE: Differentiation of Neonatal Human...CONTRACT NUMBER Differentiation of Neonatal Human Induced Pluripotent Stem Cells to Prostate Epithelial Cells: A Model to Study Prostate Cancer...13. SUPPLEMENTARY NOTES 14. ABSTRACT We set out to establish conditions for differentiation of human neonatal foreskin skin fibroblast

  14. Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview.

    Science.gov (United States)

    Mummery, Christine L; Zhang, Jianhua; Ng, Elizabeth S; Elliott, David A; Elefanty, Andrew G; Kamp, Timothy J

    2012-07-20

    Since human embryonic stem cells were first differentiated to beating cardiomyocytes a decade ago, interest in their potential applications has increased exponentially. This has been further enhanced over recent years by the discovery of methods to induce pluripotency in somatic cells, including those derived from patients with hereditary cardiac diseases. Human pluripotent stem cells have been among the most challenging cell types to grow stably in culture, but advances in reagent development now mean that most laboratories can expand both embryonic and induced pluripotent stem cells robustly using commercially available products. However, differentiation protocols have lagged behind and in many cases only produce the cell types required with low efficiency. Cardiomyocyte differentiation techniques were also initially inefficient and not readily transferable across cell lines, but there are now a number of more robust protocols available. Here, we review the basic biology underlying the differentiation of pluripotent cells to cardiac lineages and describe current state-of-the-art protocols, as well as ongoing refinements. This should provide a useful entry for laboratories new to this area to start their research. Ultimately, efficient and reliable differentiation methodologies are essential to generate desired cardiac lineages to realize the full promise of human pluripotent stem cells for biomedical research, drug development, and clinical applications.

  15. Isoliquiritigenin-induced differentiation in mouse melanoma B16F0 cell line.

    Science.gov (United States)

    Chen, Xiaoyu; Zhang, Bo; Yuan, Xuan; Yang, Fan; Liu, Jinglei; Zhao, Hong; Liu, Liangliang; Wang, Yanming; Wang, Zhenhua; Zheng, Qiusheng

    2012-01-01

    The chemotherapeutical treatment is very limited for malignant melanoma, a highly lethal disease occurs globally. Natural products derived from traditional Chinese medicine licorice are attractive in quest new treatments due to their anti-tumor activities. A new dietary flavonoid isoliquiritigenin (ISL) were thus investigated to indentify its anti-melanoma activities on mouse melanoma B16F0 cells in present study. Using biochemical and free radical biological experiments in vitro, we identified the pro-differentiated profiles of ISL and evaluated the role of reactive oxygen species (ROS) during B16F0 cell differentiation. The data showed a strong dose-response relationship between ISL exposure and the characteristics of B16F0 differentiation in terms of morphology changes and melanogenesis. The accumulated intercellular ROS during exposure are necessary to support ISL-induced differentiation, which was proven by additional redox modulators. It was confirmed further by the relative activities of enzymes and genes modulated melanogenesis in ISL-treatments with or without ROS modulators. The tumorigenicity of ISL-treated cells was limited significantly by using the colony formation assay in vitro and an animal model assay in vivo respectively. Our research demonstrated that isoliquiritigenin is a differentiation-inducing agent, and its mechanisms involve ROS accumulation facilitating melanogenesis.

  16. Isoliquiritigenin-Induced Differentiation in Mouse Melanoma B16F0 Cell Line

    Directory of Open Access Journals (Sweden)

    Xiaoyu Chen

    2012-01-01

    Full Text Available The chemotherapeutical treatment is very limited for malignant melanoma, a highly lethal disease occurs globally. Natural products derived from traditional Chinese medicine licorice are attractive in quest new treatments due to their anti-tumor activities. A new dietary flavonoid isoliquiritigenin (ISL were thus investigated to indentify its anti-melanoma activities on mouse melanoma B16F0 cells in present study. Using biochemical and free radical biological experiments in vitro, we identified the pro-differentiated profiles of ISL and evaluated the role of reactive oxygen species (ROS during B16F0 cell differentiation. The data showed a strong dose-response relationship between ISL exposure and the characteristics of B16F0 differentiation in terms of morphology changes and melanogenesis. The accumulated intercellular ROS during exposure are necessary to support ISL-induced differentiation, which was proven by additional redox modulators. It was confirmed further by the relative activities of enzymes and genes modulated melanogenesis in ISL-treatments with or without ROS modulators. The tumorigenicity of ISL-treated cells was limited significantly by using the colony formation assay in vitro and an animal model assay in vivo respectively. Our research demonstrated that isoliquiritigenin is a differentiation-inducing agent, and its mechanisms involve ROS accumulation facilitating melanogenesis.

  17. A Receptor Tyrosine Kinase Inhibitor, Dovitinib (TKI-258), Enhances BMP-2-Induced Osteoblast Differentiation In Vitro

    Science.gov (United States)

    Lee, Yura; Bae, Kyoung Jun; Chon, Hae Jung; Kim, Seong Hwan; Kim, Soon Ae; Kim, Jiyeon

    2016-01-01

    Dovitinib (TKI258) is a small molecule multi-kinase inhibitor currently in clinical phase I/II/III development for the treatment of various types of cancers. This drug has a safe and effective pharmacokinetic/pharmacodynamic profile. Although dovitinib can bind several kinases at nanomolar concentrations, there are no reports relating to osteoporosis or osteoblast differentiation. Herein, we investigated the effect of dovitinib on human recombinant bone morphogenetic protein (BMP)-2-induced osteoblast differentiation in a cell culture model. Dovitinib enhanced the BMP-2-induced alkaline phosphatase (ALP) induction, which is a representative marker of osteoblast differentiation. Dovitinib also stimulated the translocation of phosphorylated Smad1/5/8 into the nucleus and phosphorylation of mitogen-activated protein kinases, including ERK1/2 and p38. In addition, the mRNA expression of BMP-4, BMP-7, ALP, and OCN increased with dovitinib treatment. Our results suggest that dovitinib has a potent stimulating effect on BMP-2-induced osteoblast differentiation and this existing drug has potential for repositioning in the treatment of bone-related disorders. PMID:27025387

  18. The effects of luteolin on osteoclast differentiation, function in vitro and ovariectomy-induced bone loss.

    Science.gov (United States)

    Kim, Tae-Ho; Jung, Ji Won; Ha, Byung Geun; Hong, Jung Min; Park, Eui Kyun; Kim, Hyun-Ju; Kim, Shin-Yoon

    2011-01-01

    Flavonoids, a group of polyphenolic compounds abundant in plants, are known to prevent bone loss in ovariectomized (OVX) animal models. Inhibition of osteoclast differentiation and bone resorption is considered as an effective therapeutic approach in the treatment of postmenopausal bone loss. Luteolin, a plant flavonoid, has potent anti-inflammatory properties both in vivo and vitro. In this study, we found that luteolin markedly decreased the differentiation of both bone marrow mononuclear cells and Raw264.7 cells into osteoclasts. Luteolin also inhibited the bone resorptive activity of differentiated osteoclasts. We further investigated the effects of luteolin on ovariectomy-induced bone loss using micro-computed tomography, biomechanical tests and serum markers assay for bone remodeling. Oral administration of luteolin (5 and 20 mg/kg per day) to OVX mice caused significant increase in bone mineral density and bone mineral content of trabecular and cortical bones in the femur as compared to those of OVX controls, and prevented decreases of bone strength indexes induced by OVX surgery. Serum biochemical markers assays revealed that luteolin prevents OVX-induced increases in bone turnover. These data strongly suggest that luteolin has the potential for prevention of bone loss in postmenopausal osteoporosis by reducing both osteoclast differentiation and function. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. Modeling and analysis of retinoic acid induced differentiation of uncommitted precursor cells.

    Science.gov (United States)

    Tasseff, Ryan; Nayak, Satyaprakash; Song, Sang Ok; Yen, Andrew; Varner, Jeffrey D

    2011-05-01

    Manipulation of differentiation programs has therapeutic potential in a spectrum of human cancers and neurodegenerative disorders. In this study, we integrated computational and experimental methods to unravel the response of a lineage uncommitted precursor cell-line, HL-60, to Retinoic Acid (RA). HL-60 is a human myeloblastic leukemia cell-line used extensively to study human differentiation programs. Initially, we focused on the role of the BLR1 receptor in RA-induced differentiation and G1/0-arrest in HL-60. BLR1, a putative G protein-coupled receptor expressed following RA exposure, is required for RA-induced cell-cycle arrest and differentiation and causes persistent MAPK signaling. A mathematical model of RA-induced cell-cycle arrest and differentiation was formulated and tested against BLR1 wild-type (wt) knock-out and knock-in HL-60 cell-lines with and without RA. The current model described the dynamics of 729 proteins and protein complexes interconnected by 1356 interactions. An ensemble strategy was used to compensate for uncertain model parameters. The ensemble of HL-60 models recapitulated the positive feedback between BLR1 and MAPK signaling. The ensemble of models also correctly predicted Rb and p47phox regulation and the correlation between p21-CDK4-cyclin D formation and G1/0-arrest following exposure to RA. Finally, we investigated the robustness of the HL-60 network architecture to structural perturbations and generated experimentally testable hypotheses for future study. Taken together, the model presented here was a first step toward a systematic framework for analysis of programmed differentiation. These studies also demonstrated that mechanistic network modeling can help prioritize experimental directions by generating falsifiable hypotheses despite uncertainty.

  20. Inhibition of the NAD-dependent protein deacetylase SIRT2 induces granulocytic differentiation in human leukemia cells.

    Directory of Open Access Journals (Sweden)

    Yoshitaka Sunami

    Full Text Available Sirtuins, NAD-dependent protein deacetylases, play important roles in cellular functions such as metabolism and differentiation. Whether sirtuins function in tumorigenesis is still controversial, but sirtuins are aberrantly expressed in tumors, which may keep cancerous cells undifferentiated. Therefore, we investigated whether the inhibition of sirtuin family proteins induces cellular differentiation in leukemic cells. The sirtuin inhibitors tenovin-6 and BML-266 induce granulocytic differentiation in the acute promyelocytic leukemia (APL cell line NB4. This differentiation is likely caused by an inhibition of SIRT2 deacetylase activity, judging from the accumulation of acetylated α-tubulin, a major SIRT2 substrate. Unlike the clinically used differentiation inducer all-trans retinoic acid, tenovin-6 shows limited effects on promyelocytic leukemia-retinoic acid receptor α (PML-RAR-α stability and promyelocytic leukemia nuclear body formation in NB4 cells, suggesting that tenovin-6 does not directly target PML-RAR-α activity. In agreement with this, tenovin-6 induces cellular differentiation in the non-APL cell line HL-60, where PML-RAR-α does not exist. Knocking down SIRT2 by shRNA induces granulocytic differentiation in NB4 cells, which demonstrates that the inhibition of SIRT2 activity is sufficient to induce cell differentiation in NB4 cells. The overexpression of SIRT2 in NB4 cells decreases the level of granulocytic differentiation induced by tenovin-6, which indicates that tenovin-6 induces granulocytic differentiation by inhibiting SIRT2 activity. Taken together, our data suggest that targeting SIRT2 is a viable strategy to induce leukemic cell differentiation.

  1. Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Chieri [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Iwasaki, Tsuyoshi, E-mail: tsuyo-i@huhs.ac.jp [Division of Pharmacotherapy, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima, Chuo-ku, Kobe 650-8530 (Japan); Kitano, Sachie; Tsunemi, Sachi; Sano, Hajime [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer We investigated the role of S1P signaling for osteoblast differentiation. Black-Right-Pointing-Pointer Both S1P and FTY enhanced BMP-2-stimulated osteoblast differentiation by C2C12 cells. Black-Right-Pointing-Pointer S1P signaling enhanced BMP-2-stimulated Smad and ERK phosphorylation by C2C12 cells. Black-Right-Pointing-Pointer MEK/ERK signaling is a pathway underlying S1P signaling for osteoblast differentiation. -- Abstract: We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murine satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P

  2. Folate antagonist, methotrexate induces neuronal differentiation of human embryonic stem cells transplanted into nude mouse retina.

    Science.gov (United States)

    Hara, Akira; Taguchi, Ayako; Aoki, Hitomi; Hatano, Yuichiro; Niwa, Masayuki; Yamada, Yasuhiro; Kunisada, Takahiro

    2010-06-25

    Transplanted embryonic stem (ES) cells can be integrated into the retinas of adult mice as well-differentiated neuroretinal cells. However, the transplanted ES cells also have a tumorigenic activity as they have the ability for multipotent differentiation to various types of tissues. In the present study, human ES (hES) cells were transplanted into adult nude mouse retinas by intravitreal injections 20 h after intravitreal N-methyl-D-aspartate (NMDA) administration. After the transplantation of hES cells, the folate antagonist, methotrexate (MTX) was administrated in order to control the differentiation of the transplanted hES cells. Neuronal differentiation and teratogenic potential of hES cells were examined immunohistochemically 5 weeks after transplantation. The proliferative activity of transplanted cells was determined by both the mitotic index and the Ki-67 proliferative index. Disappearance of Oct-4-positive hES cells showing undifferentiated morphology was observed after intraperitoneal MTX treatment daily, for 15 days. Decreased mitotic and Ki-67 proliferative indices, and increased neuronal differentiation were detected in the surviving hES cells after the MTX treatment. These results suggest two important effects of intraperitoneal MTX treatment for hES cells transplanted into nude mouse retina: (1) MTX treatment following transplantation induces neuronal differentiation, and (2) MTX decreases proliferative activity and tumorigenic potential.

  3. Retinoic acid induces nuclear accumulation of Raf1 during differentiation of HL-60 cells.

    Science.gov (United States)

    Smith, James; Bunaciu, Rodica P; Reiterer, Gudrun; Coder, David; George, Thaddeus; Asaly, Michael; Yen, Andrew

    2009-08-01

    All trans-retinoic acid (RA) is a standard therapeutic agent used in differentiation induction therapy treatment of acute promyelocytic leukemia (APL). RA and its metabolites use a diverse set of signal transduction pathways during the differentiation program. In addition to the direct transcriptional targets of the nuclear RAR and RXR receptors, signals derived from membrane receptors and the Raf-MEK-ERK pathway are required. Raf1 phosphorylation and the prolonged activation of Raf1 persisting during the entire differentiation process are required for RA-dependent differentiation of HL-60 cells. Here we identify a nuclear redistribution of Raf1 during the RA-induced differentiation of HL-60 cells. In addition, the nuclear accumulation of Raf1 correlates with an increase in Raf1 phosphorylated at serine 621. The serine 621 phosphorylated Raf1 is predominantly localized in the nucleus. The RA-dependent nuclear accumulation of Raf1 suggests a novel nuclear role for Raf1 during the differentiation process.

  4. Retinoic acid induces nuclear accumulation of Raf1 during differentiation of HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Smith, James; Bunaciu, Rodica P.; Reiterer, Gudrun [Department of Biomedical Sciences, T4-008 VRT, Cornell University, Ithaca, NY 14853 (United States); Coder, David; George, Thaddeus [Amnis Corporation, Seattle, Washington (United States); Asaly, Michael [Department of Biomedical Sciences, T4-008 VRT, Cornell University, Ithaca, NY 14853 (United States); Yen, Andrew, E-mail: ay13@cornell.edu [Department of Biomedical Sciences, T4-008 VRT, Cornell University, Ithaca, NY 14853 (United States)

    2009-08-01

    All trans-retinoic acid (RA) is a standard therapeutic agent used in differentiation induction therapy treatment of acute promyelocytic leukemia (APL). RA and its metabolites use a diverse set of signal transduction pathways during the differentiation program. In addition to the direct transcriptional targets of the nuclear RAR and RXR receptors, signals derived from membrane receptors and the Raf-MEK-ERK pathway are required. Raf1 phosphorylation and the prolonged activation of Raf1 persisting during the entire differentiation process are required for RA-dependent differentiation of HL-60 cells. Here we identify a nuclear redistribution of Raf1 during the RA-induced differentiation of HL-60 cells. In addition, the nuclear accumulation of Raf1 correlates with an increase in Raf1 phosphorylated at serine 621. The serine 621 phosphorylated Raf1 is predominantly localized in the nucleus. The RA-dependent nuclear accumulation of Raf1 suggests a novel nuclear role for Raf1 during the differentiation process.

  5. Comparison of different protocols for neural differentiation of human induced pluripotent stem cells.

    Science.gov (United States)

    Salimi, Ali; Nadri, Samad; Ghollasi, Marzieh; Khajeh, Khosro; Soleimani, Masoud

    2014-03-01

    Although embryonic stem cells (ESCs) have enormous potentials due to their pluripotency, their therapeutic use is limited by ethical, biological and safety issues. Compared to ESCs, induced pluripotent stem cells (iPSCs) can be obtained from mouse or human fibroblasts by reprogramming. Numerous studies have established many protocols for differentiation of human iPSCs (hiPSCs) into neural lineages. However, the low differentiation efficiency of such protocols motivates researchers to design new protocols for high yield differentiation. Herein, we compared neural differentiation potential of three induction media for conversion of hiPSCs into neural lineages. In this study, hiPSCs-derived embryoid bodies were plated on laminin coated dishes and were treated with three induction media including (1) bFGF, EGF (2) RA and (3) forskolin, IBMX. Immunofluorescence staining and quantitative real-time PCR (qPCR) analysis were used to detect the expression of neural genes and proteins. qPCR analysis showed that the expression of neural genes in differentiated hiPSCs in forskolin, IBMX supplemented media was significantly higher than undifferentiated cells and those in induction media containing bFGF, EGF or RA. In conclusion, our results indicated a successful establishment protocol with high efficiency for differentiation of hiPSCs into neural lineages.

  6. microRNA-21 mediates stretch-induced osteogenic differentiation in human periodontal ligament stem cells.

    Science.gov (United States)

    Wei, Fulan; Liu, Dongxu; Feng, Cheng; Zhang, Fan; Yang, Shuangyan; Hu, Yijun; Ding, Gang; Wang, Songlin

    2015-02-01

    microRNAs (miRNAs) are short 20- to 22-nucleotide noncoding RNAs that negatively regulate the expression of target genes at the post-transcriptional level. The expression of specific miRNAs and their roles in the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) exposed to mechanical stretch remain unclear. Here, we found that stretch induced both osteogenic differentiation and the differential expression of miR-21 in PDLSCs. Furthermore, we identified activin receptor type IIB (ACVR2B) as a target gene of miR-21. Luciferase reporter assays showed that miR-21 interacts directly with the 3'-untranslated repeat sequence of ACVR2B mRNA. Mechanical stretch suppressed ACVR2B protein levels in PDLSCs, and this suppressive effect was modulated when endogenous miR-21 levels were either enhanced or inhibited. Both stretch and the expression of miR-21 altered endogenous ACVR2B protein levels and thus the osteogenic differentiation of PDLSCs. In addition, gain- and loss of function of ACVR2B mediated the osteogenic differentiation of PDLSCs. This study demonstrates that miR-21 is a mechanosensitive gene that plays an important role in the osteogenic differentiation of PDLSCs exposed to stretch.

  7. Concurrence of replicative senescence and elevated expression of p16(INK4A) with subculture-induced but not calcium-induced differentiation in normal human oral keratinocytes.

    Science.gov (United States)

    Lee, G; Park, B S; Han, S E; Oh, J E; You, Y O; Baek, J H; Kim, G S; Min, B M

    2000-10-01

    Primary normal human oral keratinocytes (NHOKs) undergo differentiation in the presence of calcium concentrations higher than 0.15 mM in vitro, which is useful in investigating the mechanisms involved in the differentiation of epithelial cells. Serial subculture of NHOKs to the postmitotic stage also induces terminal differentiation. However, the detailed mechanisms of both differentiation processes remain substantially unknown. To investigate the molecular differences in these processes, NHOKs were induced to differentiate by exposure to 1.2 mM of calcium and by serial subculture to the postmitotic stage. To study whether the cells were induced to differentiate and to undergo replicative senescence, the amount of cellular involucrin and the expression of senescence-associated beta-galactosidase (SA-beta-gal) were measured respectively. The expression of replicative senescence-associated genes and the activity of telomerase from the differentiated cells were also determined. Both calcium treatment and serial subculture to the postmitotic stage notably elevated the cellular involucrin. The percentage of SA-beta-gal-positive cells was significantly elevated by the continued subculture, but such changes were not observed in keratinocytes exposed to calcium. The concentration of cellular p16(INK4A) protein was progressively increased by the continued subculture but was not changed by calcium treatment. On the other hand, the concentrations of cellular p53 were similar in both differentiation processes. However, telomerase activity was lost in NHOKs that had undergone differentiation by both calcium treatment and serial subculture. The results indicate that calcium-induced differentiation of NHOKs has similar characteristics to their serial subculture-induced differentiation, but that the differentiation processes are not identical, because calcium-induced differentiation does not concur with either replicative senescence or the gradually increased concentration of p16

  8. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) promotes lung fibroblast proliferation, survival and differentiation to myofibroblasts.

    Science.gov (United States)

    Hasaneen, Nadia A; Cao, Jian; Pulkoski-Gross, Ashleigh; Zucker, Stanley; Foda, Hussein D

    2016-02-17

    Idiopathic pulmonary fibrosis (IPF) is a chronic progressively fatal disease. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) is a glycosylated transmembrane protein that induces the expression of some matrix metalloproteinase (MMP) in neighboring stromal cells through direct epithelial-stromal interactions. EMMPRIN is highly expressed in type II alveolar epithelial cells at the edges of the fibrotic areas in IPF lung sections. However, the exact role of EMMPRIN in IPF is unknown. To determine if EMMPRIN contributes to lung fibroblast proliferation, resistance to apoptosis, and differentiation to myofibroblasts, normal Human lung fibroblasts (NHLF) transiently transfected with either EMMPRIN/GFP or GFP were treated with TGF- β1 from 0 to 10 ng/ml for 48 h and examined for cell proliferation (thymidine incorporation), apoptosis (FACS analysis and Cell Death Detection ELISA assay), cell migration (Modified Boyden chamber) and differentiation to myofibroblasts using Western blot for α-smooth actin of cell lysates. The effect of EMMPRIN inhibition on NHLF proliferation, apoptosis, migration and differentiation to myofibroblasts after TGF- β1 treatment was examined using EMMPRIN blocking antibody. We examined the mechanism by which EMMPRIN induces its effects on fibroblasts by studying the β-catenin/canonical Wnt signaling pathway using Wnt luciferase reporter assays and Western blot for total and phosphorylated β-catenin. Human lung fibroblasts overexpressing EMMPRIN had a significant increase in cell proliferation and migration compared to control fibroblasts. Furthermore, EMMPRIN promoted lung fibroblasts resistance to apoptosis. Lung fibroblasts overexpressing EMMPRIN showed a significantly increased expression of α- smooth muscle actin, a marker of differentiation to myofibroblasts compared to control cells. TGF-β1 increased the expression of EMMPRIN in lung fibroblasts in a dose-dependent manner. Attenuation of EMMPRIN expression with the use of an

  9. Steroid sex hormone dynamics during estradiol-17β induced gonadal differentiation in Paralichthys olivaceus (Teleostei)

    Science.gov (United States)

    Sun, Peng; You, Feng; Liu, Mengxia; Wu, Zhihao; Wen, Aiyun; Li, Jun; Xu, Yongli; Zhang, Peijun

    2010-03-01

    Steroid sex hormones, such as estradiol-17β (E2) and testosterone (T), are important regulators of sex change in fish. In this study, we examined the effects of E2 treatment on the dynamics of E2 and T during gonadal differentiation in the olive flounder Paralichthys olivaceus using histology and radioimmunoassay (RIA). Flounder larvae were divided into five groups (G0-G4), and fed with 0 (control), 0.2, 2, 20 and 100 mg E2/kg feed from 35 to 110 day post hatching (dph). Fish growth in the G1 and G2 groups was not significantly different from that of the control group ( P>0.05), while fish in the G3 and G4 groups were less active and showed growth depression and high mortality. The gonads of fish in the G3 and G4 groups were smaller and surrounded by hyperplastic connective tissue. The frequency of females in the G0-G4 groups was 54.5%, 75.0%, 100%, 100% and 93.3%, respectively. The RIA analyses of E2 and T showed that T levels decreased during gonadal differentiation, and increased slightly at the onset of ovarian differentiation, while E2 levels increased gradually and peaked at the onset of ovarian differentiation in the control group. In the E2-treated groups, T levels decreased before the onset of ovarian differentiation. E2 levels were high on the 48 dph, but declined to a lower level on the 54 dph, and then increased gradually during gonadal differentiation. And a sharp increase of E2 levels were observed in all E2-treated groups at the onset of ovarian differentiation. The data suggest that T and E2 play important roles during gonadal differentiation, and an E2 dose of 2 mg/kg feed could induce sex reversal in P. olivaceus.

  10. Equine induced pluripotent stem cells have a reduced tendon differentiation capacity compared to embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Emma Patricia Bavin

    2015-11-01

    Full Text Available Tendon injuries occur commonly in horses and their repair through scar tissue formation predisposes horses to a high rate of re-injury. Pluripotent stem cells may provide a cell replacement therapy to improve tendon tissue regeneration and lower the frequency of re-injury. We have previously demonstrated that equine embryonic stem cells (ESCs differentiate into the tendon cell lineage upon injection into the damaged horse tendon and can differentiate into functional tendon cells in vitro to generate artificial tendons. Induced pluripotent stem cells (iPSCs have now been derived from horses but, to date, there are no reports on their ability to differentiate into tendon cells. As iPSCs can be produced from adult cell types, they provide a more accessible source of cells than ESCs, which require the use of horse embryos. The aim of this study was to compare tendon differentiation by ESCs and iPSCs produced through two independent methods. In 2-dimensional differentiation assays the iPSCs expressed tendon associated genes and proteins, which were enhanced by the presence of transforming growth factor-β3. However, in 3-dimensional differentiation assays the iPSCs failed to differentiate into functional tendon cells and generate artificial tendons. These results demonstrate the utility of the 3-dimensional in vitro tendon assay for measuring tendon differentiation and the need for more detailed studies to be performed on equine iPSCs to identify and understand their epigenetic differences from pluripotent ESCs prior to their clinical application.

  11. Vanadium pentoxide induces pulmonary inflammation and tumor promotion in a strain-dependent manner

    Directory of Open Access Journals (Sweden)

    Bauer Alison K

    2010-04-01

    Full Text Available Abstract Background Elevated levels of air pollution are associated with increased risk of lung cancer. Particulate matter (PM contains transition metals that may potentiate neoplastic development through the induction of oxidative stress and inflammation, a lung cancer risk factor. Vanadium pentoxide (V2O5 is a component of PM derived from fuel combustion as well as a source of occupational exposure in humans. In the current investigation we examined the influence of genetic background on susceptibility to V2O5-induced inflammation and evaluated whether V2O5 functions as a tumor promoter using a 2-stage (initiation-promotion model of pulmonary neoplasia in mice. Results A/J, BALB/cJ (BALB, and C57BL/6J (B6 mice were treated either with the initiator 3-methylcholanthrene (MCA; 10 μg/g; i.p. or corn oil followed by 5 weekly aspirations of V2O5 or PBS and pulmonary tumors were enumerated 20 weeks following MCA treatment. Susceptibility to V2O5-induced pulmonary inflammation was assessed in bronchoalveolar lavage fluid (BALF, and chemokines, transcription factor activity, and MAPK signaling were quantified in lung homogenates. We found that treatment of animals with MCA followed by V2O5 promoted lung tumors in both A/J (10.3 ± 0.9 tumors/mouse and BALB (2.2 ± 0.36 mice significantly above that observed with MCA/PBS or V2O5 alone (P 2O5 were also found to be more susceptible to V2O5-induced pulmonary inflammation and hyperpermeability (A/J>BALB>B6. Differential strain responses in inflammation were positively associated with elevated levels of the chemokines KC and MCP-1, higher NFκB and c-Fos binding activity, as well as sustained ERK1/2 activation in lung tissue. Conclusions In this study we demonstrate that V2O5, an occupational and environmentally relevant metal oxide, functions as an in vivo lung tumor promoter among different inbred strains of mice. Further, we identified a positive relationship between tumor promotion and susceptibility

  12. Cation induced differential effect on structural and functional properties of Mycobacterium tuberculosis α-Isopropylmalate synthase

    Directory of Open Access Journals (Sweden)

    Bhakuni Vinod

    2007-06-01

    Full Text Available Abstract Background α-isopropylmalate synthase (MtαIPMS, an enzyme that catalyzes the first committed step of the leucine biosynthetic pathway of Mycobacterium tuberculosis is a potential drug target for the anti-tuberculosis drugs. Cations induce differential effect of activation and inhibition of MtαIPMS. To date no concrete mechanism for such an opposite effect of similarly charged cations on the functional activity of enzyme has been presented. Results Effect of cations on the structure and function of the MtαIPMS has been studied in detail. The studies for the first time demonstrate that different cations interact specifically at different sites in the enzyme and modulate the enzyme structure differentially. The inhibitors Zn2+ and Cd2+ ions interact directly with the catalytic domain of the enzyme and induce unfolding/denaturation of the domain. The activator K+ also interacts with the catalytic TIM barrel domain however, it does not induce any significant effect on the enzyme structure. Studies with isolated catalytic TIM barrel domain showed that it can carry out the catalytic function on its own but probably requires the non-catalytic C-terminal domain for optimum functioning. An important observation was that divalent cations induce significant interaction between the regulatory and the catalytic domain of MtαIPMS thus inducing structural cooperativity in the enzyme. This divalent cation induced structural cooperativity might result in modulation of activity of the catalytic domain by regulatory domain. Conclusion The studies for the first time demonstrate that different cations bind at different sites in the enzyme leading to their differential effects on the structure and functional activity of the enzyme.

  13. Experimental study of millimeter wave-induced differentiation of bone marrow mesenchymal stem cells into chondrocytes.

    Science.gov (United States)

    Wu, Guang-Wen; Liu, Xian-Xiang; Wu, Ming-Xia; Zhao, Jin-Yan; Chen, Wen-Lie; Lin, Ru-Hui; Lin, Jiu-Mao

    2009-04-01

    Low power millimeter wave irradiation is widely used in clinical medicine. We describe the effects of this treatment on cultured mesenchymal stem cells (MSCs) and attempted to identify the underlying mechanism. Cells cultured using the whole marrow attachment culture method proliferated dispersedly or in clones. Flow cytometric analyses showed that the MSCs were CD90 positive, but negative for CD45. The negative control group (A) did not express detectable levels of Cbfa1 or Sox9 mRNA at any time point, while cells in the millimeter wave-induced groups (B and C) increasingly expressed both genes after the fourth day post-induction. Statistical analysis showed that starting on the fourth day post-induction, there were very significant differences in the expression of Cbfa1 and Sox9 mRNA between groups A and B as well as A and C at any given time point, between treated groups B and C after identical periods of induction, and within each treated group at different induction times. Transition electron microscopy analysis showed that the rough endoplasmic reticulum of cells in the induced groups was richer and more developed than in cells of the negative control group, and that the shape of cells shifted from long-spindle to near ellipse. Toluidine blue staining revealed heterochromia in the cytoplasm and extracellular matrix of cells in the induced groups, whereas no obvious heterochromia was observed in negative control cells. Induced cells also exhibited positive immunohistochemical staining of collagen II, in contrast to the negative controls. These results show that millimeter wave treatment successfully induced MSCs to differentiate as chondrocytes and the extent of differentiation increased with treatment duration. Our findings suggest that millimeter wave irradiation can be employed as a novel non-drug inducing method for the differentiation of MSCs into chondrocytes.

  14. CHD1 regulates cell fate determination by activation of differentiation-induced genes

    DEFF Research Database (Denmark)

    Baumgart, Simon J; Najafova, Zeynab; Hossan, Tareq

    2017-01-01

    . Furthermore, we observed that CHD1-dependent genes are mainly induced during osteoblast differentiation and are characterized by higher levels of CHD1 occupancy around the TSS. Interestingly, CHD1 depletion resulted in increased pausing of RNA Polymerase II (RNAPII) and decreased H2A.Z occupancy close......The coordinated temporal and spatial activation of gene expression is essential for proper stem cell differentiation. The Chromodomain Helicase DNA-binding protein 1 (CHD1) is a chromatin remodeler closely associated with transcription and nucleosome turnover downstream of the transcriptional start...... to the TSS, but not at enhancer regions. These findings reveal a novel role for CHD1 during osteoblast differentiation and provide further insights into the intricacies of epigenetic regulatory mechanisms controlling cell fate determination....

  15. Optimizing neuronal differentiation from induced pluripotent stem cells to model ASD

    Directory of Open Access Journals (Sweden)

    Dae-Sung eKim

    2014-04-01

    Full Text Available Autism spectrum disorder (ASD is an early-onset neurodevelopmental disorder characterized by deficits in social communication, and restricted and repetitive patterns of behavior. Despite its high prevalence, discovery of pathophysiological mechanisms underlying ASD has lagged due to a lack of appropriate model systems. Recent advances in induced pluripotent stem cell (iPSC technology and neural differentiation techniques allow for detailed functional analyses of neurons generated from living individuals with ASD. Refinement of cortical neuron differentiation methods from iPSCs will enable mechanistic studies of specific neuronal subpopulations that may be preferentially impaired in ASD. In this review, we summarize recent accomplishments in differentiation of cortical neurons from human pluripotent stems cells and efforts to establish in vitro model systems to study ASD using personalized neurons.

  16. N-Docosahexaenoylethanolamine ameliorates ethanol-induced impairment of neural stem cell neurogenic differentiation.

    Science.gov (United States)

    Rashid, Mohammad Abdur; Kim, Hee-Yong

    2016-03-01

    Previous studies demonstrated that prenatal exposure to ethanol interferes with embryonic and fetal development, and causes abnormal neurodevelopment. Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid highly enriched in the brain, was shown to be essential for proper brain development and function. Recently, we found that N-docosahexenoyethanolamine (synaptamide), an endogenous metabolite of DHA, is a potent PKA-dependent neurogenic factor for neural stem cell (NSC) differentiation. In this study, we demonstrate that ethanol at pharmacologically relevant concentrations downregulates cAMP signaling in NSC and impairs neurogenic differentiation. In contrast, synaptamide reverses ethanol-impaired NSC neurogenic differentiation through counter-acting on the cAMP production system. NSC exposure to ethanol (25-50 mM) for 4 days dose-dependently decreased the number of Tuj-1 positive neurons and PKA/CREB phosphorylation with a concomitant reduction of cellular cAMP. Ethanol-induced cAMP reduction was accompanied by the inhibition of G-protein activation and expression of adenylyl cyclase (AC) 7 and AC8, as well as PDE4 upregulation. In contrast to ethanol, synaptamide increased cAMP production, GTPγS binding, and expression of AC7 and AC8 isoforms in a cAMP-dependent manner, offsetting the ethanol-induced impairment in neurogenic differentiation. These results indicate that synaptamide can reduce ethanol-induced impairment of neuronal differentiation by counter-affecting shared targets in G-protein coupled receptor (GPCR)/cAMP signaling. The synaptamide-mediated mechanism observed in this study may offer a possible avenue for ameliorating the adverse impact of fetal alcohol exposure on neurodevelopment.

  17. Gene expression profiling of human neural progenitor cells following the serum-induced astrocyte differentiation.

    Science.gov (United States)

    Obayashi, Shinya; Tabunoki, Hiroko; Kim, Seung U; Satoh, Jun-ichi

    2009-05-01

    Neural stem cells (NSC) with self-renewal and multipotent properties could provide an ideal cell source for transplantation to treat spinal cord injury, stroke, and neurodegenerative diseases. However, the majority of transplanted NSC and neural progenitor cells (NPC) differentiate into astrocytes in vivo under pathological environments in the central nervous system, which potentially cause reactive gliosis. Because the serum is a potent inducer of astrocyte differentiation of rodent NPC in culture, we studied the effect of the serum on gene expression profile of cultured human NPC to identify the gene signature of astrocyte differentiation of human NPC. Human NPC spheres maintained in the serum-free culture medium were exposed to 10% fetal bovine serum (FBS) for 72 h, and processed for analyzing on a Whole Human Genome Microarray of 41,000 genes, and the microarray data were validated by real-time RT-PCR. The serum elevated the levels of expression of 45 genes, including ID1, ID2, ID3, CTGF, TGFA, METRN, GFAP, CRYAB and CSPG3, whereas it reduced the expression of 23 genes, such as DLL1, DLL3, PDGFRA, SOX4, CSPG4, GAS1 and HES5. Thus, the serum-induced astrocyte differentiation of human NPC is characterized by a counteraction of ID family genes on Delta family genes. Coimmunoprecipitation analysis identified ID1 as a direct binding partner of a proneural basic helix-loop-helix (bHLH) transcription factor MASH1. Luciferase assay indicated that activation of the DLL1 promoter by MASH1 was counteracted by ID1. Bone morphogenetic protein 4 (BMP4) elevated the levels of ID1 and GFAP expression in NPC under the serum-free culture conditions. Because the serum contains BMP4, these results suggest that the serum factor(s), most probably BMP4, induces astrocyte differentiation by upregulating the expression of ID family genes that repress the proneural bHLH protein-mediated Delta expression in human NPC.

  18. Differentially Gene Expression Profile Related to Inflammation in Endometrial Cells Induce by Lipopolysaccharide

    Institute of Scientific and Technical Information of China (English)

    Li-hua QIN; Ruo-guang WANG; Sheng LI; Chun-mei LI

    2009-01-01

    Objective To investigate differentially expressed genes related to inflammation in endometrial cells induced by Lipopolysaccharide(LPS).Methods Normal endometrium in the proliferative phase of specimen from 3 cases for the experiment was collected. The LPS group were treated with 50 μg/ml LPS. Total RNA was extracted using Trizol reagent from cells. RNA quality was assessed by determining the OD260/280 ratio by agarose gel electrophoresis, the chip was scanned by laser scanner. The acquired was analyzed. Results A total of differentially expressed genes were found, these genes were relative to many aspects. Among them, the expression of genes involved in inflammation were up-regulated by LPS, such as overexpression of lL-lβ, 8, etc.Conclusion The results indicates that inflammation-related genes may be one of the mechanisms of abnormal uterine bleeding by LPS-induced.

  19. NF1 loss induces senescence during human melanocyte differentiation in an iPSC-based model.

    Science.gov (United States)

    Larribere, Lionel; Wu, Huizi; Novak, Daniel; Galach, Marta; Bernhardt, Mathias; Orouji, Elias; Weina, Kasia; Knappe, Nathalie; Sachpekidis, Christos; Umansky, Ludmila; Beckhove, Philipp; Umansky, Viktor; De Schepper, Sofie; Kaufmann, Dieter; Ballotti, Robert; Bertolotto, Corine; Utikal, Jochen

    2015-07-01

    Neurofibromatosis type 1 (NF1) is a frequent genetic disease leading to the development of Schwann cell-derived neurofibromas or melanocytic lesions called café-au-lait macules (CALMs). The molecular mechanisms involved in CALMs formation remain largely unknown. In this report, we show for the first time pathophysiological mechanisms of abnormal melanocyte differentiation in a human NF1(+/-) -induced pluripotent stem cell (iPSC)-based model. We demonstrate that NF1 patient-derived fibroblasts can be successfully reprogrammed in NF1(+/-) iPSCs with active RAS signaling and that NF1 loss induces senescence during melanocyte differentiation as well as in patient's-derived CALMs, revealing a new role for NF1 in the melanocyte lineage.

  20. Matrix Stiffness–Induced Myofibroblast Differentiation Is Mediated by Intrinsic Mechanotransduction

    Science.gov (United States)

    Huang, Xiangwei; Yang, Naiheng; Fiore, Vincent F.; Barker, Thomas H.; Sun, Yi; Morris, Stephan W.; Ding, Qiang; Thannickal, Victor J.

    2012-01-01

    The mechanical properties of the extracellular matrix have recently been shown to promote myofibroblast differentiation and lung fibrosis. Mechanisms by which matrix stiffness regulates myofibroblast differentiation are not fully understood. The goal of this study was to determine the intrinsic mechanisms of mechanotransduction in the regulation of matrix stiffness–induced myofibroblast differentiation. A well established polyacrylamide gel system with tunable substrate stiffness was used in this study. Megakaryoblastic leukemia factor-1 (MKL1) nuclear translocation was imaged by confocal immunofluorescent microscopy. The binding of MKL1 to the α-smooth muscle actin (α-SMA) gene promoter was quantified by quantitative chromatin immunoprecipitation assay. Normal human lung fibroblasts responded to matrix stiffening with changes in actin dynamics that favor filamentous actin polymerization. Actin polymerization resulted in nuclear translocation of MKL1, a serum response factor coactivator that plays a central role in regulating the expression of fibrotic genes, including α-SMA, a marker for myofibroblast differentiation. Mouse lung fibroblasts deficient in Mkl1 did not respond to matrix stiffening with increased α-SMA expression, whereas ectopic expression of human MKL1 cDNA restored the ability of Mkl1 null lung fibroblasts to express α-SMA. Furthermore, matrix stiffening promoted production and activation of the small GTPase RhoA, increased Rho kinase (ROCK) activity, and enhanced fibroblast contractility. Inhibition of RhoA/ROCK abrogated stiff matrix–induced actin cytoskeletal reorganization, MKL1 nuclear translocation, and myofibroblast differentiation. This study indicates that actin cytoskeletal remodeling–mediated activation of MKL1 transduces mechanical stimuli from the extracellular matrix to a fibrogenic program that promotes myofibroblast differentiation, suggesting an intrinsic mechanotransduction mechanism. PMID:22461426

  1. Differential effectiveness of microbially induced resistance against herbivorous insects in Arabidopsis.

    Science.gov (United States)

    Van Oosten, Vivian R; Bodenhausen, Natacha; Reymond, Philippe; Van Pelt, Johan A; Van Loon, L C; Dicke, Marcel; Pieterse, Corné M J

    2008-07-01

    Rhizobacteria-induced systemic resistance (ISR) and pathogen-induced systemic acquired resistance (SAR) have a broad, yet partly distinct, range of effectiveness against pathogenic microorganisms. Here, we investigated the effectiveness of ISR and SAR in Arabidopsis against the tissue-chewing insects Pieris rapae and Spodoptera exigua. Resistance against insects consists of direct defense, such as the production of toxins and feeding deterrents and indirect defense such as the production of plant volatiles that attract carnivorous enemies of the herbivores. Wind-tunnel experiments revealed that ISR and SAR did not affect herbivore-induced attraction of the parasitic wasp Cotesia rubecula (indirect defense). By contrast, ISR and SAR significantly reduced growth and development of the generalist herbivore S. exigua, although not that of the specialist P. rapae. This enhanced direct defense against S. exigua was associated with potentiated expression of the defense-related genes PDF1.2 and HEL. Expression profiling using a dedicated cDNA microarray revealed four additional, differentially primed genes in microbially induced S. exigua-challenged plants, three of which encode a lipid-transfer protein. Together, these results indicate that microbially induced plants are differentially primed for enhanced insect-responsive gene expression that is associated with increased direct defense against the generalist S. exigua but not against the specialist P. rapae.

  2. Myostatin acts as an autocrine/paracrine negative regulator in myoblast differentiation from human induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Fei; Kishida, Tsunao; Ejima, Akika [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Gojo, Satoshi [Department of Cardiac Support, Kyoto Prefectural University of Medicine, Kyoto (Japan); Mazda, Osam, E-mail: mazda@koto.kpu-m.ac.jp [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan)

    2013-02-08

    Highlights: ► iPS-derived cells express myostatin and its receptor upon myoblast differentiation. ► Myostatin inhibits myoblast differentiation by inhibiting MyoD and Myo5a induction. ► Silencing of myostatin promotes differentiation of human iPS cells into myoblasts. -- Abstract: Myostatin, also known as growth differentiation factor (GDF-8), regulates proliferation of muscle satellite cells, and suppresses differentiation of myoblasts into myotubes via down-regulation of key myogenic differentiation factors including MyoD. Recent advances in stem cell biology have enabled generation of myoblasts from pluripotent stem cells, but it remains to be clarified whether myostatin is also involved in regulation of artificial differentiation of myoblasts from pluripotent stem cells. Here we show that the human induced pluripotent stem (iPS) cell-derived cells that were induced to differentiate into myoblasts expressed myostatin and its receptor during the differentiation. An addition of recombinant human myostatin (rhMyostatin) suppressed induction of MyoD and Myo5a, resulting in significant suppression of myoblast differentiation. The rhMyostatin treatment also inhibited proliferation of the cells at a later phase of differentiation. RNAi-mediated silencing of myostatin promoted differentiation of human iPS-derived embryoid body (EB) cells into myoblasts. These results strongly suggest that myostatin plays an important role in regulation of myoblast differentiation from iPS cells of human origin. The present findings also have significant implications for potential regenerative medicine for muscular diseases.

  3. Pulsed DC Electric Field-Induced Differentiation of Cortical Neural Precursor Cells.

    Directory of Open Access Journals (Sweden)

    Hui-Fang Chang

    Full Text Available We report the differentiation of neural stem and progenitor cells solely induced by direct current (DC pulses stimulation. Neural stem and progenitor cells in the adult mammalian brain are promising candidates for the development of therapeutic neuroregeneration strategies. The differentiation of neural stem and progenitor cells depends on various in vivo environmental factors, such as nerve growth factor and endogenous EF. In this study, we demonstrated that the morphologic and phenotypic changes of mouse neural stem and progenitor cells (mNPCs could be induced solely by exposure to square-wave DC pulses (magnitude 300 mV/mm at frequency of 100-Hz. The DC pulse stimulation was conducted for 48 h, and the morphologic changes of mNPCs were monitored continuously. The length of primary processes and the amount of branching significantly increased after stimulation by DC pulses for 48 h. After DC pulse treatment, the mNPCs differentiated into neurons, astrocytes, and oligodendrocytes simultaneously in stem cell maintenance medium. Our results suggest that simple DC pulse treatment could control the fate of NPCs. With further studies, DC pulses may be applied to manipulate NPC differentiation and may be used for the development of therapeutic strategies that employ NPCs to treat nervous system disorders.

  4. Behavioural differentiation induced by environmental variation when crossing a toxic zone in an amoeba

    Science.gov (United States)

    Kunita, Itsuki; Ueda, Kei-Ichi; Akita, Dai; Kuroda, Shigeru; Nakagaki, Toshiyuki

    2017-09-01

    Organisms choose from among various courses of action in response to a wide variety of environmental conditions and the mechanism by which various behaviours are induced is an open question. Interesting behaviour was recently reported: that a unicellular organism of slime mold Physarum polycephalum known as an amoeba had multiple responses (crossing, returning, etc) when the amoeba encounters a zone with toxic levels of quinine, even under carefully controlled conditions. We here examined this elegant example in more detail to obtain insight into behavioural differentiation. We found that the statistical distribution of passage times across a quinine zone switch from unimodal to bimodal (with peaks corresponding to fast crossing and no crossing) when a periodic light stimulation to modulate a biorhythm in amoeba is applied homogeneously across the space, even under the same level of chemical stimuli. Based on a mathematical model for cell movement in amoeba, we successfully reproduced the stimulation-induced differentiation, which was observed experimentally. These dynamics may be explained by a saddle structure around a canard solution. Our results imply that the differentiation of behavioural types in amoeba is modified step-by-step via the compounding of stimulation inputs. The complex behaviour like the differentiation in amoeba may provide a basis for understanding the mechanism of behaviour selection in higher animals from an ethological perspective.

  5. Pulsed DC Electric Field–Induced Differentiation of Cortical Neural Precursor Cells

    Science.gov (United States)

    Chang, Hui-Fang; Lee, Ying-Shan; Tang, Tang K.; Cheng, Ji-Yen

    2016-01-01

    We report the differentiation of neural stem and progenitor cells solely induced by direct current (DC) pulses stimulation. Neural stem and progenitor cells in the adult mammalian brain are promising candidates for the development of therapeutic neuroregeneration strategies. The differentiation of neural stem and progenitor cells depends on various in vivo environmental factors, such as nerve growth factor and endogenous EF. In this study, we demonstrated that the morphologic and phenotypic changes of mouse neural stem and progenitor cells (mNPCs) could be induced solely by exposure to square-wave DC pulses (magnitude 300 mV/mm at frequency of 100-Hz). The DC pulse stimulation was conducted for 48 h, and the morphologic changes of mNPCs were monitored continuously. The length of primary processes and the amount of branching significantly increased after stimulation by DC pulses for 48 h. After DC pulse treatment, the mNPCs differentiated into neurons, astrocytes, and oligodendrocytes simultaneously in stem cell maintenance medium. Our results suggest that simple DC pulse treatment could control the fate of NPCs. With further studies, DC pulses may be applied to manipulate NPC differentiation and may be used for the development of therapeutic strategies that employ NPCs to treat nervous system disorders. PMID:27352251

  6. Differentiation of Human Induced Pluripotent Stem Cells to Mammary-like Organoids

    Directory of Open Access Journals (Sweden)

    Ying Qu

    2017-02-01

    Full Text Available Human induced pluripotent stem cells (iPSCs can give rise to multiple cell types and hold great promise in regenerative medicine and disease-modeling applications. We have developed a reliable two-step protocol to generate human mammary-like organoids from iPSCs. Non-neural ectoderm-cell-containing spheres, referred to as mEBs, were first differentiated and enriched from iPSCs using MammoCult medium. Gene expression profile analysis suggested that mammary gland function-associated signaling pathways were hallmarks of 10-day differentiated mEBs. We then generated mammary-like organoids from 10-day mEBs using 3D floating mixed gel culture and a three-stage differentiation procedure. These organoids expressed common breast tissue, luminal, and basal markers, including estrogen receptor, and could be induced to produce milk protein. These results demonstrate that human iPSCs can be directed in vitro toward mammary lineage differentiation. Our findings provide an iPSC-based model for studying regulation of normal mammary cell fate and function as well as breast disease development.

  7. Differentiation of Human Induced Pluripotent Stem Cells to Mammary-like Organoids.

    Science.gov (United States)

    Qu, Ying; Han, Bingchen; Gao, Bowen; Bose, Shikha; Gong, Yiping; Wawrowsky, Kolja; Giuliano, Armando E; Sareen, Dhruv; Cui, Xiaojiang

    2017-02-14

    Human induced pluripotent stem cells (iPSCs) can give rise to multiple cell types and hold great promise in regenerative medicine and disease-modeling applications. We have developed a reliable two-step protocol to generate human mammary-like organoids from iPSCs. Non-neural ectoderm-cell-containing spheres, referred to as mEBs, were first differentiated and enriched from iPSCs using MammoCult medium. Gene expression profile analysis suggested that mammary gland function-associated signaling pathways were hallmarks of 10-day differentiated mEBs. We then generated mammary-like organoids from 10-day mEBs using 3D floating mixed gel culture and a three-stage differentiation procedure. These organoids expressed common breast tissue, luminal, and basal markers, including estrogen receptor, and could be induced to produce milk protein. These results demonstrate that human iPSCs can be directed in vitro toward mammary lineage differentiation. Our findings provide an iPSC-based model for studying regulation of normal mammary cell fate and function as well as breast disease development.

  8. Rapid differentiation of superficial urothelial cells after chitosan-induced desquamation.

    Science.gov (United States)

    Veranic, Peter; Erman, Andreja; Kerec-Kos, Mojca; Bogataj, Marija; Mrhar, Ales; Jezernik, Kristijan

    2009-01-01

    Superficial cell desquamation followed by differentiation of newly exposed superficial cells induces regeneration of the urinary bladder epithelium, urothelium. In the present work, chitosan was evaluated as a new inducer of urothelial cell desquamation, in order to study the regeneration of mouse urothelial cells in vivo. Intravesical application of chitosan dispersion caused complete removal of only the superficial layer of cells within 20 min of treatment. Differentiation of the new superficial layer was followed by the appearance and distribution of three urothelial differentiation markers, tight junction protein ZO1, cytokeratin 20 and the maturation of the apical plasma membrane. The arrangement of ZO1 into continuous lines in individual cells of the intermediate layer was already found after 10 min of chitosan application, when desquamation had just started. The appearance of the apical membrane changed from microvillar to typically scalloped within 20 min of regeneration, while complete arrangement of the cytokeratin 20 network took 60 min. These findings provide a new perspective on the rate of the differentiation process in the urothelium and make chitosan a new and a very controllable tool for studies on urothelial regeneration.

  9. Directed neuronal differentiation of mouse embryonic and induced pluripotent stem cells and their gene expression profiles.

    Science.gov (United States)

    Chen, Xuesong; Gu, Qi; Wang, Xiang; Ma, Qingwen; Tang, Huixiang; Yan, Xiaoshuang; Guo, Xinbing; Yan, Hao; Hao, Jie; Zeng, Fanyi

    2013-07-01

    Embryonic stem cells (ESCs) may be useful as a therapeutic source of cells for the production of healthy tissue; however, they are associated with certain challenges including immunorejection as well as ethical issues. Induced pluripotent stem cells (iPSCs) are a promising substitute since a patient's own adult cells would serve as tissue precursors. Ethical concerns prevent a full evaluation of the developmental potency of human ESCs and iPSCs, therefore, mouse iPSC models are required for protocol development and safety assessments. We used a modified culturing protocol to differentiate pluripotent cells from a mouse iPS cell line and two mouse ES cell lines into neurons. Our results indicated that all three pluripotent stem cell lines underwent nearly the same differentiation process when induced to form neurons in vitro. Genomic expression microarray profiling and single-cell RT-qPCR were used to analyze the neural lineage differentiation process, and more than one thousand differentially expressed genes involved in multiple molecular processes relevant to neural development were identified.

  10. Differential Effects of Stress-induced Cortisol Responses on Recollection and Familiarity-based Recognition Memory

    OpenAIRE

    McCullough, Andrew M.; Ritchey, Maureen; Ranganath, Charan; Yonelinas, Andrew

    2015-01-01

    Stress-induced changes in cortisol can impact memory in various ways. However, the precise relationship between cortisol and recognition memory is still poorly understood. For instance, there is reason to believe that stress could differentially affect recollection-based memory, which depends on the hippocampus, and familiarity-based recognition, which can be supported by neocortical areas alone. Accordingly, in the current study we examined the effects of stress-related changes in cortisol o...

  11. The tyrosine kinase inhibitor dasatinib induces a marked adipogenic differentiation of human multipotent mesenchymal stromal cells.

    Directory of Open Access Journals (Sweden)

    Adriana Borriello

    Full Text Available BACKGROUND: The introduction of specific BCR-ABL inhibitors in chronic myelogenous leukemia therapy has entirely mutated the prognosis of this hematologic cancer from being a fatal disorder to becoming a chronic disease. Due to the probable long lasting treatment with tyrosine-kinase inhibitors (TKIs, the knowledge of their effects on normal cells is of pivotal importance. DESIGN AND METHODS: We investigated the effects of dasatinib treatment on human bone marrow-derived mesenchymal stromal cells (MSCs. RESULTS: Our findings demonstrate, for the first time, that dasatinib induces MSCs adipocytic differentiation. Particularly, when the TKI is added to the medium inducing osteogenic differentiation, a high MSCs percentage acquires adipocytic morphology and overexpresses adipocytic specific genes, including PPARγ, CEBPα, LPL and SREBP1c. Dasatinib also inhibits the activity of alkaline phosphatase, an osteogenic marker, and remarkably reduces matrix mineralization. The increase of PPARγ is also confirmed at protein level. The component of osteogenic medium required for dasatinib-induced adipogenesis is dexamethasone. Intriguingly, the increase of adipocytic markers is also observed in MSCs treated with dasatinib alone. The TKI effect is phenotype-specific, since fibroblasts do not undergo adipocytic differentiation or PPARγ increase. CONCLUSIONS: Our data demonstrate that dasatinib treatment affects bone marrow MSCs commitment and suggest that TKIs therapy might modify normal phenotypes with potential significant negative consequences.

  12. The aryl hydrocarbon receptor ligand ITE inhibits TGFβ1-induced human myofibroblast differentiation.

    Science.gov (United States)

    Lehmann, Geniece M; Xi, Xia; Kulkarni, Ajit A; Olsen, Keith C; Pollock, Stephen J; Baglole, Carolyn J; Gupta, Shikha; Casey, Ann E; Huxlin, Krystel R; Sime, Patricia J; Feldon, Steven E; Phipps, Richard P

    2011-04-01

    Fibrosis can occur in any human tissue when the normal wound healing response is amplified. Such amplification results in fibroblast proliferation, myofibroblast differentiation, and excessive extracellular matrix deposition. Occurrence of these sequelae in organs such as the eye or lung can result in severe consequences to health. Unfortunately, medical treatment of fibrosis is limited by a lack of safe and effective therapies. These therapies may be developed by identifying agents that inhibit critical steps in fibrotic progression; one such step is myofibroblast differentiation triggered by transforming growth factor-β1 (TGFβ1). In this study, we demonstrate that TGFβ1-induced myofibroblast differentiation is blocked in human fibroblasts by a candidate endogenous aryl hydrocarbon receptor (AhR) ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Our data show that ITE disrupts TGFβ1 signaling by inhibiting the nuclear translocation of Smad2/3/4. Although ITE functions as an AhR agonist, and biologically persistent AhR agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, cause severe toxic effects, ITE exhibits no toxicity. Interestingly, ITE effectively inhibits TGFβ1-driven myofibroblast differentiation in AhR(-/-) fibroblasts: Its ability to inhibit TGFβ1 signaling is AhR independent. As supported by the results of this study, the small molecule ITE inhibits myofibroblast differentiation and may be useful clinically as an antiscarring agent.

  13. Lead Poisoning Disturbs Oligodendrocytes Differentiation Involved in Decreased Expression of NCX3 Inducing Intracellular Calcium Overload

    Directory of Open Access Journals (Sweden)

    Teng Ma

    2015-08-01

    Full Text Available Lead (Pb poisoning has always been a serious health concern, as it permanently damages the central nervous system. Chronic Pb accumulation in the human body disturbs oligodendrocytes (OLs differentiation, resulting in dysmyelination, but the molecular mechanism remains unknown. In this study, Pb at 1 μM inhibits OLs precursor cells (OPCs differentiation via decreasing the expression of Olig 2, CNPase proteins in vitro. Moreover, Pb treatment inhibits the sodium/calcium exchanger 3 (NCX3 mRNA expression, one of the major means of calcium (Ca2+ extrusion at the plasma membrane during OPCs differentiation. Also addition of KB-R7943, NCX3 inhibitor, to simulate Pb toxicity, resulted in decreased myelin basic protein (MBP expression and cell branching. Ca2+ response trace with Pb and KB-R7943 treatment did not drop down in the same recovery time as the control, which elevated intracellular Ca2+ concentration reducing MBP expression. In contrast, over-expression of NCX3 in Pb exposed OPCs displayed significant increase MBP fluorescence signal in positive regions and CNPase expression, which recovered OPCs differentiation to counterbalance Pb toxicity. In conclusion, Pb exposure disturbs OLs differentiation via affecting the function of NCX3 by inducing intracellular calcium overload.

  14. Sphingosine 1-Phosphate Induces Differentiation of Mesoangioblasts towards Smooth Muscle. A Role for GATA6

    Science.gov (United States)

    Donati, Chiara; Marseglia, Giuseppina; Magi, Alberto; Serratì, Simona; Cencetti, Francesca; Bernacchioni, Caterina; Nannetti, Genni; Benelli, Matteo; Brunelli, Silvia; Torricelli, Francesca; Cossu, Giulio; Bruni, Paola

    2011-01-01

    Different cells can contribute to repair following vascular injury by differentiating into smooth muscle (SM) cells; however the extracellular signals involved are presently poorly characterized. Mesoangioblasts are progenitor cells capable of differentiating into various mesoderm cell types including SM cells. In this study the biological action exerted by the pleiotropic sphingolipid sphingosine 1-phosphate (S1P) in human mesoangioblasts has been initially investigated by cDNA microarray analysis. Obtained data confirmed the anti-apoptotic action of this sphingolipid and identified for the first time a strong differentiating action toward SM cells. Quantitative mRNA and protein analysis corroborated the microarray results demonstrating enhanced expression of myogenic marker proteins and regulation of the expression of transcription factor GATA6 and its co-regulator, LMCD1. Importantly, GATA6 up-regulation induced by S1P was responsible for the enhanced expression of SM-specific contractile proteins. Moreover, by specific gene silencing experiments GATA6 was critical in the pro-differentiating activity of the cytokine TGFβ. Finally, the pharmacological inhibition of endogenous S1P formation in response to TGFβ abrogated GATA6 up-regulation, supporting the view that the S1P pathway plays a physiological role in mediating the pro-myogenic effect of TGFβ. This study individuates GATA6 as novel player in the complex transcriptional regulation of mesoangioblast differentiation into SM cells and highlights a role for S1P to favour vascular regeneration. PMID:21629665

  15. Rapamycin induces differentiation of glioma stem/progenitor cells by activating autophagy

    Institute of Scientific and Technical Information of China (English)

    Wen-Zhuo Zhuang; Lin-Mei Long; Wen-Jun Ji; Zhong-Qin Liang

    2011-01-01

    Glioma stem/progenitor cells(GSPCs) are considered to be responsible for the initiation,propagation,and recurrence of gliomas.The factors determining their differentiation remain poorly defined.Accumulating evidences indicate that alterations in autophagy may influence cell fate during mammalian development and differentiation.Here,we investigated the role of autophagy in GSPC differentiation.SU-2 cells were treated with rapamycin,3-methyladenine (3-MA) plus rapamycin,E64d plus rapamycin,or untreated as control.SU-2 cell xenografts in nude mice were treated with rapamycin or 3-MA plus rapamycin,or untreated as control.Western blotting and immunocytochemistry showed up-regulation of microtubule-associated protein light chain-3(LC3)-II in rapamycin-treated cells.The neurosphere formation rate and the number of cells in each neurosphere were significantly lower in the rapamycin treatment group than in other groups.Real-time PCR and immunocytochemistry showed down-regulation of stem/progenitor cell markers and up-regulation of differentiation markers in rapamycin-treated cells.Transmission electron microscopy revealed autophagy activation in rapamycin-treated tumor cells in mice.Immunohistochemistry revealed decreased Nestin-positive cells and increased GFAP-positive cells in rapamycin-treated tumor sections.These results indicate that rapamycin induces differentiation of GSPCs by activating autophagy.

  16. Radiation-induced glioblastoma signaling cascade regulates viability, apoptosis and differentiation of neural stem cells (NSC).

    Science.gov (United States)

    Ivanov, Vladimir N; Hei, Tom K

    2014-12-01

    Ionizing radiation alone or in combination with chemotherapy is the main treatment modality for brain tumors including glioblastoma. Adult neurons and astrocytes demonstrate substantial radioresistance; in contrast, human neural stem cells (NSC) are highly sensitive to radiation via induction of apoptosis. Irradiation of tumor cells has the potential risk of affecting the viability and function of NSC. In this study, we have evaluated the effects of irradiated glioblastoma cells on viability, proliferation and differentiation potential of non-irradiated (bystander) NSC through radiation-induced signaling cascades. Using media transfer experiments, we demonstrated significant effects of the U87MG glioblastoma secretome after gamma-irradiation on apoptosis in non-irradiated NSC. Addition of anti-TRAIL antibody to the transferred media partially suppressed apoptosis in NSC. Furthermore, we observed a dramatic increase in the production and secretion of IL8, TGFβ1 and IL6 by irradiated glioblastoma cells, which could promote glioblastoma cell survival and modify the effects of death factors in bystander NSC. While differentiation of NSC into neurons and astrocytes occurred efficiently with the corresponding differentiation media, pretreatment of NSC for 8 h with medium from irradiated glioblastoma cells selectively suppressed the differentiation of NSC into neurons, but not into astrocytes. Exogenous IL8 and TGFβ1 increased NSC/NPC survival, but also suppressed neuronal differentiation. On the other hand, IL6 was known to positively affect survival and differentiation of astrocyte progenitors. We established a U87MG neurosphere culture that was substantially enriched by SOX2(+) and CD133(+) glioma stem-like cells (GSC). Gamma-irradiation up-regulated apoptotic death in GSC via the FasL/Fas pathway. Media transfer experiments from irradiated GSC to non-targeted NSC again demonstrated induction of apoptosis and suppression of neuronal differentiation of NSC. In

  17. Ketamine induces toxicity in human neurons differentiated from embryonic stem cells via mitochondrial apoptosis pathway

    Science.gov (United States)

    Bosnjak, Zeljko J.; Yan, Yasheng; Canfield, Scott; Muravyeva, Maria Y.; Kikuchi, Chika; Wells, Clive; Corbett, John; Bai, Xiaowen

    2013-01-01

    Ketamine is widely used for anesthesia in pediatric patients. Growing evidence indicates that ketamine causes neurotoxicity in a variety of developing animal models. Our understanding of anesthesia neurotoxicity in humans is currently limited by difficulties in obtaining neurons and performing developmental toxicity studies in fetal and pediatric populations. It may be possible to overcome these challenges by obtaining neurons from human embryonic stem cells (hESCs) in vitro. hESCs are able to replicate indefinitely and differentiate into every cell type. In this study, we investigated the toxic effect of ketamine on neurons differentiated from hESCs. Two-week-old neurons were treated with different doses and durations of ketamine with or without the reactive oxygen species (ROS) scavenger, Trolox. Cell viability, ultrastructure, mitochondrial membrane potential (ΔΨm), cytochrome c distribution within cells, apoptosis, and ROS production were evaluated. Here we show that ketamine induced ultrastructural abnormalities and dose- and time-dependently caused cell death. In addition, ketamine decreased ΔΨm and increased cytochrome c release from mitochondria. Ketamine also increased ROS production and induced differential expression of oxidative stress-related genes. Specifically, abnormal ultrastructural and ΔΨm changes occurred earlier than cell death in the ketamine-induced toxicity process. Furthermore, Trolox significantly decreased ROS generation and attenuated cell death caused by ketamine in a dose-dependent manner. In conclusion, this study illustrates that ketamine time- and dose-dependently induces human neurotoxicity via ROS-mediated mitochondrial apoptosis pathway and that these side effects can be prevented by the antioxidant agent Trolox. Thus, hESC-derived neurons might provide a promising tool for studying anesthetic-induced developmental neurotoxicity and prevention strategies. PMID:22873495

  18. Crocin suppresses tumor necrosis factor-alpha-induced cell death of neuronally differentiated PC-12 cells.

    Science.gov (United States)

    Soeda, S; Ochiai, T; Paopong, L; Tanaka, H; Shoyama, Y; Shimeno, H

    2001-11-01

    Crocus sativus L. is used in Chinese traditional medicine to treat some disorders of the central nervous system. Crocin is an ethanol-extractable component of Crocus sativus L.; it is reported to prevent ethanol-induced impairment of learning and memory in mice. In this study, we demonstrate that crocin suppresses the effect of tumor necrosis factor (TNF)-alpha on neuronally differentiated PC-12 cells. PC-12 cells dead from exposure to TNF-alpha show apoptotic morphological changes and DNA fragmentation. These hallmark features of cell death did not appear in cells treated in the co-presence of 10 microM crocin. Moreover, crocin suppressed the TNF-alpha-induced expression of Bcl-Xs and LICE mRNAs and simultaneously restored the cytokine-induced reduction of Bcl-X(L) mRNA expression. The modulating effects of crocin on the expression of Bcl-2 family proteins led to a marked reduction of a TNF-alpha-induced release of cytochrome c from the mitochondria. Crocin also blocked the cytochrome c-induced activation of caspase-3. To learn how crocin exhibits these anti-apoptotic actions in PC-12 cells, we tested the effect of crocin on PC-12 cell death induced by daunorubicin. We found that crocin inhibited the effect of daunorubicin as well. Our findings suggest that crocin inhibits neuronal cell death induced by both internal and external apoptotic stimuli.

  19. Uremic Toxins Enhance Statin-Induced Cytotoxicity in Differentiated Human Rhabdomyosarcoma Cells

    Directory of Open Access Journals (Sweden)

    Hitoshi Uchiyama

    2014-09-01

    Full Text Available The risk of myopathy and rhabdomyolysis is considerably increased in statin users with end-stage renal failure (ESRF. Uremic toxins, which accumulate in patients with ESRF, exert cytotoxic effects that are mediated by various mechanisms. Therefore, accumulation of uremic toxins might increase statin-induced cytotoxicity. The purpose of this study was to determine the effect of four uremic toxins—hippuric acid, 3-carboxy-4-methyl-5-propyl-2-furanpropionate, indole-3-acetic acid, and 3-indoxyl sulfate—on statin-induced myopathy. Differentiated rhabdomyosarcoma cells were pre-treated with the uremic toxins for seven days, and then the cells were treated with pravastatin or simvastatin. Cell viability and apoptosis were assessed by viability assays and flow cytometry. Pre-treatment with uremic toxins increased statin- but not cisplatin-induced cytotoxicity (p < 0.05 vs. untreated. In addition, the pre-treatment increased statin-induced apoptosis, which is one of the cytotoxic factors (p < 0.05 vs. untreated. However, mevalonate, farnesol, and geranylgeraniol reversed the effects of uremic toxins and lowered statin-induced cytotoxicity (p < 0.05 vs. untreated. These results demonstrate that uremic toxins enhance statin-induced apoptosis and cytotoxicity. The mechanism underlying this effect might be associated with small G-protein geranylgeranylation. In conclusion, the increased severity of statin-induced rhabdomyolysis in patients with ESRF is likely due to the accumulation of uremic toxins.

  20. Two-stage induced differentiation of OCT4+/Nanog+ stem-like cells in lung adenocarcinoma.

    Science.gov (United States)

    Li, Rong; Huang, Jinsu; Ma, Meili; Lou, Yuqing; Zhang, Yanwei; Wu, Lixia; Chang, David W; Zhao, Picheng; Dong, Qianggang; Wu, Xifeng; Han, Baohui

    2016-10-18

    Stem-like cells in solid tumors are purported to contribute to cancer development and poor treatment outcome. The abilities to self-renew, differentiate, and resist anticancer therapies are hallmarks of these rare cells, and steering them into lineage commitment may be one strategy to curb cancer development or progression. Vitamin D is a prohormone that can alter cell growth and differentiation and may induce the differentiation cancer stem-like cells. In this study, octamer-binding transcription factor 4 (OCT4)-positive/Nanog homeobox (Nanog)- positive lung adenocarcinoma stem-like cells (LACSCs) were enriched from spheroid cultured SPC-A1 cells and differentiated by a two-stage induction (TSI) method, which involved knockdown of hypoxia-inducible factor 1-alpha (HIF1α) expression (first stage) followed by sequential induction with 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3, VD3) and suberoylanilide hydroxamic acid (SAHA) treatment (second stage). The results showed the HIF1α-knockdowned cells displayed diminished cell invasion and clonogenic activities. Moreover, the TSI cells highly expressed tumor suppressor protein p63 (P63) and forkhead box J1 (FOXJ1) and lost stem cell characteristics, including absent expression of OCT4 and Nanog. These cells regained sensitivity to cisplatin in vitro while losing tumorigenic capacity and decreased tumor cell proliferation in vivo. Our results suggest that induced transdifferentiation of LACSCs by vitamin D and SAHA may become novel therapeutic avenue to alter tumor cell phenotypes and improve patient outcome.The development and progression of lung cancer may involve rare population of stem-like cells that have the ability to grow, differentiate, and resist drug treatment. However, current therapeutic strategies have mostly focused on tumor characteristics and neglected the potential source of cells that may contribute to poor clinical outcome. We generated lung adenocarcinoma stem-like cells from spheroid culture and

  1. Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused Three-Dimensional Multicompartment Bioreactor

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    Nora Freyer

    2016-08-01

    Full Text Available The hepatic differentiation of human induced pluripotent stem cells (hiPSC holds great potential for application in regenerative medicine, pharmacological drug screening, and toxicity testing. However, full maturation of hiPSC into functional hepatocytes has not yet been achieved. In this study, we investigated the potential of a dynamic three-dimensional (3D hollow fiber membrane bioreactor technology to improve the hepatic differentiation of hiPSC in comparison to static two-dimensional (2D cultures. A total of 100 × 106 hiPSC were seeded into each 3D bioreactor (n = 3. Differentiation into definitive endoderm (DE was induced by adding activin A, Wnt3a, and sodium butyrate to the culture medium. For further maturation, hepatocyte growth factor and oncostatin M were added. The same differentiation protocol was applied to hiPSC maintained in 2D cultures. Secretion of alpha-fetoprotein (AFP, a marker for DE, was significantly (p < 0.05 higher in 2D cultures, while secretion of albumin, a typical characteristic for mature hepatocytes, was higher after hepatic differentiation of hiPSC in 3D bioreactors. Functional analysis of multiple cytochrome P450 (CYP isoenzymes showed activity of CYP1A2, CYP2B6, and CYP3A4 in both groups, although at a lower level compared to primary human hepatocytes (PHH. CYP2B6 activities were significantly (p < 0.05 higher in 3D bioreactors compared with 2D cultures, which is in line with results from gene expression. Immunofluorescence staining showed that the majority of cells was positive for albumin, cytokeratin 18 (CK18, and hepatocyte nuclear factor 4-alpha (HNF4A at the end of the differentiation process. In addition, cytokeratin 19 (CK19 staining revealed the formation of bile duct-like structures in 3D bioreactors similar to native liver tissue. The results indicate a better maturation of hiPSC in the 3D bioreactor system compared to 2D cultures and emphasize the potential of dynamic 3D culture

  2. Corticosteroids reverse cytokine-induced block of survival and differentiation of oligodendrocyte progenitor cells from rats

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    Marx Romy

    2008-09-01

    Full Text Available Abstract Background Periventricular leukomalacia (PVL is a frequent complication of preterm delivery. Proinflammatory cytokines, such as interferon-γ (IFN-γ and tumor necrosis factor α (TNF-α released from astrocytes and microglia activated by infection or ischemia have previously been shown to impair survival and maturation of oligodendrocyte progenitors and could thus be considered as potential factors contributing to the generation of this disease. The first goal of the present study was to investigate whether exposure of oligodendrocyte precursors to these cytokines arrests the maturation of ion currents in parallel to its effects on myelin proteins and morphological maturation. Secondly, in the search for agents, that can protect differentiating oligodendrocyte precursor cells from cytokine-induced damage we investigated effects of coapplications of corticosteroids with proinflammatory cytokines on the subsequent survival and differentiation of oligodendrocyte progenitor cells. Methods To exclude influences from factors released from other cell types purified cultures of oligodendrocyte precursors were exposed to cytokines and/or steroids and allowed to differentiate for further 6 days in culture. Changes in membrane surface were investigated with capacitance recordings and Scanning Ion Conductance Microscopy. Na+- and K+- currents were investigated using whole cell patch clamp recordings. The expression of myelin specific proteins was investigated using western blots and the precursor cells were identified using immunostaining with A2B5 antibodies. Results Surviving IFN-γ and TNF-α treated cells continued to maintain voltage-activated Na+- and K+ currents characteristic for the immature cells after 6 days in differentiation medium. Corticosterone, dihydrocorticosterone and, most prominently dexamethasone, counteracted the deleterious effects of IFN-γ and TNF-α on cell survival, A2B5-immunostaining and expression of myelin basic

  3. Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused Three-Dimensional Multicompartment Bioreactor

    Science.gov (United States)

    Freyer, Nora; Knöspel, Fanny; Strahl, Nadja; Amini, Leila; Schrade, Petra; Bachmann, Sebastian; Damm, Georg; Seehofer, Daniel; Jacobs, Frank; Monshouwer, Mario; Zeilinger, Katrin

    2016-01-01

    Abstract The hepatic differentiation of human induced pluripotent stem cells (hiPSC) holds great potential for application in regenerative medicine, pharmacological drug screening, and toxicity testing. However, full maturation of hiPSC into functional hepatocytes has not yet been achieved. In this study, we investigated the potential of a dynamic three-dimensional (3D) hollow fiber membrane bioreactor technology to improve the hepatic differentiation of hiPSC in comparison to static two-dimensional (2D) cultures. A total of 100 × 106 hiPSC were seeded into each 3D bioreactor (n = 3). Differentiation into definitive endoderm (DE) was induced by adding activin A, Wnt3a, and sodium butyrate to the culture medium. For further maturation, hepatocyte growth factor and oncostatin M were added. The same differentiation protocol was applied to hiPSC maintained in 2D cultures. Secretion of alpha-fetoprotein (AFP), a marker for DE, was significantly (p < 0.05) higher in 2D cultures, while secretion of albumin, a typical characteristic for mature hepatocytes, was higher after hepatic differentiation of hiPSC in 3D bioreactors. Functional analysis of multiple cytochrome P450 (CYP) isoenzymes showed activity of CYP1A2, CYP2B6, and CYP3A4 in both groups, although at a lower level compared to primary human hepatocytes (PHH). CYP2B6 activities were significantly (p < 0.05) higher in 3D bioreactors compared with 2D cultures, which is in line with results from gene expression. Immunofluorescence staining showed that the majority of cells was positive for albumin, cytokeratin 18 (CK18), and hepatocyte nuclear factor 4-alpha (HNF4A) at the end of the differentiation process. In addition, cytokeratin 19 (CK19) staining revealed the formation of bile duct-like structures in 3D bioreactors similar to native liver tissue. The results indicate a better maturation of hiPSC in the 3D bioreactor system compared to 2D cultures and emphasize the potential of dynamic 3D culture

  4. Dendritic cells enhance UHMWPE wear particle-induced osteoclast differentiation of macrophages.

    Science.gov (United States)

    Cang, Dingwei; Guo, Kaijin; Zhao, Fengchao

    2015-10-01

    Ultra-high molecular weight polyethylene (UHMWPE) has been widely used in large joint replacement. Osteolysis induced by the UHMWPE wear particles is one of the main causes of replacement failure. This study aims to elucidate whether dendritic cells play a role in UHMWPE particle-induced osteolysis. An in vitro Raw 264.7 and DC 2.4 coculture system was employed to examine the effects of dendritic cells on the inflammatory and osteoclastogenic responses of Raw 264.7 toward UHMWPE particles. The expression of cytokines, NF-κB, and osteoclast marker genes was analyzed by ELISA, western blot, or quantitative PCR. The osteoclast differentiation was measured by TRAP staining and flow cytometry. UHMWPE particles induced Raw 264.7 cells to differentiate into osteoclasts, which was enhanced by coculturing with DC 2.4 cells. DC 2.4 cells augmented UHMWPE particle-elicited activation of NF-κB signaling, higher levels of TNF-α and MCP-1, and an increased expression of MMP-9, Calcr, and Ctsk, though DC 2.4 coculture alone did not significantly cause the aforementioned changes. These results suggest that dendritic cells, among other immune cells recruited by UHMWPE particle induced inflammation, could further exacerbate inflammation and osteolysis.

  5. Betulinic Acid Induces Apoptosis in Differentiated PC12 Cells Via ROS-Mediated Mitochondrial Pathway.

    Science.gov (United States)

    Wang, Xi; Lu, Xiaocheng; Zhu, Ronglan; Zhang, Kaixin; Li, Shuai; Chen, Zhongjun; Li, Lixin

    2017-01-25

    Betulinic acid (BA), a pentacyclic triterpene of natural origin, has been demonstrated to have varied biologic activities including anti-viral, anti-inflammatory, and anti-malarial effects; it has also been found to induce apoptosis in many types of cancer. However, little is known about the effect of BA on normal cells. In this study, the effects of BA on normal neuronal cell apoptosis and the mechanisms involved were studied using differentiated PC12 cells as a model. Treatment with 50 μM BA for 24 h apparently induced PC12 cell apoptosis. In the early stage of apoptosis, the level of intracellular reactive oxygen species (ROS) increased. Afterwards, the loss of the mitochondrial membrane potential, the release of cytochrome c and the activation of caspase-3 occurred. Treatment with antioxidants could significantly reduce BA-induced PC12 cell apoptosis. In conclusion, we report for the first time that BA induced the mitochondrial apoptotic pathway in differentiated PC12 cells through ROS.

  6. Role of ROCK Isoforms in Regulation of Stiffness Induced Myofibroblast Differentiation in Lung Fibrosis.

    Science.gov (United States)

    Htwe, Su S; Cha, Byung H; Yue, Kan; Khademhosseini, Ali; Knox, Alan J; Ghaemmaghami, Amir M

    2017-02-22

    Fibrosis is a major cause of progressive organ dysfunction in several chronic pulmonary diseases. Rho associated coiled-coil forming kinase (ROCK) has shown to be involved in myofibroblast differentiation driven by altered matrix stiffness in fibrotic state. There are two known ROCK isoforms in human, ROCK1 (ROKβ) and ROCK2 (ROKα), but specific role of each isoform in myofibroblast differentiation in lung fibrosis remains unknown. To study this, we developed a Gelatin methacryloyl (GelMA) hydrogel based culture system with different stiffness levels relevant to healthy and fibrotic lungs. We have shown that stiff matrix and not soft matrix, can induce myofibroblast differentiation with high αSMA expression. Furthermore, our data confirm that the inhibition of ROCK signalling by a pharmacological inhibitor (i.e. Y27632) attenuates stiffness induced αSMA expression and fibre assembly in myofibroblasts. To assess the role of ROCK isoforms in this process we used siRNA to knock down the expression of each isoform. Our data showed that knocking down either ROCK1 or ROCK2 did not result in a reduction in αSMA expression in myofibroblasts on stiff matrix as opposed to soft matrix where αSMA expression was reduced significantly. Paradoxically, on stiff matrix, the absence of one isoform (particularly ROCK2) exaggerated αSMA expression and led to thick fibre assembly. Moreover complete loss of αSMA fibre assembly was seen only in the absence of both ROCK isoforms suggesting that both isoforms are implicated in this process. Overall our results indicate the differential role of ROCK isoforms in myofibroblast differentiation on soft and stiff matrices.

  7. NOTCH SIGNALLING MODULATES HYPOXIA-INDUCED NEUROENDOCRINE DIFFERENTIATION OF HUMAN PROSTATE CANCER CELLS

    Science.gov (United States)

    Danza, Giovanna; Di Serio, Claudia; Rosati, Fabiana; Lonetto, Giuseppe; Sturli, Niccolò; Kacer, Doreen; Pennella, Antonio; Ventimiglia, Giuseppina; Barucci, Riccardo; Piscazzi, Annamaria; Prudovsky, Igor; Landriscina, Matteo; Marchionni, Niccolò; Tarantini, Francesca

    2012-01-01

    Prostate carcinoma is among the most common causes of cancer-related death in men, representing 15% of all male malignancies in developed countries. Neuroendocrine differentiation has been associated with tumor progression, poor prognosis and with the androgen-independent status. Currently, no successful therapy exists for advanced, castration-resistant disease. Because hypoxia has been linked to prostate cancer progression and unfavourable outcome, we sought to determine whether hypoxia would impact the degree of neuroendocrine differentiation of prostate cancer cells, in vitro. Results exposure of LNCaP cells to low oxygen tension induced a neuroendocrine phenotype, associated with an increased expression of the transcription factor neurogenin3 and neuroendocrine markers, such as neuron-specific enolase, chromogranin A and β3-tubulin. Moreover, hypoxia triggered a significant decrease of Notch 1 and Notch 2 mRNA and protein expression, with subsequent down regulation of Notch-mediated signalling, as demonstrated by reduced levels of the Notch target genes, Hes1 and Hey1. Neuroendocrine differentiation was promoted by attenuation of Hes1 transcription, as cells expressing a dominant negative form of Hes1 displayed increased levels of neuroendocrine markers under normoxic conditions. Although hypoxia down regulated Notch 1 and Notch 2 mRNA transcription and receptor activation also in the androgen independent cell lines, PC3 and Du145, it did not change the extent of NE differentiation in these cultures, suggesting that androgen sensitivity may be required for transdifferentiation to occur. Conclusions hypoxia induces neuroendocrine differentiation of LNCaP cells in vitro, which appears to be driven by the inhibition of Notch signalling with subsequent down-regulation of Hes1 transcription. PMID:22172337

  8. Dopamine inhibits proliferation, induces differentiation and apoptosis of K562 leukaemia cells

    Institute of Scientific and Technical Information of China (English)

    HE Qun; YUAN Lin-bo

    2007-01-01

    Background Dopamine exerts its effects mainly in nervous system through D1, D2 or D3 receptors. There are few reports dealing with the effects of dopamine on leukaemia cells. However, some dopamine agonists or antagonists do show biological effects on some types of leukaemia cells. Here, we report the effects of dopamine on the proliferation,differentiation and apoptosis of K562 leukaemia cells.Methods Proliferation was determined by MTT assay and cell counting both in liquid and semisolid cultures.Differentiation was verified by morphology, benzidine staining and flow cytometry. Apoptosis was checked by Hoechst 33258 staining and flow cytometry. The two groups were untreated group and treated group (dopamine 10-9 mol/L-10-4mol/L).Results In liquid culture, MTT assay and colony assay, dopamine inhibited proliferation of K562 cells. Inhibition rate was 29.28% at 10-6 mol/L and 36.10% at 10-5 mol/L after culture for 5 days in MTT assay. In benzidine staining and CD71 expression, dopamine induced K562 cells toward erythroid differentiation by increased 155% at 10-6 mol/L and by 171% at 10-5 mol/L after culture for 5 days in benzidine staining. In Hoechst 33258 staining and flow cytometry,dopamine induced K562 cells toward apoptosis. The sub G1 peak stained by PI was 14.23% at 10-4 mol/L dopamine after culture for 3 days compared with the control (0.81%) in flow cytometry.Conclusion Dopamine inhibites proliferation and induces both differentiation and apoptosis of K562 leukaemia cells.

  9. Scoparone attenuates RANKL-induced osteoclastic differentiation through controlling reactive oxygen species production and scavenging

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Sang-Hyun; Jang, Hae-Dong, E-mail: haedong@hnu.kr

    2015-02-15

    Scoparone, one of the bioactive components of Artemisia capillaris Thunb, has various biological properties including immunosuppressive, hepatoprotective, anti-allergic, anti-inflammatory, and antioxidant effects. This study aims at evaluating the anti-osteoporotic effect of scoparone and its underlying mechanism in vitro. Scoparone demonstrated potent cellular antioxidant capacity. It was also found that scoparone inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and suppressed cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression via c-jun N-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK)/p38-mediated c-Fos–nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling pathway. During osteoclast differentiation, the production of general reactive oxygen species (ROS) and superoxide anions was dose-dependently attenuated by scoparone. In addition, scoparone diminished NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 1 (Nox1) expression and activation via the tumor necrosis factor receptor-associated factor 6 (TRAF6)–cSrc–phosphatidylinositol 3-kinase (PI3k) signaling pathway and prevented the disruption of mitochondrial electron transport chain system. Furthermore, scoparone augmented the expression of superoxide dismutase 1 (SOD1) and catalase (CAT). The overall results indicate that the inhibitory effect of scoparone on RANKL-induced osteoclast differentiation is attributed to the suppressive effect on ROS and superoxide anion production by inhibiting Nox1 expression and activation and protecting the mitochondrial electron transport chain system and the scavenging effect of ROS resulting from elevated SOD1 and CAT expression. - Highlights: • Scoparone dose-dependently inhibited RANKL-induced osteoclast differentiation. • Scoparone diminished general ROS and superoxide anions in a dose-dependent manner. • Scoparone inhibited Nox1 expression and

  10. Isolation, Culture and Induced Differentiation of Fetal Porcine Islet Derived Pancreatic Stem Cell

    Institute of Scientific and Technical Information of China (English)

    FENG Ruo-peng; ZHANG Hui-ru; WANG Yun; QIAO Hai; ZHAO Ting; SHEN Wen-zheng; DOU Zhong-ying

    2007-01-01

    To isolate and culture the porcine pancreatic stem cells and investigate their function, the fetal porcine pancreatic stem cells were isolated by the method of suspending plus adhering culture. The isolated cells were then identified by irnmunohistochemical staining, and their culture viability measured through the MTT method in vitro. This induced them to differentiate into endocrine cells and detect their function. The isolated IPSCS did not express nestin, but expressed CK-19, a marker of ductal epithelia cells and oc-actin, a smooth muscle marker, demonstrating the growth characteristics of ES-like cells, and strong proliferative ability, after 18 passages. They could excrete insulin, and showed ultrastructure changes after being induced. Porcine pancreatic stem cells can be isolated by this method, induced to form islet-like clusters, and can secret insulin.

  11. Cobalt protoporphyrin induces differentiation of monocytic THP-1 cells through regulation of cytoplasmic Ref-1-related NADPH oxidase activity.

    Science.gov (United States)

    Song, Ju Dong; Lee, Sang Kwon; Park, Si Eun; Kim, Kang Mi; Kim, Koanhoi; Park, Yeong Min; Park, Young Chul

    2011-11-01

    Cobalt protoporphyrin (CoPP) is a potent and effective metalloporphyrin inducer of heme oxygenase-1 (HO-1) activity in many tissues. Here, we report that CoPP induces differentiation of monocytic THP-1 cells into macrophage-like cells. CoPP induced a marked growth inhibition with a slight reduction in viability, and increased adhesion and spreading of THP-1 cells. However, other protoporphyrins did not. CoPP also resulted in expression of CD11b, MMP9, MSR1, CD14 and ICAM-1, which are differentiation markers for macrophages. Interestingly, we observed a decrease of cytoplasmic redox factor-1 (Ref-1) levels in the process of CoPP-induced differentiation of THP-1 cells. In addition, knockdown of Ref-1 by siRNA enhanced cell adhesion induced by CoPP. Furthermore, an inhibitor of NADPH oxidase, diphenyleneiodonium (DPI), completely abolished CoPP-induced adhesion of Ref-1-deficient cells using an siRNA. A cytosolic factor for NADPH oxidase activity, p47phox, was significantly increased in THP-1 cells by CoPP treatment. Κnockdown of Ref-1 increased CoPP-induced p47phox expression in THP-1 cells. Taken together, these results suggest that CoPP induces differentiation of monocytic THP-1 cells, and that the CoPP-induced differentiation is associated with cytoplasmic Ref-1-related NADPH oxidase activity.

  12. Differential regulation of peripheral IL-1β-induced mechanical allodynia and thermal hyperalgesia in rats.

    Science.gov (United States)

    Kim, Min J; Lee, Sang Y; Yang, Kui Y; Nam, Soon H; Kim, Hyun J; Kim, Young J; Bae, Yong C; Ahn, Dong K

    2014-04-01

    This study examined the differential mechanisms of mechanical allodynia and thermal hyperalgesia after injection of interleukin (IL) 1β into the orofacial area of male Sprague-Dawley rats. The subcutaneous administration of IL-1β produced both mechanical allodynia and thermal hyperalgesia. Although a pretreatment with iodoresiniferatoxin (IRTX), a transient receptor potential vanilloid 1 (TRPV1) antagonist, did not affect IL-1β-induced mechanical allodynia, it significantly abolished IL-1β-induced thermal hyperalgesia. On the other hand, a pretreatment with D-AP5, an N-methyl-d-aspartate (NMDA) receptor antagonist, and NBQX, an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, blocked IL-1β-induced mechanical allodynia. Pretreatment with H89, a protein kinase A (PKA) inhibitor, blocked IL-1β-induced mechanical allodynia but not thermal hyperalgesia. In contrast, pretreatment with chelerythrine, a protein kinase C (PKC) inhibitor, inhibited IL-1β-induced thermal hyperalgesia. Subcutaneous injections of 2% lidocaine, a local anesthetic agent, blocked IL-1β-induced thermal hyperalgesia but not IL-1β-induced mechanical allodynia. In the resiniferatoxin (RTX)-pretreated rats, a subcutaneous injection of IL-1β did not produce thermal hyperalgesia due to the depletion of TRPV1 in the primary afferent fibers. Double immunofluorescence revealed the colocalization of PKA with neurofilament 200 (NF200) and of PKC with the calcitonin gene-related peptide (CGRP) in the trigeminal ganglion. Furthermore, NMDA receptor 1 (NR1) and TRPV1 predominantly colocalize with PKA and PKC, respectively, in the trigeminal ganglion. These results suggest that IL-1β-induced mechanical allodynia is mediated by sensitized peripheral NMDA/AMPA receptors through PKA-mediated signaling in the large-diameter primary afferent nerve fibers, whereas IL-1β-induced thermal hyperalgesia is mediated by sensitized peripheral TRPV1 receptors through PKC

  13. ESAT6-induced IFNgamma and CXCL9 can differentiate severity of tuberculosis.

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    Zahra Hasan

    Full Text Available BACKGROUND: Protective responses against Mycobacterium tuberculosis are dependent on appropriate T cell and macrophage activation. Mycobacterial antigen six kDa early secreted antigenic target (ESAT6 and culture filtrate protein 10 (CFP10 can detect M. tuberculosis specific IFNgamma responses. However, most studies have been performed in non-endemic regions and to study pulmonary tuberculosis (PTB. We have studied ESAT6 and CFP10 induced cytokine and chemokines responses in PTB and extrapulmonary (EPul TB. METHODOLOGY: IFNgamma, IL10, CXCL9 and CCL2 responses were determined using an ex vivo whole blood assay system in PTB (n = 30 and EPulTB patients with limited (LNTB, n = 24 or severe (SevTB, n = 22 disease, and in healthy endemic controls (ECs. Responses to bacterial LPS were also determined. PRINCIPAL FINDINGS: ESAT6- and CFP10-induced IFNgamma was comparable between ECs and TB patients. Both ESAT6- and CFP10-induced IFNgamma secretion was greater in LNTB than PTB. ESAT6-induced CXCL9 was greater in EPulTB as compared with PTB, with an increase in SevTB as compared with LNTB. CFP10-induced CCL2 was higher in PTB than LNTB patients. LPS-stimulated CXCL9 was greatest in SevTB and LPS-induced CCL2 was increased in PTB as compared with LNTB patients. A positive correlation between ESAT6-induced IFNgamma and CXCL9 was present in all TB patients, but IFNgamma and CCL2 was only correlated in LNTB. ESAT-induced CCL2 and CXCL9 were significantly associated in LNTB while correlation in response to LPS was only present in SevTB. CONCLUSIONS: ESAT6 induced IFNgamma and CXCL9 can differentiate between limited and severe TB infections.

  14. Tissue transglutaminase is involved in mechanical load-induced osteogenic differentiation of human ligamentum flavum cells.

    Science.gov (United States)

    Chao, Yuan-Hung; Huang, Shih-Yung; Yang, Ruei-Cheng; Sun, Jui-Sheng

    2016-07-01

    Mechanical load-induced osteogenic differentiation might be the key cellular event in the calcification and ossification of ligamentum flavum. The aim of this study was to investigate the influence of tissue transglutaminase (TGM2) on mechanical load-induced osteogenesis of ligamentum flavum cells. Human ligamentum flavum cells were obtained from 12 patients undergoing lumbar spine surgery. Osteogenic phenotypes of ligamentum flavum cells, such as alkaline phosphatase (ALP), Alizarin red-S stain, and gene expression of osteogenic makers were evaluated following the administration of mechanical load and BMP-2 treatment. The expression of TGM2 was evaluated by real-time PCR, Western blotting, and enzyme-linked immunosorbent assay (ELISA) analysis. Our results showed that mechanical load in combination with BMP-2 enhanced calcium deposition and ALP activity. Mechanical load significantly increased ALP and OC gene expression on day 3, whereas BMP-2 significantly increased ALP, OPN, and Runx2 on day 7. Mechanical load significantly induced TGM2 gene expression and enzyme activity in human ligamentum flavum cells. Exogenous TGM2 increased ALP and OC gene expression; while, inhibited TG activity significantly attenuated mechanical load-induced and TGM2-induced ALP activity. In summary, mechanical load-induced TGM2 expression and enzyme activity is involved in the progression of the calcification of ligamentum flavum.

  15. Cell population structure prior to bifurcation predicts efficiency of directed differentiation in human induced pluripotent cells

    Science.gov (United States)

    Bargaje, Rhishikesh; Trachana, Kalliopi; Shelton, Martin N.; McGinnis, Christopher S.; Zhou, Joseph X.; Chadick, Cora; Cook, Savannah; Cavanaugh, Christopher; Huang, Sui; Hood, Leroy

    2017-01-01

    Steering the differentiation of induced pluripotent stem cells (iPSCs) toward specific cell types is crucial for patient-specific disease modeling and drug testing. This effort requires the capacity to predict and control when and how multipotent progenitor cells commit to the desired cell fate. Cell fate commitment represents a critical state transition or “tipping point” at which complex systems undergo a sudden qualitative shift. To characterize such transitions during iPSC to cardiomyocyte differentiation, we analyzed the gene expression patterns of 96 developmental genes at single-cell resolution. We identified a bifurcation event early in the trajectory when a primitive streak-like cell population segregated into the mesodermal and endodermal lineages. Before this branching point, we could detect the signature of an imminent critical transition: increase in cell heterogeneity and coordination of gene expression. Correlation analysis of gene expression profiles at the tipping point indicates transcription factors that drive the state transition toward each alternative cell fate and their relationships with specific phenotypic readouts. The latter helps us to facilitate small molecule screening for differentiation efficiency. To this end, we set up an analysis of cell population structure at the tipping point after systematic variation of the protocol to bias the differentiation toward mesodermal or endodermal cell lineage. We were able to predict the proportion of cardiomyocytes many days before cells manifest the differentiated phenotype. The analysis of cell populations undergoing a critical state transition thus affords a tool to forecast cell fate outcomes and can be used to optimize differentiation protocols to obtain desired cell populations. PMID:28167799

  16. miR-142-3p prevents macrophage differentiation during cancer-induced myelopoiesis.

    Science.gov (United States)

    Sonda, Nada; Simonato, Francesca; Peranzoni, Elisa; Calì, Bianca; Bortoluzzi, Stefania; Bisognin, Andrea; Wang, Ena; Marincola, Francesco M; Naldini, Luigi; Gentner, Bernhard; Trautwein, Christian; Sackett, Sara Dutton; Zanovello, Paola; Molon, Barbara; Bronte, Vincenzo

    2013-06-27

    Tumor progression is accompanied by an altered myelopoiesis causing the accumulation of immunosuppressive cells. Here, we showed that miR-142-3p downregulation promoted macrophage differentiation and determined the acquisition of their immunosuppressive function in tumor. Tumor-released cytokines signaling through gp130, the common subunit of the interleukin-6 cytokine receptor family, induced the LAP∗ isoform of C/EBPβ transcription factor, promoting macrophage generation. miR-142-3p downregulated gp130 by canonical binding to its messenger RNA (mRNA) 3' UTR and repressed C/EBPβ LAP∗ by noncanonical binding to its 5' mRNA coding sequence. Enforced miR expression impaired macrophage differentiation both in vitro and in vivo. Mice constitutively expressing miR-142-3p in the bone marrow showed a marked increase in survival following immunotherapy with tumor-specific T lymphocytes. By modulating a specific miR in bone marrow precursors, we thus demonstrated the feasibility of altering tumor-induced macrophage differentiation as a potent tool to improve the efficacy of cancer immunotherapy.

  17. Preferential Lineage-Specific Differentiation of Osteoblast-Derived Induced Pluripotent Stem Cells into Osteoprogenitors

    Science.gov (United States)

    Roberts, Casey L.; Chen, Silvia S.; Murchison, Angela C.; Ogle, Rebecca A.; Francis, Michael P.; Ogle, Roy C.

    2017-01-01

    While induced pluripotent stem cells (iPSCs) hold great clinical promise, one hurdle that remains is the existence of a parental germ-layer memory in reprogrammed cells leading to preferential differentiation fates. While it is problematic for generating cells vastly different from the reprogrammed cells' origins, it could be advantageous for the reliable generation of germ-layer specific cell types for future therapeutic use. Here we use human osteoblast-derived iPSCs (hOB-iPSCs) to generate induced osteoprogenitors (iOPs). Osteoblasts were successfully reprogrammed and demonstrated by endogenous upregulation of Oct4, Sox2, Nanog, TRA-1-81, TRA-16-1, SSEA3, and confirmatory hPSC Scorecard Algorithmic Assessment. The hOB-iPSCs formed embryoid bodies with cells of ectoderm and mesoderm but have low capacity to form endodermal cells. Differentiation into osteoprogenitors occurred within only 2–6 days, with a population doubling rate of less than 24 hrs; however, hOB-iPSC derived osteoprogenitors were only able to form osteogenic and chondrogenic cells but not adipogenic cells. Consistent with this, hOB-iOPs were found to have higher methylation of PPARγ but similar levels of methylation on the RUNX2 promoter. These data demonstrate that iPSCs can be generated from human osteoblasts, but variant methylation patterns affect their differentiation capacities. Therefore, epigenetic memory can be exploited for efficient generation of clinically relevant quantities of osteoprogenitor cells. PMID:28250775

  18. [The heat shock protein 90 inhibitor induces apoptosis and differentiation of Kasumi-1 and its mechanisms].

    Science.gov (United States)

    Yu, Wen-juan; Rao, Qing; Wang, Min; Tian, Zheng; Liu, Xiang-rong; Lin, Dong; Wang, Jian-xiang

    2005-12-01

    To explore the effect of 17-allylamide-17-demethoxygeldanamycin (17AAG), a heat shock protein 90 (HSP90) inhibitor, on the growth, differentiation and apoptosis of leukemic Kasumi-1 cells. Kasumi-1 cells were treated with 17AAG at different concentrations in suspension culture. Cell proliferation was analysed by MTT assay, expression of myeloid-specific differentiation antigen and cell cycle by flow cytometry, cell apoptosis by annexin V staining, agarose gel electrophoresis and flow cytometry. KIT protein was analysed by Western blot and c-kit mRNA by RT-PCR. 17AAG treatment caused a dose-dependent inhibition of the cell proliferation with the IC(50) of 0.62 micromol/L. A dose-dependent increase in early apoptosis occurred at 24 hours treatment and in late apoptosis at 48 hours treatment. 17AAG induced a time- and dose-dependent increase in expression of myeloid cell surface protein CD11b and CD15, a progressive decline in S-phase cell fraction and an increase in G(0)/G(1) cells. When Kasumi-1 cells were incubated with 1 micromol/L of 17AAG, KIT protein began to decrease at 2 hours and KIT protein could hardly be detected at 20 hours, but c-kit mRNA was not decreased. 17AAG treatment of Kasumi-1 cells could lower KIT protein expression, inhibit cell proliferation, induce cell partial differentiation, apoptosis and accumulation in G(0)/G(1) phase.

  19. c-MYC-Induced Sebaceous Gland Differentiation Is Controlled by an Androgen Receptor/p53 Axis

    Directory of Open Access Journals (Sweden)

    Denny L. Cottle

    2013-02-01

    Full Text Available Although the sebaceous gland (SG plays an important role in skin function, the mechanisms regulating SG differentiation and carcinoma formation are poorly understood. We previously reported that c-MYC overexpression stimulates SG differentiation. We now demonstrate roles for the androgen receptor (AR and p53. MYC-induced SG differentiation was reduced in mice lacking a functional AR. High levels of MYC triggered a p53-dependent DNA damage response, leading to accumulation of proliferative SG progenitors and inhibition of AR signaling. Conversely, testosterone treatment or p53 deletion activated AR signaling and restored MYC-induced differentiation. Poorly differentiated human sebaceous carcinomas exhibited high p53 and low AR expression. Thus, the consequences of overactivating MYC in the SG depend on whether AR or p53 is activated, as they form a regulatory axis controlling proliferation and differentiation.

  20. Structurally distinct polycyclic aromatic hydrocarbons induce differential transcriptional responses in developing zebrafish

    Energy Technology Data Exchange (ETDEWEB)

    Goodale, Britton C. [Department of Environmental and Molecular Toxicology, The Environmental Health Sciences Center, Oregon State University, Corvallis, OR (United States); Tilton, Susan C. [Computational Biology and Bioinformatics, Pacific Northwest National Laboratory (United States); Corvi, Margaret M.; Wilson, Glenn R. [Department of Environmental and Molecular Toxicology, The Environmental Health Sciences Center, Oregon State University, Corvallis, OR (United States); Janszen, Derek B. [Computational Biology and Bioinformatics, Pacific Northwest National Laboratory (United States); Anderson, Kim A. [Department of Environmental and Molecular Toxicology, The Environmental Health Sciences Center, Oregon State University, Corvallis, OR (United States); Waters, Katrina M. [Computational Biology and Bioinformatics, Pacific Northwest National Laboratory (United States); Tanguay, Robert L., E-mail: tanguay.robert@oregonstate.edu [Department of Environmental and Molecular Toxicology, The Environmental Health Sciences Center, Oregon State University, Corvallis, OR (United States)

    2013-11-01

    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment as components of fossil fuels and by-products of combustion. These multi-ring chemicals differentially activate the aryl hydrocarbon receptor (AHR) in a structurally dependent manner, and induce toxicity via both AHR-dependent and -independent mechanisms. PAH exposure is known to induce developmental malformations in zebrafish embryos, and recent studies have shown cardiac toxicity induced by compounds with low AHR affinity. Unraveling the potentially diverse molecular mechanisms of PAH toxicity is essential for understanding the hazard posed by complex PAH mixtures present in the environment. We analyzed transcriptional responses to PAH exposure in zebrafish embryos exposed to benz(a)anthracene (BAA), dibenzothiophene (DBT) and pyrene (PYR) at concentrations that induced developmental malformations by 120 h post-fertilization (hpf). Whole genome microarray analysis of mRNA expression at 24 and 48 hpf identified genes that were differentially regulated over time and in response to the three PAH structures. PAH body burdens were analyzed at both time points using GC–MS, and demonstrated differences in PAH uptake into the embryos. This was important for discerning dose-related differences from those that represented unique molecular mechanisms. While BAA misregulated the least number of transcripts, it caused strong induction of cyp1a and other genes known to be downstream of the AHR, which were not induced by the other two PAHs. Analysis of functional roles of misregulated genes and their predicted regulatory transcription factors also distinguished the BAA response from regulatory networks disrupted by DBT and PYR exposure. These results indicate that systems approaches can be used to classify the toxicity of PAHs based on the networks perturbed following exposure, and may provide a path for unraveling the toxicity of complex PAH mixtures. - Highlights: • Defined global mRNA expression

  1. Epigenetic Modulation of Human Induced Pluripotent Stem Cell Differentiation to Oligodendrocytes

    Directory of Open Access Journals (Sweden)

    Panagiotis Douvaras

    2016-04-01

    Full Text Available Pluripotent stem cells provide an invaluable tool for generating human, disease-relevant cells. Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system, characterized by myelin damage. Oligodendrocytes are the myelinating cells of the central nervous system (CNS; they differentiate from progenitor cells, and their membranes ensheath axons, providing trophic support and allowing fast conduction velocity. The current understanding of oligodendrocyte biology was founded by rodent studies, where the establishment of repressive epigenetic marks on histone proteins, followed by activation of myelin genes, leads to lineage progression. To assess whether this epigenetic regulation is conserved across species, we differentiated human embryonic and induced pluripotent stem cells to oligodendrocytes and asked whether similar histone marks and relative enzymatic activities could be detected. The transcriptional levels of enzymes responsible for methylation and acetylation of histone marks were analyzed during oligodendrocyte differentiation, and the post-translational modifications on histones were detected using immunofluorescence. These studies showed that also in human cells, differentiation along the oligodendrocyte lineage is characterized by the acquisition of multiple repressive histone marks, including deacetylation of lysine residues on histone H3 and trimethylation of residues K9 and K27. These data suggest that the epigenetic modulation of oligodendrocyte identity is highly conserved across species.

  2. Inhibitory Effect of Chrysanthemum zawadskii Herbich var. latilobum Kitamura Extract on RANKL-Induced Osteoclast Differentiation

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    Dong Ryun Gu

    2013-01-01

    Full Text Available Chrysanthemum zawadskii Herbich var. latilobum Kitamura, known as “Gujulcho” in Korea, has been used in traditional medicine to treat various inflammatory diseases, including rheumatoid arthritis. However, these effects have not been tested on osteoclasts, the bone resorbing cells that regulate bone metabolism. Here, we investigated the effects of C. zawadskii Herbich var. latilobum Kitamura ethanol extract (CZE on osteoclast differentiation induced by treatment with the receptor activator of NF-κB ligand (RANKL. CZE inhibited osteoclast differentiation and formation in a dose-dependent manner. The inhibitory effect of CZE on osteoclastogenesis was due to the suppression of ERK activation and the ablation of RANKL-stimulated Ca2+-oscillation via the inactivation of PLCγ2, followed by the inhibition of CREB activation. These inhibitory effects of CZE resulted in a significant repression of c-Fos expression and a subsequent reduction of NFATc1, a key transcription factor for osteoclast differentiation, fusion, and activation in vitro and in vivo. These results indicate that CZE negatively regulates osteoclast differentiation and may be a therapeutic candidate for the treatment of various bone diseases, such as postmenopausal osteoporosis, rheumatoid arthritis, and periodontitis.

  3. Influence of suppressor gene p16 on retinoic acid inducing cancer cell A549 differentiation

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To investigate the role of suppressor gene p16 in the process of differential regulation of retinoic acid (RA) on the A549 lung cancer cells.Methods Tumor suppressor gene p16 was transferred into A549 cells and the cells were treated with all-trans retinoic acid (ATR) at the dosage of 5×10-6 mol/L for 4 d. After that, the proliferation and differentiation of A549 cells were examined by growth curve and cytometry analysis, the change of lung lineage-specific marker MUC1 was tested by immunohistochemical staining. Meanwhile, Western blot was used to observe the change of p16 protein expression in A549 cells treated with ATRA.Results ATRA could obviously inhibit the growth and induce the differentiation of A549 Cells that were transferred with p16 gene. There were more cells arrested in G1/G0 phase and the expression of MUG1 was markedly down-regulated than in control cells. The expression of p16 protein was up-regulated in A549 cells treated with ATRA.Conclusion Suppressor gene p16 could enhance the effects of RA and proliferated suppression and differential induction of A549 cells.

  4. Pig Induced Pluripotent Stem Cell-Derived Neural Rosettes Parallel Human Differentiation Into Sensory Neural Subtypes.

    Science.gov (United States)

    Webb, Robin L; Gallegos-Cárdenas, Amalia; Miller, Colette N; Solomotis, Nicholas J; Liu, Hong-Xiang; West, Franklin D; Stice, Steven L

    2017-04-01

    The pig is the large animal model of choice for study of nerve regeneration and wound repair. Availability of porcine sensory neural cells would conceptually allow for analogous cell-based peripheral nerve regeneration in porcine injuries of similar severity and size to those found in humans. After recently reporting that porcine (or pig) induced pluripotent stem cells (piPSCs) differentiate into neural rosette (NR) structures similar to human NRs, here we demonstrate that pig NR cells could differentiate into neural crest cells and other peripheral nervous system-relevant cell types. Treatment with either bone morphogenetic protein 4 or fetal bovine serum led to differentiation into BRN3A-positive sensory cells and increased expression of sensory neuron TRK receptor gene family: TRKA, TRKB, and TRKC. Porcine sensory neural cells would allow determination of parallels between human and porcine cells in response to noxious stimuli, analgesics, and reparative mechanisms. In vitro differentiation of pig sensory neurons provides a novel model system for neural cell subtype specification and would provide a novel platform for the study of regenerative therapeutics by elucidating the requirements for innervation following injury and axonal survival.

  5. Small Molecule-Induced Complement Factor D (Adipsin) Promotes Lipid Accumulation and Adipocyte Differentiation.

    Science.gov (United States)

    Song, No-Joon; Kim, Suji; Jang, Byung-Hyun; Chang, Seo-Hyuk; Yun, Ui Jeong; Park, Ki-Moon; Waki, Hironori; Li, Dean Y; Tontonoz, Peter; Park, Kye Won

    2016-01-01

    Adipocytes are differentiated by various transcriptional cascades integrated on the master regulator, Pparγ. To discover new genes involved in adipocyte differentiation, preadipocytes were treated with three newly identified pro-adipogenic small molecules and GW7845 (a Pparγ agonist) for 24 hours and transcriptional profiling was analyzed. Four genes, Peroxisome proliferator-activated receptor γ (Pparγ), human complement factor D homolog (Cfd), Chemokine (C-C motif) ligand 9 (Ccl9), and GIPC PDZ Domain Containing Family Member 2 (Gipc2) were induced by at least two different small molecules but not by GW7845. Cfd and Ccl9 expressions were specific to adipocytes and they were altered in obese mice. Small hairpin RNA (shRNA) mediated knockdown of Cfd in preadipocytes inhibited lipid accumulation and expression of adipocyte markers during adipocyte differentiation. Overexpression of Cfd promoted adipocyte differentiation, increased C3a production, and led to induction of C3a receptor (C3aR) target gene expression. Similarly, treatments with C3a or C3aR agonist (C4494) also promoted adipogenesis. C3aR knockdown suppressed adipogenesis and impaired the pro-adipogenic effects of Cfd, further suggesting the necessity for C3aR signaling in Cfd-mediated pro-adipogenic axis. Together, these data show the action of Cfd in adipogenesis and underscore the application of small molecules to identify genes in adipocytes.

  6. Modulation of Gene Expression Networks underlying Realgar-Induced Differentiation of Acute Promyelocytic Leukemia Cells

    Institute of Scientific and Technical Information of China (English)

    王怀宇; 刘陕西

    2002-01-01

    Objective: To elucidate the molecular mechanism of the differentiation of acute promyelocytic leukemia (APL) cell line NB4 induced by realgar. Methods: The response of NB4 cell to realgar was explored with a cDNA microarray representing 1003 different human genes. Results: The analysis of gene expression profiles indicated that 8 genes were up-regulated and 33 genes were down-regulated 48 hrs after realgar treatment. Among the 8 up-regulated genes, 2 genes were involved in ubiquitin proteasome degradation pathway. Some genes related to RNA processing, protein synthesis and signal transduction were down-regulated. Conclusion: The ubiquitin-proteasome degradation pathway may play an important role in the degradation of PML/RAR α fusion protein and the differentiation of NB4 cells.

  7. The nucleus of differentiated root plant cells: modifications induced by arbuscular mycorrhizal fungi

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    G Lingua

    2009-12-01

    Full Text Available The nuclei of plant cells show marked differences in chromatin organisation, related to their DNA content, which ranges from the type with large strands of condensed chromatin (reticulate or chromonematic nuclei to one with mostly decondensed chromatin (chromocentric or diffuse nuclei. A loosening of the chromatin structure generally occurs in actively metabolising cells, such as differentiating and secretory cells, in relation to their high transcriptional activity. Endoreduplication may occur, especially in plants with a small genome, which increases the availability of nuclear templates, the synthesis of DNA, and probably regulates gene expression. Here we describe structural and quantitative changes of the chromatin and their relationship with transcription that occur in differentiated cells following an increase of their metabolism. The nuclei of root cortical cells of three plants with different 2C DNA content (Allium porrum, Pisum sativum and Lycopersicon esculentm and their modifications induced by arbuscular mycorrhization, which strongly increase the metabolic activity of colonised cells, are taken as examples.

  8. Milk-derived ribonuclease 5 preparations induce myogenic differentiation in vitro and muscle growth in vivo.

    Science.gov (United States)

    Knight, Matthew I; Tester, Angus M; McDonagh, Matthew B; Brown, Andrew; Cottrell, Jeremy; Wang, Jianghui; Hobman, Peter; Cocks, Benjamin G

    2014-12-01

    Ribonuclease 5, also known as angiogenin, is a stable and abundant ribonuclease in milk whey protein, which is able to regulate several cellular functions, including capillary formation, neuron survival, and epithelial cell growth. Ribonuclease 5 is important for protein synthesis directly stimulating rRNA synthesis in the nucleolus. Here, we show that biologically active RNase5 can be purified from bovine milk. Furthermore, we show that milk-derived RNase5 directly stimulates muscle cell differentiation in vitro, inducing C2C12 cell differentiation and myogenesis. When supplemented into the diet of healthy adult mice, milk-derived RNase5 preparations promoted muscle weight gain and grip strength. Collectively, these data indicate that milk-derived RNase5 preparations exhibit a novel role in skeletal muscle cell function.

  9. Algal Toxin Azaspiracid-1 Induces Early Neuronal Differentiation and Alters Peripherin Isoform Stoichiometry

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    Linda V. Hjørnevik

    2015-12-01

    Full Text Available Azaspiracid-1 is an algal toxin that accumulates in edible mussels, and ingestion may result in human illness as manifested by vomiting and diarrhoea. When injected into mice, it causes neurotoxicological symptoms and death. Although it is well known that azaspiracid-1 is toxic to most cells and cell lines, little is known about its biological target(s. A rat PC12 cell line, commonly used as a model for the peripheral nervous system, was used to study the neurotoxicological effects of azaspiracid-1. Azaspiracid-1 induced differentiation-related morphological changes followed by a latter cell death. The differentiated phenotype showed peripherin-labelled neurite-like processes simultaneously as a specific isoform of peripherin was down-regulated. The precise mechanism behind this down-regulation remains uncertain. However, this study provides new insights into the neurological effects of azaspiracid-1 and into the biological significance of specific isoforms of peripherin.

  10. Safrole oxide induced neuronal differentiation of rat bone-marrow mesenchymal stem cells by elevating Hsp70.

    Science.gov (United States)

    Zhao, YanChun; Xin, Jie; Sun, ChunHui; Zhao, BaoXiang; Zhao, Jing; Su, Le

    2012-11-01

    In a previous study, we found that at low concentrations, safrole oxide (SFO) could induce vascular endothelial cell (VEC) transdifferentiation into neuron-like cells; however, whether SFO could induce bone-marrow mesenchymal stem cell (BMSC) neural differentiation was unknown. Here, we found that SFO could effectively induce BMSC neural differentiation in the presence of serum and fibroblast growth factor 2 and did not affect cell viability at low concentrations. The levels of neuron-specific enolase and neurofilament-L were increased greatly, but that of glial fibrillary acidic protein was absent with SFO treatment for 48h. Furthermore, SFO could increase the level of heat shock protein 70 (Hsp70), an important factor in neuronal differentiation. Knockdown of Hsp70 by its small interfering RNA blocked SFO-induced BMSC differentiation. Thus, SFO is a novel inducer of BMSC differentiation to neuron-like cells and Hsp70 is implicated in the differentiation process. We provide a new tool for obtaining neuron-like cells from BMSCs and for further investigating the new effect of Hsp70 on BMSC neuronal differentiation.

  11. Histone deacetylase inhibitor valproic acid promotes the differentiation of human induced pluripotent stem cells into hepatocyte-like cells.

    Directory of Open Access Journals (Sweden)

    Yuki Kondo

    Full Text Available In this study, we aimed to elucidate the effects and mechanism of action of valproic acid on hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells. Human induced pluripotent stem cells were differentiated into endodermal cells in the presence of activin A and then into hepatic progenitor cells using dimethyl sulfoxide. Hepatic progenitor cells were matured in the presence of hepatocyte growth factor, oncostatin M, and dexamethasone with valproic acid that was added during the maturation process. After 25 days of differentiation, cells expressed hepatic marker genes and drug-metabolizing enzymes and exhibited drug-metabolizing enzyme activities. These expression levels and activities were increased by treatment with valproic acid, the timing and duration of which were important parameters to promote differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells into hepatocytes. Valproic acid inhibited histone deacetylase activity during differentiation of human induced pluripotent stem cells, and other histone deacetylase inhibitors also enhanced differentiation into hepatocytes. In conclusion, histone deacetylase inhibitors such as valproic acid can be used to promote hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells.

  12. Monocytic differentiation of K562 cells induced by proanthocyanidins from grape seeds.

    Science.gov (United States)

    Wang, Min; Wang, Li; Pan, Xiao-Jing; Zhang, Hong

    2012-01-01

    Grape seeds procyanidins can inhibit the proliferation of some cancer cell lines and have strong antioxidant activity. The purpose of this study was to investigate whether grape seeds procyanidins affect the proliferation and redifferentiation in K562 cells. The sulforhodamine B colorimetric assay and trypan blue staining were used to measure cell proliferation and survival. Morphological changes, NBT reductive activity, and surface antigens were used to detect redifferentiation of K562 cells. Intracellular reactive oxygen species (iROS) were detected by a fluorescent probe. Grape seeds procyanidins inhibited cell proliferation but the treatment did not appreciably increase lethality. After treatment with grape seeds procyanidins, a typical differentiated morphology was observed. The positive rate of CD11b and CD14 cells and NBT reductive activities increased significantly. As antioxidants, grape seeds procyanidins can induce arrest in the phase G1 and decrease iROS formation. All results indicate that the antioxidant grape seeds procyanidins are likely to induce monocytic differentiation in leukemia cells, mostly through decreasing iROS formation and inducing phase G1 arrest.

  13. Differential expression of microRNAs in avian leukosis virus subgroup J-induced tumors.

    Science.gov (United States)

    Wang, Qi; Gao, Yulong; Ji, Xiaolin; Qi, Xiaole; Qin, Liting; Gao, Honglei; Wang, Yongqiang; Wang, Xiaomei

    2013-02-22

    Avian leukosis virus subgroup J (ALV-J) has become pandemic and induced serious clinical outbreaks in chickens in China. In particular, ALV-J induced various clinical tumors in infected chickens, which caused enormous economic losses to poultry. In this study, an infectious clone from an epidemic ALV-J Chinese isolate designated HLJ09SH01 was constructed and rescued. The rescued virus (named rHLJ09SH01) was inoculated into specific-pathogen-free (SPF) layer chickens, and infected chickens were observed for 238 days to explore the oncogenicity of rHLJ09SH01. As a result, 57.9% of rHLJ09SH01-infected chickens produced tumors. Accumulating evidence shows that microRNAs (miRNAs) have a close relationship with tumorigenesis. To gain more insight into the tumorigenesis of ALV-J, a miRNA microarray was performed as part of an investigation of changes in host miRNA expression in a liver tumor from ALV-J infected chickens. The results showed that four miRNAs were significantly differentially expressed; these data were verified using real-time PCR. Bioinformatics analysis showed the differentially expressed miRNAs to be involved in some tumorigenesis-related signaling pathways, such as the MAPK signaling pathway and the Wnt signaling pathway, which may represent a possible signaling pathway that was involved in the ALV-J-induced tumorigenesis.

  14. Salidroside induces rat mesenchymal stem cells to differentiate into dopaminergic neurons.

    Science.gov (United States)

    Zhao, Hong-Bin; Ma, Hui; Ha, Xiao-Qin; Zheng, Ping; Li, Xiao-Yun; Zhang, Ming; Dong, Ju-Zi; Yang, Yin-Shu

    2014-04-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterised by the loss of substantia nigra dopaminergic neurons that leads to a reduction in striatal dopamine (DA) levels. Replacing lost cells by transplanting dopaminergic neurons has potential value to repair the damaged brain. Salidroside (SD), a phenylpropanoid glycoside isolated from plant Rhodiola rosea, is neuroprotective. We examined whether salidroside can induce mesenchymal stem cells (MSCs) to differentiate into neuron-like cells, and convert MSCs into dopamine neurons that can be applied in clinical use. Salidroside induced rMSCs to adopt a neuronal morphology, upregulated the expression of neuronal marker molecules, such as gamma neuronal enolase 2 (Eno2/NSE), microtubule-associated protein 2 (Map2), and beta 3 class III tubulin (Tubb3/β-tubulin III). It also increased expression of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and nerve growth factor (NGF) mRNAs, and promoted the secretion of these growth factors. The expression of dopamine neurons markers, such as dopamine-beta-hydroxy (DBH), dopa decarboxylase (DDC) and tyrosine hydroxylase (TH), was significantly upregulated after treatment with salidroside for 1-12 days. DA steadily increased after treatment with salidroside for 1-6 days. Thus salidroside can induce rMSCs to differentiate into dopaminergic neurons. © 2014 The Authors Cell Biology International Published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology.

  15. Structurally Distinct Polycyclic Aromatic Hydrocarbons Induce Differential Transcriptional Responses in Developing Zebrafish

    Energy Technology Data Exchange (ETDEWEB)

    Goodale, Britton; Tilton, Susan C.; Corvi, Margaret M.; Wilson, Glenn V.; Janszen, Derek B.; Anderson, Kim A.; Waters, Katrina M.; Tanguay, Robert

    2013-11-01

    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment as components of fossil fuels and by-products of combustion. These multi-ring chemicals differentially activate the aryl hydrocarbon receptor (AHR) in a structurally dependent manner, and induce toxicity via both AHR-dependent and -independent mechanisms. PAH exposure is known to induce developmental malformations in zebrafish embryos, and recent studies have shown cardiac toxicity induced by compounds with low AHR affinity. Unraveling the potentially diverse molecular mechanisms of PAH toxicity is essential for understanding the hazard posed by complex PAH mixtures present in the environment. We analyzed transcriptional responses to PAH exposure in zebrafish embryos exposed to benz(a)anthracene (BAA), dibenzothiophene (DBT) and pyrene (PYR) at concentrations that induced developmental malformations by 120 h post-fertilization (hpf). Whole genome microarray analysis of mRNA expression at 24 and 48 hpf identified genes that were differentially regulated over time and in response to the three PAH structures. PAH body burdens were analyzed at both time points using GC-MS, and demonstrated differences in PAH uptake into the embryos. This was important for discerning dose-related differences from those that represented unique molecular mechanisms. While BAA misregulated the least number of transcripts, it caused strong induction of cyp1a and other genes known to be downstream of the AHR, which were not induced by the other two PAHs. Analysis of functional roles of misregulated genes and their predicted regulatory transcription factors also distinguished the BAA response from regulatory networks disrupted by DBT and PYR exposure. These results indicate that systems approaches can be used to classify the toxicity of PAHs based on the networks perturbed following exposure, and may provide a path for unraveling the toxicity of complex PAH mixtures.

  16. Hydrogen peroxide induces adaptive response and differential gene expression in human embryo lung fibroblast cells.

    Science.gov (United States)

    Wei, Qinzhi; Huang, Haiyan; Yang, Linqing; Yuan, Jianhui; Yang, Xiaohua; Liu, Yungang; Zhuang, Zhixiong

    2014-04-01

    Hydrogen peroxide (H2 O2 ), a substance involved in cellular oxidative stress, has been observed to induce an adaptive response, which is characterized by a protection against the toxic effect of H2 O2 at higher concentrations. However, the molecular mechanism for the adaptive response remains unclear. In particular, the existing reports on H2 O2 -induced adaptive response are limited to animal cells and human tumor cells, and relatively normal human cells have never been observed for an adaptive response to H2 O2 . In this study, a human embryo lung fibroblast (MRC-5) cell line was used to model an adaptive response to H2 O2 , and the relevant differential gene expressions by using fluoro mRNA differential display RT-PCR. The results showed significant suppression of cytotoxicity of H2 O2 (1100 μM, 1 h) after pretreatment of the cells with H2 O2 at lower concentrations (0.088-8.8 μM, 24 h), as indicated by cell survival, lactate dehydrogenase release, and the rate of apoptotic cells. Totally 60 mRNA components were differentially expressed compared to untreated cells, and five of them (sizing 400-600 bp) which demonstrated the greatest increase in expression were cloned and sequenced. They showed identity with known genes, such as BCL-2, eIF3S5, NDUFS4, and RPS10. Real time RT-PCR analysis of the five genes displayed a pattern of differential expression consistent with that by the last method. These five genes may be involved in the induction of adaptive response by H2 O2 in human cells, at least in this particular cell type. Copyright © 2012 Wiley Periodicals, Inc.

  17. Creating an Animal Model of Tendinopathy by Inducing Chondrogenic Differentiation with Kartogenin.

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    Ting Yuan

    Full Text Available Previous animal studies have shown that long term rat treadmill running induces over-use tendinopathy, which manifests as proteoglycan accumulation and chondrocytes-like cells within the affected tendons. Creating this animal model of tendinopathy by long term treadmill running is however time-consuming, costly and may vary among animals. In this study, we used a new approach to develop an animal model of tendinopathy using kartogenin (KGN, a bio-compound that can stimulate endogenous stem/progenitor cells to differentiate into chondrocytes. KGN-beads were fabricated and implanted into rat Achilles tendons. Five weeks after implantation, chondrocytes and proteoglycan accumulation were found at the KGN implanted site. Vascularity as well as disorganization in collagen fibers were also present in the same site along with increased expression of the chondrocyte specific marker, collagen type II (Col. II. In vitro studies confirmed that KGN was released continuously from KGN-alginate in vivo beads and induced chondrogenic differentiation of tendon stem/progenitor cells (TSCs suggesting that chondrogenesis after KGN-bead implantation into the rat tendons is likely due to the aberrant differentiation of TSCs into chondrocytes. Taken together, our results showed that KGN-alginate beads can be used to create a rat model of tendinopathy, which, at least in part, reproduces the features of over-use tendinopathy model created by long term treadmill running. This model is mechanistic (stem cell differentiation, highly reproducible and precise in creating localized tendinopathic lesions. It is expected that this model will be useful to evaluate the effects of various topical treatments such as NSAIDs and platelet-rich plasma (PRP for the treatment of tendinopathy.

  18. The endocrine disruptor diethylstilbestrol induces adipocyte differentiation and promotes obesity in mice

    Energy Technology Data Exchange (ETDEWEB)

    Hao, Chan-Juan; Cheng, Xue-Jia; Xia, Hong-Fei, E-mail: hongfeixia@yahoo.com.cn; Ma, Xu

    2012-08-15

    Epidemiology studies indicate that exposure to endocrine disruptors during developmental “window” contributes to adipogenesis and the development of obesity. Implication of endocrine disruptor such as diethylstilbestrol (DES) on adipose tissue development has been poorly investigated. Here we evaluated the effects of DES on adipocyte differentiation in vitro and in vivo, and explored potential mechanism involved in its action. DES induced 3T3-L1 preadipocyte differentiation in a dose-dependent manner, and activated the expression of estrogen receptor (ER) and peroxisome proliferator-acivated receptor (PPAR) γ as well as its target genes required for adipogenesis in vitro. ER mediated the enhancement of DES-induced PPARγ activity. Moreover, DES perturbed key regulators of adipogenesis and lipogenic pathway in vivo. In utero exposure to low dose of DES significantly increased body weight, liver weight and fat mass in female offspring at postnatal day (PND) 60. In addition, serum triglyceride and glucose levels were also significantly elevated. These results suggest that perinatal exposure to DES may be expected to increase the incidence of obesity in a sex-dependent manner and can act as a potential chemical stressor for obesity and obesity-related disorders. -- Highlights: ► DES induced adipocyte differentiation in a dose-dependent manner in 3T3-L1 cells. ► DES activated adipogenic critical regulators and markers in vitro and in vivo. ► Perinatal exposure to DES led to the obese phenotype in female offspring. ► DES might be a potential chemical stressor for obesity and obesity-related disorders.

  19. Interleukin-27 induces the endothelial differentiation in Sca-1+ cardiac resident stem cells.

    Science.gov (United States)

    Tanaka, Tomohiro; Obana, Masanori; Mohri, Tomomi; Ebara, Masaki; Otani, Yuta; Maeda, Makiko; Fujio, Yasushi

    2015-10-01

    Cytokines play important roles in cardiac repair and regeneration. Recently, we demonstrated that interleukin (IL)-6 family cytokines induce the endothelial differentiation of Sca-1+ cardiac resident stem cells through STAT3/Pim-1 signaling pathway. In contrast, the biological functions of IL-12 family cytokines in heart remain to be elucidated, though they show structural homology with IL-6. In the present study, we examined the effects of IL-12 family cytokines on the transdifferentiation of cardiac Sca-1+ cells into cardiac cells. RT-PCR analyses revealed that IL-27 receptor α (IL-27Rα), but not IL-12R or IL-23R, was expressed in cardiac Sca-1+ cells. The transcript expression of IL-27 was elevated in murine hearts in cardiac injury models. Intriguingly, IL-27 stimulation for 14 days induced the endothelial cell (EC) marker genes, such as CD-31 and VE-cadherin. Immunoblot analyses clarified that IL-27 treatment rapidly phosphorylated STAT3. IL-27 upregulated the expression of Pim-1, but the overexpression of dominant negative STAT3 abrogated the induction of Pim-1 by IL-27. Finally, adenoviral transfection of dominant negative Pim-1 inhibited IL-27-induced EC differentiation of cardiac Sca-1+ cells. These findings demonstrated that IL-27 promoted the commitment of cardiac stem cells into the EC lineage, possibly leading to neovascularization as a novel biological function. IL-27 could not only regulate the inflammation but also contribute to the maintenance of the tissue homeostasis through stem cell differentiation at inflammatory sites. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  20. 10e12z CLA alters adipocyte differentiation and adipocyte cytokine expression and induces macrophage proliferation.

    Science.gov (United States)

    Belda, Benjamin J; Thompson, Jerry T; Eser, Pinar O; Vanden Heuvel, John P

    2012-05-01

    The trans-10, cis-12 (10e12z) conjugated linoleic acid (CLA) isomer of CLA is responsible for loss of lipid storage or adipose tissue in vitro or in vivo. This isomer also induces inflammatory signaling in both mouse and human adipocytes in vitro. However, when these events occur and whether they are significant enough to affect other cell types are unclear. In these experiments, the 3T3-L1 cell line has been used to examine the interaction between inflammatory signaling and decreased differentiation or lipid storage induced by 10e12z CLA. In assays measuring both lipid accumulation and gene expression, differentiating 3T3-L1 cells exhibit concurrent induction of inflammatory signaling, as measured by cyclooxygenase-2 expression, and a decrease in adipocyte marker gene expression. Furthermore, in fully differentiated adipocytes, as identified in microarray assays and confirmed with real-time polymerase chain reaction, 10e12z CLA also significantly affected expression of both matrix metalloprotein-3 (MMP-3), collagen VI α 3 ColVI alpha 3 (VIα3) and the cytokine epiregulin, demonstrating that the effects of 10e12z broadly impact adipocyte function. In agreement with other experimental systems, 10e12z CLA inhibited RAW 264.7 cell proliferation; however, in response to adipocyte-conditioned media, 10e12z-CLA-treated adipocytes induced proliferation of this cell line, suggesting that the effect of 10e12z CLA is context dependent. These results are largely consistent with the known activation of the inflammatory mediator nuclear factor-κB in adipocytes in vitro and in vivo by 10e12z CLA treatment and demonstrate that adipose is an important target tissue of this isomer that impacts other cell types.

  1. [5-Aza-2'-deoxycytidine enhances differentiation and apoptosis induced by phenylbutyrate in Kasumi-1 cells].

    Science.gov (United States)

    Hao, Chang-lai; Tang, Ke-jing; Chen, Sen; Xing, Hai-yan; Wang, Min; Wang, Jian-xiang

    2005-03-01

    To investigate whether phenylbutyrate (PB) combined with 5-aza-2'-deoxycytidine (5-Aza-CdR)could inhibit transcription repression and induce t(8;21) acute myelogenous leukemia (AML) Kasumi-1 cells to differentiate and undergo apoptosis. Kasumi-1 cells were treated with PB and 5-Aza-CdR at different concentrations in suspension culture. Cellular proliferation was determined by the MTT assay, expression of myeloid-specific differentiation antigen and cell cycles were analyzed by flow cytometry. Cell apoptosis were assessed using AnnexinV/PI staining and flow cytometry. Treatment of Kasumi-1 cells with PB caused a dose-dependent inhibition of proliferation, with an IC(50) of 2.3 mmol/L. When combined with 5-Aza-CdR, PB resulted in a greater growth inhibition with an IC(50) of 1.95 mmol/L. Treatment of Kasumi-1 cells with PB resulted in cell cycle arrest at G(0)/G(1), while combined treatment with PB and 5-Aza-CdR led to cell cycle arrest at G(2)/M. Expression of myeloid cell differentiation antigens CD11b and CD13 induced by PB was enhanced when Kasumi-1 cells were pretreated with low dose of 5-Aza-CdR. High, but not low, concentrations of 5-Aza-CdR could enhance early apoptosis of Kasumi-1 cells induced by PB. Phenylbuty rate, when combined with 5-Aza-CdR, inhibits AML cell in vitro proliferation and increases apoptosis in a synergistic fashion.

  2. Prenatal exposure of ethanol induces increased glutamatergic neuronal differentiation of neural progenitor cells

    Directory of Open Access Journals (Sweden)

    Han Seol-Heui

    2010-11-01

    Full Text Available Abstract Background Prenatal ethanol exposure during pregnancy induces a spectrum of mental and physical disorders called fetal alcohol spectrum disorder (FASD. The central nervous system is the main organ influenced by FASD, and neurological symptoms include mental retardation, learning abnormalities, hyperactivity and seizure susceptibility in childhood along with the microcephaly. In this study, we examined whether ethanol exposure adversely affects the proliferation of NPC and de-regulates the normal ratio between glutamatergic and GABAergic neuronal differentiation using primary neural progenitor culture (NPC and in vivo FASD models. Methods Neural progenitor cells were cultured from E14 embryo brain of Sprague-Dawley rat. Pregnant mice and rats were treated with ethanol (2 or 4 g/kg/day diluted with normal saline from E7 to E16 for in vivo FASD animal models. Expression level of proteins was investigated by western blot analysis and immunocytochemical assays. MTT was used for cell viability. Proliferative activity of NPCs was identified by BrdU incorporation, immunocytochemistry and FACS analysis. Results Reduced proliferation of NPCs by ethanol was demonstrated using BrdU incorporation, immunocytochemistry and FACS analysis. In addition, ethanol induced the imbalance between glutamatergic and GABAergic neuronal differentiation via transient increase in the expression of Pax6, Ngn2 and NeuroD with concomitant decrease in the expression of Mash1. Similar pattern of expression of those transcription factors was observed using an in vivo model of FASD as well as the increased expression of PSD-95 and decreased expression of GAD67. Conclusions These results suggest that ethanol induces hyper-differentiation of glutamatergic neuron through Pax6 pathway, which may underlie the hyper-excitability phenotype such as hyperactivity or seizure susceptibility in FASD patients.

  3. Leishmania donovani infection induces anemia in hamsters by differentially altering erythropoiesis in bone marrow and spleen.

    Directory of Open Access Journals (Sweden)

    William P Lafuse

    Full Text Available Leishmania donovani is a parasite that causes visceral leishmaniasis by infecting and replicating in macrophages of the bone marrow, spleen, and liver. Severe anemia and leucopenia is associated with the disease. Although immune defense mechanisms against the parasite have been studied, we have a limited understanding of how L. donovani alters hematopoiesis. In this study, we used Syrian golden hamsters to investigate effects of L. donovani infection on erythropoiesis. Infection resulted in severe anemia and leucopenia by 8 weeks post-infection. Anemia was associated with increased levels of serum erythropoietin, which indicates the hamsters respond to the anemia by producing erythropoietin. We found that infection also increased numbers of BFU-E and CFU-E progenitor populations in the spleen and bone marrow and differentially altered erythroid gene expression in these organs. In the bone marrow, the mRNA expression of erythroid differentiation genes (α-globin, β-globin, ALAS2 were inhibited by 50%, but mRNA levels of erythroid receptor (c-kit, EpoR and transcription factors (GATA1, GATA2, FOG1 were not affected by the infection. This suggests that infection has a negative effect on differentiation of erythroblasts. In the spleen, erythroid gene expression was enhanced by infection, indicating that the anemia activates a stress erythropoiesis response in the spleen. Analysis of cytokine mRNA levels in spleen and bone marrow found that IFN-γ mRNA is highly increased by L. donovani infection. Expression of the IFN-γ inducible cytokine, TNF-related apoptosis-inducing ligand (TRAIL, was also up-regulated. Since TRAIL induces erythroblasts apoptosis, apoptosis of bone marrow erythroblasts from infected hamsters was examined by flow cytometry. Percentage of erythroblasts that were apoptotic was significantly increased by L. donovani infection. Together, our results suggest that L. donovani infection inhibits erythropoiesis in the bone marrow by

  4. Cell-Type-Specific Effects of Silibinin on Vitamin D-Induced Differentiation of Acute Myeloid Leukemia Cells Are Associated with Differential Modulation of RXRα Levels

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    Rina Wassermann

    2012-01-01

    Full Text Available Plant polyphenols have been shown to enhance the differentiation of acute myeloid leukemia (AML cells induced by the hormonal form of vitamin D3 (1α,25-dihydroxyvitamin D3; 1,25D. However, how these agents modulate 1,25D effects in different subtypes of AML cells remains poorly understood. Here, we show that both carnosic acid (CA and silibinin (SIL synergistically enhancd 1,25D-induced differentiation of myeloblastic HL60 cells. However, in promonocytic U937 cells, only CA caused potentiation while SIL attenuated 1,25D effect. The enhanced effect of 1,25D+CA was accompanied by increases in both the vitamin D receptor (VDR and retinoid X receptor alpha (RXRα protein levels and vitamin D response element (VDRE transactivation in both cell lines. Similar increases were observed in HL60 cells treated with 1,25D + SIL. In U937 cells, however, SIL inhibited 1,25D-induced VDRE transactivation concomitant with downregulation of RXRα at both transcriptional and posttranscriptional levels. These inhibitory effects correlated with the inability of SIL, with or without 1,25D, to activate the Nrf2/antioxidant response element signaling pathway in U937 cells. These results suggest that opposite effects of SIL on 1,25D-induced differentiation of HL60 and U937 cells may be determined by cell-type-specific signaling and transcriptional responses to this polyphenol resulting in differential modulation of RXRα expression.

  5. Radix Astragali-induced differentiation of rat bone marrow-derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Xinsheng Wang; Haifeng Li; Ying Zhao; Xiaoli Zhang; Aihua Bo

    2009-01-01

    BACKGROUND: Chemical induction has been shown to be effective at promoting the differentiation of bone marrow-derived mesenchymal stem cells (MSCs). However, these inductors have cytotoxicity side effects that may damage cells over time. Traditional Chinese medicines avoid this disadvantage while still producing effective induction.OBJECTIVE: To investigate the influence of Radix Astragali (Huangqi) on the differentiation of MSCs.DESIGN, TIME AND SETTING: In vitro study of traditional Chinese medicine in neural stem cell differentiation. The experiment was performed at the Central Laboratory of Hebei North University between April and June 2007.MATERIALS: Radix Astragafi solution (lot No. 060105; license No. Z53021585) was purchased from Daii Pharmaceutical Co., Ltd., China; rabbit anti-rat nestin, rabbit anti-rat neuron-specific enolase (NSE), mouse anti-rat microtubule-associated protein 2, and rabbit anti-rat glial fibrillary acidic protein were purchased from Wuhan Boster, China.METHODS: Whole bone marrow was isolated from the femur and tibia of 6-week-old male Wistar rats and subcultured. The fourth passage of MSCs were harvested and induced by different concentrations (50, 100, 200, 400 g/L) of Radix Astragali.MAIN OUTCOME MEASURES: Hematoxylin-eosin staining was used to observe MSC morphology after 24 hours of induction. Immunocytochemistry was employed to observe the expression of NSE (specific neuronal marker), nestin (marker of neural stem cell), glial fibrillary acidic protein and microtubule-associated protein 2 (markers of astrocytes).RESULTS: Following Radix Astragafi treatment, changes occurred in cell morphology including: cell body pyknosis; thin and long processes formed in some cells, with growth corresponding to drug concentration and induction time; and the formation of network-like connections between some cells.With increasing drug concentration and induction time, nestin expression was upregulated, and the number of positive cells increased

  6. 1,25-dihydroxycholecalciferol-induced differentiation of myelomonocytic leukemic cells unresponsive to colony stimulating factors and phorbol esters

    Energy Technology Data Exchange (ETDEWEB)

    Bettens, F.; Schlick, E.; Farrar, W.; Ruscetti, F.

    1986-12-01

    The murine myelomonocytic leukemia cell line WEHI-3B D/sup +/, which differentiates in response to granulocyte colony stimulating factor (G-CSF), can also be induced to differentiate into monocyte-macrophages by phorbol myristate acetate (PMA) treatment, whereas the WEHI-3B D/sup -/ subline, which is unresponsive to G-CSF and PMA, can be induced to differentiate to granulocytes as well as monocytes by 1,25-dihydroxycholecalciferol (1,25-(OH)/sub 2/ D3), the biologically active metabolite of vitamin D3. A newly developed variant of the WEHI-3B D/sup +/ line, named WEHI-3B D/sup +/G, which was responsive to G-CSF but not to PMA, was also differentiated to granulocytes by 1,25-(OH)/sub 2/ D3. Although vitamin D3 has been reported to induce macrophage differentiation in responsive tumor cells, this is the first demonstration that 1,25-(OH)/sub 2/ D3 can induce granulocyte differentiation. In both differentiation pathways, cessation of cellular proliferation accompanies changes in morphologic and cytochemical properties of the cells. This suggests that leukemic cell lines unresponsive to differentiation agents acting at the cell surface retain their ability to differentiate in response to agents that do not act via the plasma membrane such as 1,25-(OH)/sub 2/ D3, which has cytosolic/nuclear receptors. These results suggest that low doses of 1,25-(OH)/sub 2/ D3 may be useful in combination with hemopoietic growth factors (CSFs) as therapeutic agent to induce leukemic cell differentiation in vivo.

  7. Carbon nanotube array inducing osteogenic differentiation of human mesenchymal stem cells.

    Science.gov (United States)

    Xu, Baiyao; Ju, Yang; Cui, Yanbin; Song, Guanbin

    2015-06-01

    Carbon nanotubes (CNTs) are a kind of nanomaterials which have been shown a promising application for biomedicine. There are a lot of studies to use CNTs to induce the differentiation of mesenchymal stem cells (MSCs). However, the cellular behavior of MSCs on the top layer of CNT array was still not well understood. In this study, we evaluated the morphology, the gene expressions of the osteogenic differentiation related markers, and the gene expressions of collagen type II (Col II, a marker of chondrogenesis), PPARγ (a marker of adipogenesis) and scleraxis (SCX, a marker of tenogenesis) in human mesenchymal stem cells (hMSCs) cultured on multi-walled carbon nanotube (MWCNT) array. The effect of MWCNT array on the mineralization of hMSCs which were cultured in osteogenic differentiation medium (ODM) was further assayed. Our results showed that the hMSCs cultured on MWCNT array spread well, formed numerous spiral shaped cell colons and showed perinuclear morphology. Compared to hMSCs cultured on dish, the gene expression of osteocalcin (OCN) was increased while the gene expressions of collagen type II (Col II), PPARγ and scleraxis (SCX) were decreased in hMSCs which were cultured on MWCNT array without any differentiation factors. Furthermore, compared with hMSCs on dish, the gene expressions of collagen type I (Col I), osteocalcin (OCN), osteopontin (OPN) and RUNX2, and the mineralization of hMSCs on MWCNT array were enhanced when they were cultured in osteogenic differentiation medium (ODM). Our results indicated that MWCNT array was able to promote the osteogenesis of hMSCs.

  8. Flap loop of GluD2 binds to Cbln1 and induces presynaptic differentiation

    OpenAIRE

    Kuroyanagi, Tomoaki; Hirano, Tomoo

    2010-01-01

    Glutamate receptor delta2 (GluD2) is selectively expressed on the postsynaptic spines at parallel-fiber (PF)-Purkinje neuron (PN) synapses. GluD2 knockout mice show a reduced number of PF-PN synapses, suggesting that GluD2 is involved in synapse formation. Recent studies revealed that GluD2 induces presynaptic differentiation in a manner dependent on its N-terminal domain (NTD) through binding of Cbln1 secreted from cerebellar granule neurons. However, the underlying mechanism of the specific...

  9. Differentiation of Human Induced Pluripotent Stem Cells to Mammary-like Organoids

    OpenAIRE

    Ying Qu; Bingchen Han; Bowen Gao; Shikha Bose; Yiping Gong; Kolja Wawrowsky; Giuliano, Armando E.; Dhruv Sareen; Xiaojiang Cui

    2017-01-01

    Human induced pluripotent stem cells (iPSCs) can give rise to multiple cell types and hold great promise in regenerative medicine and disease-modeling applications. We have developed a reliable two-step protocol to generate human mammary-like organoids from iPSCs. Non-neural ectoderm-cell-containing spheres, referred to as mEBs, were first differentiated and enriched from iPSCs using MammoCult medium. Gene expression profile analysis suggested that mammary gland function-associated signaling ...

  10. Differentiation of Human Induced Pluripotent Stem Cells to Mammary-like Organoids

    OpenAIRE

    Qu, Ying; Han, Bingchen; Gao, Bowen; Bose, Shikha; Gong, Yiping; Wawrowsky, Kolja; Giuliano, Armando E.; Sareen, Dhruv; Cui, Xiaojiang

    2017-01-01

    Summary Human induced pluripotent stem cells (iPSCs) can give rise to multiple cell types and hold great promise in regenerative medicine and disease-modeling applications. We have developed a reliable two-step protocol to generate human mammary-like organoids from iPSCs. Non-neural ectoderm-cell-containing spheres, referred to as mEBs, were first differentiated and enriched from iPSCs using MammoCult medium. Gene expression profile analysis suggested that mammary gland function-associated si...

  11. Ganoderma lucidum Polysaccharides Induce Macrophage-Like Differentiation in Human Leukemia THP-1 Cells via Caspase and p53 Activation

    Directory of Open Access Journals (Sweden)

    Jia-Wei Hsu

    2011-01-01

    Full Text Available Differentiation therapy by induction of tumor cells is an important method in the treatment of hematological cancers such as leukemia. Tumor cell differentiation ends cancer cells' immortality, thus stopping cell growth and proliferation. In our previous study, we found that fucose-containing polysaccharide fraction F3 extracted from Ganoderma lucidum can bring about cytokine secretion and cell death in human leukemia THP-1 cells. This prompted us to further investigate on how F3 induces the differentiation in human leukemia cells. We integrated time-course microarray analysis and network modeling to study the F3-induced effects on THP-1 cells. In addition, we determined the differentiation effect using Liu's staining, nitroblue tetrazolium (NBT reduction assay, flow cytometer, western blotting and Q-PCR. We also examined the modulation and regulation by F3 during the differentiation process. Dynamic gene expression profiles showed that cell differentiation was induced in F3-treated THP-1 cells. Furthermore, F3-treated THP-1 cells exhibited enhanced macrophage differentiation, as demonstrated by changes in cell adherence, cell cycle arrest, NBT reduction and expression of differentiation markers including CD11b, CD14, CD68, matrix metalloproteinase-9 and myeloperoxidase. In addition, caspase cleavage and p53 activation were found to be significantly enhanced in F3-treated THP-1 cells. We unraveled the role of caspases and p53 in F3-induced THP-1 cells differentiation into macrophages. Our results provide a molecular explanation for the differentiation effect of F3 on human leukemia THP-1 cells and offer a prospect for a potential leukemia differentiation therapy.

  12. Effects of Melatonin on Differentiation Potential of Ito Cells in Mice with Induced Fibrosis of the Liver.

    Science.gov (United States)

    Nalobin, D S; Suprunenko, E A; Golichenkov, V A

    2016-10-01

    We studied the effects of melatonin on differentiation potential of Ito cells during atypical regeneration of mouse liver under conditions of CCl4-induced fibrosis. The dynamics of fibrosis was traced at the histological level and the effects of melatonin on the differentiation potential of mouse Ito cells were evaluated. Melatonin alleviated fibrotic changes in the liver tissue and reduced differentiation of Ito cells into myofibroblasts under conditions of atypical regeneration of the liver in induced fibrosis. The hepatoprotective role of melatonin was shown.

  13. Pycnogenol induces differentiation and apoptosis in human promyeloid leukemia HL-60 cells.

    Science.gov (United States)

    Huang, W W; Yang, J S; Lin, C F; Ho, W J; Lee, M R

    2005-06-01

    Pycnogenol, rich of many phytochemicals of medical value, is a commercialized nutrient supplement extracted from the bark of European coastal pine. In this study, we investigated the anti-tumor effects of Pycnogenol on HL-60, U937 and K562 human leukemia cell lines. We found that Pycnogenol inhibited cell proliferation dose- and time-dependently, and the IC(50)s of Pycnogenol on HL-60, U937 and K562 cells were 150, 40 and 100 microg/ml, respectively. When HL-60 cells were incubated with low concentrations of Pycnogenol (50, 100 and 125 microg/ml) for 24 h, a prominent G0/G1 arrest was observed, followed by gradual accumulation of sub-G0/G1 nuclei. At 48 h of treatment, 50-70% of HL-60 cells differentiated, as evidenced by morphological changes, NBT reduction, induction of NSE activity, and increases of cell surface expression of CD11b. However, results from Annexin V/PI staining, DAPI staining and DNA fragmentation assay indicated that Pycnogenol induced HL-60, U937 and K562 cell apoptosis at their respective IC(50)s after 24 h of treatments. Pretreatment of z-DEVD-fmk, a caspase-3 specific inhibitor, not only decreased caspase-3 activity but also reduced the percentage of apoptotic cells induced by Pycnogenol. This indicated that caspase-3 activation was involved in Pycnogenol induced-apoptosis. In conclusion, Pycnogenol induced differentiation and apoptosis in leukemia cells. Our data suggest that Pycnogenol could serve as a potent cancer chemopreventive or chemotherapeutic agent for human leukemia.

  14. Non-coding RNAs change their expression profile after Retinoid induced differentiation of the promyelocytic cell line NB4

    Directory of Open Access Journals (Sweden)

    Caporaso Maria G

    2010-01-01

    Full Text Available Abstract Background The importance of non-coding RNAs (ncRNAs as fine regulators of eukaryotic gene expression has emerged by several studies focusing on microRNAs (miRNAs. miRNAs represent a newly discovered family of non coding-RNAs. They are thought to be crucial players of human hematopoiesis and related tumorigenesis and to represent a potential tool to detect the early stages of cancer. More recently, the expression regulation of numerous long ncRNAs has been linked to cell growth, differentiation and cancer although the molecular mechanism of their function is still unknown. NB4 cells are promyelocytic cells that can be induced to differentiation upon retinoic acid (ATRA treatment and represent a feasible model to study changes of non coding RNAs expression between cancer cells and their terminally differentiated counterpart. Findings we screened, by microarray analysis, the expression of 243 miRNAs and 492 human genes transcribing for putative long ncRNAs different from miRNAs in NB4 cells before and after ATRA induced differentiation. Our data show that 8 miRNAs, and 58 long ncRNAs were deregulated by ATRA induced NB4 differentiation. Conclusion our data suggest that ATRA-induced differentiation lead to deregulation of a large number of the ncRNAs that can play regulatory roles in both tumorigenesis and differentiation.

  15. Coleusin factor, a novel anticancer diterpenoid, inhibits osteosarcoma growth by inducing bone morphogenetic protein-2-dependent differentiation.

    Science.gov (United States)

    Geng, Shuo; Sun, Bo; Lu, Ran; Wang, Jingze

    2014-06-01

    Coleusin factor is a diterpenoid compound isolated from the root of a tropical plant, Coleus forskohlii. Although Coleusin factor has been reported to suppress proliferation of and induce apoptosis in several types of cancer cells, the effects of Coleusin factor on osteosarcoma and the underlying mechanism are still not fully understood. In this study, we show that Coleusin factor treatment potently inhibits the growth of osteosarcoma cells associated with G(1) cell-cycle arrest. Interestingly, apoptosis and cell death are not induced. Instead, Coleusin factor causes osteosarcoma cells to exhibit typical properties of differentiated osteoblasts, including a morphologic alteration resembling osteoblasts, the expression of osteoblast differentiation markers, elevated alkaline phosphatase activity, and increased cellular mineralization. Coleusin factor treatment significantly increases the expression of bone morphogenetic protein-2 (BMP-2), a crucial osteogenic regulator, and runt-related transcription factor 2 (RUNX2), one of the key transcription factors of the BMP pathway. When BMP-2 signaling is blocked, Coleusin factor fails to inhibit cell proliferation and to induce osteoblast differentiation. Thus, upregulation of BMP-2 autocrine is critical for Coleusin factor to induce osteoblast differentiation and exert its anticancer effects on osteosarcoma. Importantly, administration of Coleusin factor inhibits the growth of osteosarcoma xenografted in nude mice without systemic or immunologic toxicity. Osteosarcoma is a highly aggressive cancer marked by the loss of normal differentiation. Coleusin factor represents a new type of BMP-2 inducer that restores differentiation in osteosarcoma cells. It may provide a promising therapeutic strategy against osteosarcoma with minimal side effects.

  16. Orphan nuclear receptor Nur77 is required for the differentiation of C6 glioma cells induced by cholera toxin

    Institute of Scientific and Technical Information of China (English)

    Dong XU; Yi-jun HUANG; Yan LI; Wei YIN; Guang-mei YAN

    2009-01-01

    Aim: To investigate a possible regulator gene involved in the cholera toxin-induced differentiation of rat C6 glioma cells. Methods: The global changes in the mRNA expression pattern induced by cholera toxin were analyzed using gene chip microarray. The selected gene was then silenced by RNA interference or overexpressed with an ORF plasmid to determine its necessity in this process. Results: Nur77, a member of the orphan nuclear receptor family (NR4A), was markedly up-regulated during the process of differentiation. Furthermore, RNAi of nur77 attenuated the induction effect of cholera toxin on C6 cells, whereas overexpression of nur77 led to similarly differentiated behavior, including morphologic and biomarker changes, as well as cell cycle arrest. Conclusion: Nur77 participated actively and essentially as an important regulator in the cholera toxin-induced differentiation of C6 cells.

  17. Centrifuge model test on earthquake-induced differential settlement of foundation on cohesive ground

    Institute of Scientific and Technical Information of China (English)

    SHAMOTO; Yasuhiro; HOTTA; Hiroyuki

    2009-01-01

    Dynamic centrifuge model test was conducted to study the earthquake-induced differential settlement of foundation on cohesive ground, and the influence of asymmetry of building was investigated. During the experiment, the overconsolidated kaolin clay ground with a three-dimensional asymmetrical structure model was shaken by a basically balanced input motion, and bender elements were used to measure shear wave velocities of model ground to reveal the soil fabric evolution during and after shaking. The test results show that, the total seismic settlement of foundation is composed of instantaneous and long-term post-earthquake settlements, and most of the differential settlement occurs immediately after the earthquake while the post-earthquake settlement is relatively uniform despite its large amplitude. The asymmetry of building affects the settlement behavior considerably. Compared with 1-or 2-dimensional structures, more evident differential settlement occurs under threedimensional asymmetrical building during shaking, which accounts for one-half of the total seismic settlements and results in complex spatial tilting effects of foundation.

  18. Mechanisms of all-trans retinoic acid-induced differentiation of acute promyelocytic leukemia cells

    Indian Academy of Sciences (India)

    Ji-Wang Zhang; Jian Gu; Zhen-Yi Wang; Sai-Juan Chen; Zhu Chen

    2000-09-01

    Retinoic acids (RA) play a key role in myeloid differentiation through their agonistic nuclear receptors (RAR/RXR) to modulate the expression of target genes. In acute promyelocytic leukemia (APL) cells with rearrangement of retinoic acid receptor (RAR) (including: PML-RAR, PLZF-RAR, NPM-RAR, NuMA-RAR or STAT5b-RAR) as a result of chromosomal translocations, the RA signal pathway is disrupted and myeloid differentiation is arrested at the promyelocytic stage. Pharmacologic dosage of all-trans retinoic acid (ATRA) directly modulates PML-RAR and its interaction with the nuclear receptor co-repressor complex, which restores the wild-type RAR/RXR regulatory pathway and induces the transcriptional expression of downstream genes. Analysing gene expression profiles in APL cells before and after ATRA treatment represents a useful approach to identify genes whose functions are involved in this new cancer treatment. A chronologically well coordinated modulation of ATRA-regulated genes has thus been revealed which seems to constitute a balanced functional network underlying decreased cellular proliferation, initiation and progression of maturation, and maintenance of cell survival before terminal differentiation.

  19. Detection of differentially expressed genes in methylnitrosourea-induced rat mammary adenocarcinomas.

    Science.gov (United States)

    Hu, L; Lin, L; Crist, K A; Kelloff, G J; Steele, V E; Lubet, R A; You, M; Wang, Y

    1997-01-01

    In this study, altered gene expression in five methylnitrosourea (MNU)-induced rat mammary adenocarcinomas was investigated using a newly developed competitive cDNA library screening assay. In order to detect the differentially expressed cDNA transcripts, three cDNA libraries (rat mammary, rat liver, and rat kidney) with over 18,000 clones were differentially screened with competing normal and neoplastic mammary cDNA probes. Ninety-eight clones indicated by competitive hybridization to be differentially expressed in tumors were verified by dot-blot hybridization analysis. Of these clones, 45 were found to be overexpressed while 53 were underexpressed in tumors. Forty-five of the confirmed clones were further analyzed by single-pass cDNA sequence determination. Four clones showed homology with cytochrome oxidase subunit I, polyoma virus PTA noncoding region, cytoplasmic beta-actin, and mouse secretory protein containing thrombospondin motifs. Further investigation into the potential roles of these identified genes should contribute significantly to our understanding of the molecular mechanism(s) of rat mammary tumorigenesis.

  20. Marmoset induced pluripotent stem cells: Robust neural differentiation following pretreatment with dimethyl sulfoxide

    Directory of Open Access Journals (Sweden)

    Zhifang Qiu

    2015-07-01

    Full Text Available The marmoset is an important nonhuman primate model for regenerative medicine. For experimental autologous cell therapy based on induced pluripotent (iPS cells in the marmoset, cells must be able to undergo robust and reliable directed differentiation that will not require customization for each specific iPS cell clone. When marmoset iPS cells were aggregated in a hanging drop format for 3 days, followed by exposure to dual SMAD inhibitors and retinoic acid in monolayer culture for 3 days, we found substantial variability in the response of different iPS cell clones. However, when clones were pretreated with 0.05–2% dimethyl sulfoxide (DMSO for 24 hours, all clones showed a very similar maximal response to the directed differentiation scheme. Peak responses were observed at 0.5% DMSO in two clones and at 1% DMSO in a third clone. When patterns of gene expression were examined by microarray analysis, hierarchical clustering showed very similar responses in all 3 clones when they were pretreated with optimal DMSO concentrations. The change in phenotype following exposure to DMSO and the 6 day hanging drop/monolayer treatment was confirmed by immunocytochemistry. Analysis of DNA content in DMSO-exposed cells indicated that it is unlikely that DMSO acts by causing cells to exit from the cell cycle. This approach should be generally valuable in the directed neural differentiation of pluripotent cells for experimental cell therapy.

  1. Donor Dependent Variations in Hematopoietic Differentiation among Embryonic and Induced Pluripotent Stem Cell Lines.

    Directory of Open Access Journals (Sweden)

    Olivier Féraud

    Full Text Available Hematopoiesis generated from human embryonic stem cells (ES and induced pluripotent stem cells (iPS are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could not be predicted based on phenotype or gene expression of the undifferentiated cells. These results demonstrate the influence of genetic background in variation of hematopoietic potential rather than the reprogramming process.

  2. Five Transcription Factors and FGF Pathway Inhibition Efficiently Induce Erythroid Differentiation in the Epiblast

    Directory of Open Access Journals (Sweden)

    Wei Weng

    2014-03-01

    Full Text Available Primitive erythropoiesis follows a stereotypic developmental program of mesoderm ventralization and internalization, hemangioblast formation and migration, and erythroid lineage specification. Induction of erythropoiesis is inefficient in either ES/iPS cells in vitro or nonhemangioblast cell populations in vivo. Using the chick model, we report that epiblast cells can be directly and efficiently differentiated into the erythroid lineage by expressing five hematopoietic transcription regulators (SCL+LMO2+GATA2+LDB1+E2A and inhibiting the FGF pathway. We show that these five genes are expressed with temporal specificity during normal erythropoiesis. Initiation of SCL and LMO2 expression requires FGF activity, whereas erythroid differentiation is enhanced by FGF inhibition. The lag between hematopoiesis and erythropoiesis is attributed to sequential coregulator expression and hemangioblast migration. Globin gene transcription can be ectopically and prematurely induced by manipulating the availability of these factors and the FGF pathway activity. We propose that similar approaches can be taken for efficient erythroid differentiation in vitro.

  3. Flap loop of GluD2 binds to Cbln1 and induces presynaptic differentiation.

    Science.gov (United States)

    Kuroyanagi, Tomoaki; Hirano, Tomoo

    2010-07-30

    Glutamate receptor delta2 (GluD2) is selectively expressed on the postsynaptic spines at parallel-fiber (PF)-Purkinje neuron (PN) synapses. GluD2 knockout mice show a reduced number of PF-PN synapses, suggesting that GluD2 is involved in synapse formation. Recent studies revealed that GluD2 induces presynaptic differentiation in a manner dependent on its N-terminal domain (NTD) through binding of Cbln1 secreted from cerebellar granule neurons. However, the underlying mechanism of the specific binding of the NTD to Cbln1 remains elusive. Here, we have identified the flap loop (Arg321-Trp339) in the NTD of GluD2 (GluD2-NTD) as a crucial region for the binding to Cbln1 and the induction of presynaptic differentiation. Both induction of presynaptic differentiation and binding of Cbln1 were abolished in the HEK cells expressing not wild-type GluD2 but GluD2 with mutations in the flap loop. Especially, single amino acid substitution of either Arg321 or Trp323 to alanine was sufficient to disable the GluD2 function. Finally, a homology model of GluD2-NTD suggested that the flap loop is located at the distal end, which appears consistent with an interaction with Cbln1 and a presynaptic varicosity.

  4. Centrifuge model test on earthquake-induced differential settlement of foundation on cohesive ground

    Institute of Scientific and Technical Information of China (English)

    ZHOU YanGuo; CHEN YunMin; SHAMOTO Yasuhiro; HOTTA Hiroyuki

    2009-01-01

    Dynamic centrifuge model test was conducted to study the earthquake-induced differential settlement of foundation on cohesive ground, and the influence of asymmetry of building was investigated. During the experiment, the overconsolidated kaolin clay ground with a three-dimensional asymmetrical structure model was shaken by a basically balanced input motion, and bender elements were used to measure shear wave velocities of model ground to reveal the soil fabric evolution during and after shaking. The test results show that, the total seismic settlement of foundation is composed of instantaneous and long-term post-earthquake settlements, and most of the differential settlement occurs immediately after the earthquake while the post-earthquake settlement is relatively uniform despite its large amplitude. The asymmetry of building affects the settlement behavior considerably. Compared with 1- or 2-dimensional structures, more evident differential settlement occurs under three-dimensional asymmetrical building during shaking, which accounts for one-half of the total seismic settlements and results in complex spatial tilting effects of foundation.

  5. Macrophage microvesicles induce macrophage differentiation and miR-223 transfer.

    Science.gov (United States)

    Ismail, Noura; Wang, Yijie; Dakhlallah, Duaa; Moldovan, Leni; Agarwal, Kitty; Batte, Kara; Shah, Prexy; Wisler, Jon; Eubank, Tim D; Tridandapani, Susheela; Paulaitis, Michael E; Piper, Melissa G; Marsh, Clay B

    2013-02-07

    Microvesicles are small membrane-bound particles comprised of exosomes and various-sized extracellular vesicles. These are released by several cell types. Microvesicles have a variety of cellular functions from communication to mediating growth and differentiation. Microvesicles contain proteins and nucleic acids. Previously, we showed that plasma microvesicles contain microRNAs (miRNAs). Based on our previous report, the majority of peripheral blood microvesicles are derived from platelets, while mononuclear phagocytes, including macrophages, are the second most abundant population. Here, we characterized macrophage-derived microvesicles and explored their role in the differentiation of naive monocytes. We also identified the miRNA content of the macrophage-derived microvesicles. We found that RNA molecules contained in the macrophage-derived microvesicles were transported to target cells, including mono cytes, endothelial cells, epithelial cells, and fibroblasts. Furthermore, we found that miR-223 was transported to target cells and was functionally active. Based on our observations, we hypothesize that microvesicles bind to and activate target cells. Furthermore, we find that microvesicles induce the differentiation of macrophages. Thus, defining key components of this response may identify novel targets to regulate host defense and inflammation.

  6. Wnt signaling induces differentiation of progenitor cells in organotypic keratinocyte cultures

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    Liu Bob Y

    2007-02-01

    Full Text Available Abstract Background Interfollicular skin develops normally only when the activity of the progenitor cells in the basal layer is counterbalanced by the exit of cells into the suprabasal layers, where they differentiate and cornify to establish barrier function. Distinct stem and progenitor compartments have been demonstrated in hair follicles and sebaceous glands, but there are few data to describe the control of interfollicular progenitor cell activity. Wnt signaling has been shown to be an important growth-inducer of stem cell compartments in skin and many other tissues. Results Here, we test the effect of ectopic Wnt1 expression on the behavior of interfollicular progenitor cells in an organotypic culture model, and find that Wnt1 signaling inhibits their growth and promotes terminal differentiation. Conclusion These results are consistent with the phenotypes reported for transgenic mice engineered to have gain or loss of function of Wnt signaling in skin, which would recommend our culture model as an accurate one for molecular analysis. Since it is known that canonical ligands are expressed in skin, it is likely that this pathway normally regulates the balance of growth and differentiation, and suggests it could be important to pathogenesis.

  7. Tregs promote the differentiation of Th17 cells in silica-induced lung fibrosis in mice.

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    Laiyu Song

    Full Text Available BACKGROUND: Silicosis is an occupational lung disease caused by inhalation of silica dust and characterized by lung inflammation and fibrosis. Previous study showed that Tregs regulate the process of silicosis by modulating the maintenance of immune homeostasis in the lung. Th17 cells share reciprocal developmental pathway with Tregs and play a pivotal role in the immunopathogenesis of many lung diseases by recruiting and activating neutrophils, but the regulatory function of Tregs on Th17 response in silica induced lung fibrosis remains to be explored. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the role of Th17 and IL-17 in the development of silicosis and their interaction with Tregs, Treg-depleted mice model was generated and exposed to silica to establish experimental model of silica-induced lung fibrosis. Here we showed that silica increased Th17 response in lung fibrosis. Tregs depletion enhanced the neutrophils accumulation and attenuated Th17 response in silica induced lung fibrosis. Both mRNA and protein results showed that Tregs exerted its modulatory function on Th17 cells and IL-17 by regulating TGF-β1 and IL-1β. CONCLUSION/SIGNIFICANCE: Our study suggested that Tregs could promote Th17 cells differentiation by regulating TGF-β1 and IL-1β in silica induced lung fibrosis of mice, which further the understanding of the progress of silicosis and provide a new insight in the regulatory mechanism of Th17 by Tregs in lung inflammation.

  8. Effect of amyloid peptides on serum withdrawal-induced cell differentiation and cell viability

    Institute of Scientific and Technical Information of China (English)

    Yi Peng WANG; Ze Fen WANG; Ying Chun ZHANG; Qing TIAN; Jian Zhi WANG

    2004-01-01

    Abnormal deposition of amyloid-β(Aβ) peptides and formation of neuritic plaques are recognized as pathological processes in Alzheimer's disease (AD) brain. By using amyloid precursor protein (APP) transfected cells, this study aims to investigate the effect of overproduction of Aβ on cell differentiation and cell viability. It was shown that after serum withdrawal, untransfected cell (N2a/Wt) and vector transfected cells (N2a/vector) extended long and branched cell processes, whereas no neurites was induced in wild type APP (N2a/APP695) and Swedish mutant APP (N2a/APPswe) transfected N2a cells. After differentiation by serum withdrawal, the localization of APP/Aβ and neurofilament was extended to neurites, whereas those of APP-transfected cells were still restricted within the cell body. Levels of both APP and Aβ were significantly higher in N2a/APP695 and N2a/APPswe than in N2a/Wt, as determined by Western blot and Sandwich ELISA, respectively. To further investigate the effect of Aβ on the inhibition of cell differentiation,we added exogenously the similar level or about 10-times of the Aβ level produced by N2a/APP695 and N2a/APPswe to the culture medium and co-cultured with N2a/Wt for 12 h, and we found that the inhibition of serum withdrawalinduced differentiation observed in N2a/APP695 and N2a/APPswe could not be reproduced by exogenous administration of Aβ into N2a/Wt. We also observed that neither endogenous production nor exogenous addition of Aβ1-40 or Aβ1-42, even to hundreds fold of the physiological concentration, affected obviously the cell viability. These results suggest that the overproduction of Aβ could not arrest cell differentiation induced by serum deprivation and that, at least to a certain degree and in a limited time period, is not toxic to cell viability.

  9. BMP12 induces tenogenic differentiation of adipose-derived stromal cells.

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    Hua Shen

    Full Text Available Adipose-derived stromal cells (ASCs are pluripotent cells that have the capacity to differentiate into tendon fibroblasts (TFs. They are abundant in adults, easy to access, and are therefore an ideal cell source for tendon tissue engineering. Despite this potential, the molecular cues necessary for tenogenic differentiation of ASCs are unknown. Unlike other bone morphogenetic proteins (BMPs, BMP12, BMP13, and BMP14 have been reported to be less osteo-chondrogenic and to induce tendon rather than bone formation in vivo. This study investigated the effects of BMP12 and BMP14 on ASC differentiation in vitro. In canine ASCs, BMP12 effectively increased the expression of the tendon markers scleraxis and tenomodulin at both mRNA and protein levels. Consistent with these results, BMP12 induced scleraxis promoter driven-GFP and tenomodulin protein expression in mouse ASCs. Although BMP12 also enhanced the expression of the cartilage matrix gene aggrecan in ASCs, the resulting levels remained considerably lower than those detected in tendon fibroblasts. In addition, BMP12 reduced expression of the bone marker osteocalcin, but not the osteogenic transcription factor runx-2. BMP14 exhibited similar, but marginally less potent and selective effects, compared to BMP12. BMPs are known to signal through the canonical Smad pathway and the non-canonical mitogen-activated protein kinase (MAPK pathway. BMP12 triggered robust phosphorylation of Smad1/5/8 but not Smad2/3 or p38 MAPK in ASCs. The effect was likely conveyed by type I receptors ALK2/3/6, as phosphorylation of Smad1/5/8 was blocked by the ALK2/3/6 inhibitor LDN-193189 but not by the ALK4/5/7 inhibitor SB-505124. Moreover, ALK6 was found to be the most abundant type I receptor in ASCs, with mRNA expression 100 to 10,000 times that of any other type I receptor. Collectively, results support the conclusion that BMP12 induces tenogenic differentiation of ASCs via the Smad1/5/8 pathway.

  10. Blockade of LFA-1 augments in vitro differentiation of antigen-induced Foxp3+ Treg cells

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    Verhagen, Johan; Wraith, David C.

    2014-01-01

    Adoptive transfer of antigen-specific, in vitro-induced Foxp3+ Treg (iTreg) cells protects against autoimmune disease. To generate antigen-specific iTreg cells at high purity, however, remains a challenge. Whereas polyclonal T cell stimulation with anti-CD3 and anti-CD28 antibody yields Foxp3+ iTreg cells at a purity of 90–95%, antigen-induced iTreg cells typically do not exceed a purity of 65–75%, even in a TCR-transgenic model. In a similar vein to thymic Treg cell selection, iTreg cell differentiation is influenced not only by antigen recognition and the availability of TGF-β but also by co-factors including costimulation and adhesion molecules. In this study, we demonstrate that blockade of the T cell integrin Leukocyte Function-associated Antigen-1 (LFA-1) during antigen-mediated iTreg cell differentiation augments Foxp3 induction, leading to approximately 90% purity of Foxp3+ iTreg cells. This increased efficacy not only boosts the yield of Foxp3+ iTreg cells, it also reduces contamination with activated effector T cells, thus improving the safety of adoptive transfer immunotherapy. PMID:25108241

  11. Two-step differentiation of mast cells from induced pluripotent stem cells.

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    Yamaguchi, Tomoko; Tashiro, Katsuhisa; Tanaka, Satoshi; Katayama, Sumie; Ishida, Waka; Fukuda, Ken; Fukushima, Atsuki; Araki, Ryoko; Abe, Masumi; Mizuguchi, Hiroyuki; Kawabata, Kenji

    2013-03-01

    Mast cells play important roles in the pathogenesis of allergic diseases. They are generally classified into 2 phenotypically distinct populations: connective tissue-type mast cells (CTMCs) and mucosal-type mast cells (MMCs). The number of mast cells that can be obtained from tissues is limited, making it difficult to study the function of mast cells. Here, we report the generation and characterization of CTMC-like mast cells derived from mouse induced pluripotent stem (iPS) cells. iPS cell-derived mast cells (iPSMCs) were generated by the OP9 coculture method or embryoid body formation method. The number of Safranin O-positive cells, expression levels of CD81 protein and histidine decarboxylase mRNA, and protease activities were elevated in the iPSMCs differentiated by both methods as compared with those in bone marrow-derived mast cells (BMMCs). Electron microscopic analysis revealed that iPSMCs contained more granules than BMMCs. Degranulation was induced in iPSMCs after stimulation with cationic secretagogues or vancomycin. In addition, iPSMCs had the ability to respond to stimulation with the IgE/antigen complex in vitro and in vivo. Moreover, when iPSMCs generated on OP9 cells were cocultured with Swiss 3T3 fibroblasts, protease activities as maturation index were more elevated, demonstrating that mature mast cells were differentiated from iPS cells. iPSMCs can be used as an in vitro model of CTMCs to investigate their functions.

  12. Wogonin Attenuates Ovalbumin Antigen-Induced Neutrophilic Airway Inflammation by Inhibiting Th17 Differentiation

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    Rie Takagi

    2014-01-01

    Full Text Available Allergic airway inflammation is generally considered to be a Th2-type immune response. Recent studies, however, have demonstrated that Th17-type immune responses also play important roles in this process, particularly in the pathogenesis of neutrophilic airway inflammation, a hallmark of severe asthma. We scrutinized several Kampo extracts that reportedly exhibit anti-inflammatory activity by using in vitro differentiation system of human and mouse naïve T cells. We found that hange-shashin-to (HST and oren-gedoku-to (OGT possess inhibitory activity for Th17 responses in vitro. Indeed, wogonin and berberine, major components common to HST and OGT, exhibit Th17-inhibitory activities in both murine and human systems in vitro. We therefore evaluated whether wogonin suppresses OVA-induced neutrophilic airway inflammation in OVA TCR-transgenic DO11.10 mice. Consequently, oral administration of wogonin significantly improved OVA-induced neutrophilic airway inflammation. Wogonin suppressed the differentiation of naïve T cells to Th17 cells, while showing no effects on activated Th17 cells.

  13. Pinoresinol inhibits proliferation and induces differentiation on human HL60 leukemia cells.

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    Sepporta, Maria Vittoria; Mazza, Teresa; Morozzi, Guido; Fabiani, Roberto

    2013-01-01

    Pinoresinol (PIN), one of the simplest lignans, is the precursor of other dietary lignans that are present in whole-grain cereals, legumes, fruits, and other vegetables. Several experimental and epidemiological evidences suggest that lignans may prevent human cancer in different organs. In this study we investigated the chemopreventive properties of PIN on cell lines derived from different sites either expressing or not the functional tumor suppressor protein p53. It was found that PIN inhibited the proliferation of p53 wild type colon and prostate tumor cells (HCT116 and LNCaP) while in breast cells the inhibition of growth was observed only in p53 mutant cells (MDA-MB-231). A potent antiproliferative activity of PIN was also observed on p53 null cells HL60 (IC50% 8 μM), their multidrug resistant variant HL60R (IC50% 32 μM) and K562. On HL60 cells, PIN caused a block of cell cycle in the G0/G1 phase, induced a weak proapoptotic effect but it was a good trigger of differentiation (NBT reduction and CD11b expression). PIN caused an upregulation of the CDK inhibitor p21(WAF1/Cip1) both at mRNA and protein levels so suggesting that this could be a mechanism by which PIN reduced proliferation and induced differentiation on HL60 cells.

  14. The inhibitory effect of vitamin K on RANKL-induced osteoclast differentiation and bone resorption.

    Science.gov (United States)

    Wu, Wei-Jie; Kim, Min Seuk; Ahn, Byung-Yong

    2015-10-01

    To further understand the correlation between vitamin K and bone metabolism, the effects of vitamins K1, menaquinone-4 (MK-4), and menaquinone-7 (MK-7) on RANKL-induced osteoclast differentiation and bone resorption were comparatively investigated. Vitamin K2 groups (MK-4 and MK-7) were found to significantly inhibit RANKL-medicated osteoclast cell formation of bone marrow macrophages (BMMs) in a dose-dependent manner, without any evidence of cytotoxicity. The mRNA expression of specific osteoclast differentiation markers, such as c-Fos, NFATc1, OSCAR, and TRAP, as well as NFATc1 protein expression and TRAP activity in RANKL-treated BMMs were inhibited by vitamin K2, although MK-4 exhibited a significantly greater efficiency compared to MK-7. In contrast, the same dose of vitamin K1 had no inhibitory effect on RANKL-induced osteoclast cell formation, but increased the expression of major osteoclastogenic genes. Interestingly, vitamins K1, MK-4 and MK-7 all strongly inhibited osteoclastic bone resorption (p vitamins K1, MK-4 and MK-7 have anti-osteoporotic properties, while their regulation effects on osteoclastogenesis are somewhat different.

  15. Spirulina phycocyanin induces differential protein expression and apoptosis in SKOV-3 cells.

    Science.gov (United States)

    Pan, Ruowang; Lu, Rongmao; Zhang, Ying; Zhu, Mei; Zhu, Wen; Yang, Rongrong; Zhang, Enyong; Ying, Jun; Xu, Teng; Yi, Huiguang; Li, Jinsong; Shi, Mengru; Zhou, Li; Xu, Zuyuan; Li, Peizhen; Bao, Qiyu

    2015-11-01

    The present study was designed to determine the effects of phycocyanin (PC) on Human ovarian cancer SKOV-3 cells and the underlying molecular mechanisms of action. The inhibitory effects of PC on the cell proliferation were detected by MTT assay. The IC50 values of PC were 182.0μM and 133.6μM for 24h and 48h exposure, respectively. PC induced apoptosis in SKOV-3 cells was observed by electron microscopy and flow cytometry. The apoptosis rate was increased from 1.6% to 19.8% after PC exposure. The fluorescence intensity of ROS and the activities of Caspase-3, Caspase-8, and Caspase-9 were increased. Differentiated expression protein spots were selected and identified using proteomic techniques. There were 698±73 and 683±79 protein spots resolved in untreated and PC-treated cells, respectively. Forty five differential protein spots were analyzed by MALDI-TOF-MS, including mtSSB, PSME3, and nucleolin. The mRNA expression profiles determined by RT-PCR were consistent with that of the two-dimensional electrophoresis. The decreased proteins such as HSP60, nucleolin, PPase, peroxiredoxin-4 and the increased protein (mtSSB) were identified in SKOV-3 cells after PC treatment, indicating that the effects of PC on tumor cell apoptosis may be relate to multiple target proteins. And the mitochondrial pathway may be the main pathway for PC-induced apoptosis.

  16. Differentiation of bone marrow mesenchymal stem cells induced by myocardial medium under hypoxic conditions

    Institute of Scientific and Technical Information of China (English)

    Xiao-jie XIE; Jian-an WANG; Jiang CAO; Xing ZHANG

    2006-01-01

    Aim: To explore whether bone marrow mesenchymal stem cells (MSC) can differentiate into myocardial-like cells induced by myocardial medium, especially the hypoxia/reoxygenation-conditioned medium of cardiomyocytes. Methods: Myocardial cells obtained from neonatal Sprague-Dawley rat ventricles were isolated and cultured in vitro and a hypoxia reoxygenation model was established. The MSC isolated from adult Sprague-Dawley rats were purified and then incubated with 3 different mediums: medium A - the conditioned medium of normal cardiomyocytes; medium B - the conditioned medium of cardiomyocytes after hypoxia reoxygenation; and the control medium - ordinary medium. The expressions of the cardiac myosin heavy chain (MHC), troponin T(TnT) and connexin 43 were investigated in the MSC after 24 h, 48 h and 72 h cultivation, respectively. Results: The MSC expressed MHC and TnT when incubated with the conditioned medium of cardiomyocytes after hypoxia reoxygenation, but did not express connexin 43. None of MHC, TnT and connexin 43 was detected in the MSC incubated with the conditioned medium of normal cardiomyocytes. Conclusion: The results indicate for the first time that myocardial medium for hypoxic preconditioning can induce MSC differentiation into myocardial-like cells.

  17. Nanotopography Promotes Pancreatic Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.

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    Kim, Jong Hyun; Kim, Hyung Woo; Cha, Kyoung Je; Han, Jiyou; Jang, Yu Jin; Kim, Dong Sung; Kim, Jong-Hoon

    2016-03-22

    Although previous studies suggest that nanotopographical features influence properties and behaviors of stem cells, only a few studies have attempted to derive clinically useful somatic cells from human pluripotent stem cells using nanopatterned surfaces. In the present study, we report that polystyrene nanopore-patterned surfaces significantly promote the pancreatic differentiation of human embryonic and induced pluripotent stem cells. We compared different diameters of nanopores and showed that 200 nm nanopore-patterned surfaces highly upregulated the expression of PDX1, a critical transcription factor for pancreatic development, leading to an approximately 3-fold increase in the percentage of differentiating PDX1(+) pancreatic progenitors compared with control flat surfaces. Furthermore, in the presence of biochemical factors, 200 nm nanopore-patterned surfaces profoundly enhanced the derivation of pancreatic endocrine cells producing insulin, glucagon, or somatostatin. We also demonstrate that nanopore-patterned surface-induced upregulation of PDX1 is associated with downregulation of TAZ, suggesting the potential role of TAZ in nanopore-patterned surface-mediated mechanotransduction. Our study suggests that appropriate cytokine treatments combined with nanotopographical stimulation could be a powerful tool for deriving a high purity of desired cells from human pluripotent stem cells.

  18. Canthin-6-one induces cell death, cell cycle arrest and differentiation in human myeloid leukemia cells.

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    Vieira Torquato, Heron F; Ribeiro-Filho, Antonio C; Buri, Marcus V; Araújo Júnior, Roberto T; Pimenta, Renata; de Oliveira, José Salvador R; Filho, Valdir C; Macho, Antonio; Paredes-Gamero, Edgar J; de Oliveira Martins, Domingos T

    2017-04-01

    Canthin-6-one is a natural product isolated from various plant genera and from fungi with potential antitumor activity. In the present study, we evaluate the antitumor effects of canthin-6-one in human myeloid leukemia lineages. Kasumi-1 lineage was used as a model for acute myeloid leukemia. Cells were treated with canthin-6-one and cell death, cell cycle and differentiation were evaluated in both total cells (Lin(+)) and leukemia stem cell population (CD34(+)CD38(-)Lin(-/low)). Among the human lineages tested, Kasumi-1 was the most sensitive to canthin-6-one. Canthin-6-one induced cell death with apoptotic (caspase activation, decrease of mitochondrial potential) and necrotic (lysosomal permeabilization, double labeling of annexin V/propidium iodide) characteristics. Moreover, canthin-6-one induced cell cycle arrest at G0/G1 (7μM) and G2 (45μM) evidenced by DNA content, BrdU incorporation and cyclin B1/histone 3 quantification. Canthin-6-one also promoted differentiation of Kasumi-1, evidenced by an increase in the expression of myeloid markers (CD11b and CD15) and the transcription factor PU.1. Furthermore, a reduction of the leukemic stem cell population and clonogenic capability of stem cells were observed. These results show that canthin-6-one can affect Kasumi-1 cells by promoting cell death, cell cycle arrest and cell differentiation depending on concentration used. Canthin-6-one presents an interesting cytotoxic activity against leukemic cells and represents a promising scaffold for the development of molecules for anti-leukemic applications, especially by its anti-leukemic stem cell activity. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. [Differentiation of human promyelocytic leukemia HL-60 cells induced by proanthocyanidin and its mechanism].

    Science.gov (United States)

    Xie, Zhao-Yang; Wu, Bin-Hua; Yang, Zhi-Gang; Chen, Xiao-Fang; Chen, Qiu-Shen

    2013-08-01

    This study was purposed to investigate the proliferation, differentiation and apoptosis of human promyelocytic leukemia HL-60 cells induced by proanthocyanidin (PAC). HL-60 cells were incubated with 20 mg/L PAC for 24 h, the cell growth was evaluated by CCK-8 assay. the effect of PAC on HL-60 cells was evaluated and the cells morphology was observed by optical microscopy. Expression of CD14 and CD11b, and cell cycle were analyzed by flow cytometry. The results showed that the growth of HL-60 cells was inhibited after treatment with PAC of different concentration in a dose-dependent manner (P HL-60 cells with inhibition ratio (72.3 ± 1.8)% for 24 h. Microscopy displayed that some cells differentiated to relative mature cells after treating for 48 h. Expression of CD14 increased and the expression of CD11b increased a little after treating with 20 mg/L PAC for 24 h, the ratio of cells in G0/G1 phase increased, but the ratio of cells in S phase decreased. The mRNA and protein expression of P21 gene increased, but the protein expression of CDK4 and Cyclin D1 decreased. It is concluded that PAC may inhibit the proliferation of HL-60 cells in vitro, induces the differentiation of HL-60 cells, and arrests the cells in G0/G1 phase. The possible mechanism may be related to up-regulation of P21 gene expression and down-regulation of the protein expression of CDK4 and Cyclin D1.

  20. BMP7 transfection induces in-vitro osteogenic differentiation of dental pulp mesenchymal stem cells

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    Ka Po John Yau

    2013-01-01

    Full Text Available Objective: To assess whether in-vitro osteogenic differentiation of human dental pulp mesenchymal stem cells can be induced by transient transfection with the gene encoding human bone morphogenic protein 7 (BMP7. Materials and Methods: A mesenchymal stem cell population was isolated from the dental pulp of two extracted permanent premolars, expanded and characterized. The human BMP7 gene, as a recombinant pcDNA3.1/V5-His-TOPO-BMP7 plasmid, was transfected into the cells. Three negative controls were used: No plasmid, empty vector, and an unrelated vector encoding green fluorescent protein. After the interval of 24 and 48 h, mRNA levels of alkaline phosphatase and osteocalcin as markers of in-vitro osteogenic differentiation were measured by real-time polymerase chain reaction and standardized against β-actin mRNA levels. Results: The level of alkaline phosphatase mRNA was significantly higher for the BMP7 group than for all three negative controls 48 h after transfection (706.9 vs. 11.24 for untransfected cells, 78.05 for empty vector, and 73.10 for green fluorescent protein vector. The level of osteocalcin mRNA was significantly higher for the BMP7 group than for all three negative controls 24 h after transfection (1.0, however, decreased after another 24 h. Conclusions: In-vitro osteoblastic differentiation of human dental pulp mesenchymal stem cells, as indicated by expression of alkaline phosphatase and osteocalcin, can be induced by transient transfection with the BMP7 gene.

  1. DNA-damage response gene GADD45A induces differentiation in hematopoietic stem cells without inhibiting cell cycle or survival.

    Science.gov (United States)

    Wingert, Susanne; Thalheimer, Frederic B; Haetscher, Nadine; Rehage, Maike; Schroeder, Timm; Rieger, Michael A

    2016-03-01

    Hematopoietic stem cells (HSCs) maintain blood cell production life-long by their unique abilities of self-renewal and differentiation into all blood cell lineages. Growth arrest and DNA-damage-inducible 45 alpha (GADD45A) is induced by genotoxic stress in HSCs. GADD45A has been implicated in cell cycle control, cell death and senescence, as well as in DNA-damage repair. In general, GADD45A provides cellular stability by either arresting the cell cycle progression until DNA damage is repaired or, in cases of fatal damage, by inducing apoptosis. However, the function of GADD45A in hematopoiesis remains controversial. We revealed the changes in murine HSC fate control orchestrated by the expression of GADD45A at single cell resolution. In contrast to other cellular systems, GADD45A expression did not cause a cell cycle arrest or an alteration in the decision between cell survival and apoptosis in HSCs. Strikingly, GADD45A strongly induced and accelerated the differentiation program in HSCs. Continuous tracking of individual HSCs and their progeny via time-lapse microscopy elucidated that once GADD45A was expressed, HSCs differentiate into committed progenitors within 29 hours. GADD45A-expressing HSCs failed to long-term reconstitute the blood of recipients by inducing multilineage differentiation in vivo. Importantly, γ-irradiation of HSCs induced their differentiation by upregulating endogenous GADD45A. The differentiation induction by GADD45A was transmitted by activating p38 Mitogen-activated protein kinase (MAPK) signaling and allowed the generation of megakaryocytic-erythroid, myeloid, and lymphoid lineages. These data indicate that genotoxic stress-induced GADD45A expression in HSCs prevents their fatal transformation by directing them into differentiation and thereby clearing them from the system.

  2. Effects of cellular origin on differentiation of human induced pluripotent stem cell-derived endothelial cells.

    Science.gov (United States)

    Hu, Shijun; Zhao, Ming-Tao; Jahanbani, Fereshteh; Shao, Ning-Yi; Lee, Won Hee; Chen, Haodong; Snyder, Michael P; Wu, Joseph C

    2016-06-02

    Human induced pluripotent stem cells (iPSCs) can be derived from various types of somatic cells by transient overexpression of 4 Yamanaka factors (OCT4, SOX2, C-MYC, and KLF4). Patient-specific iPSC derivatives (e.g., neuronal, cardiac, hepatic, muscular, and endothelial cells [ECs]) hold great promise in drug discovery and regenerative medicine. In this study, we aimed to evaluate whether the cellular origin can affect the differentiation, in vivo behavior, and single-cell gene expression signatures of human iPSC-derived ECs. We derived human iPSCs from 3 types of somatic cells of the same individuals: fibroblasts (FB-iPSCs), ECs (EC-iPSCs), and cardiac progenitor cells (CPC-iPSCs). We then differentiated them into ECs by sequential administration of Activin, BMP4, bFGF, and VEGF. EC-iPSCs at early passage (10 < P < 20) showed higher EC differentiation propensity and gene expression of EC-specific markers (PECAM1 and NOS3) than FB-iPSCs and CPC-iPSCs. In vivo transplanted EC-iPSC-ECs were recovered with a higher percentage of CD31(+) population and expressed higher EC-specific gene expression markers (PECAM1, KDR, and ICAM) as revealed by microfluidic single-cell quantitative PCR (qPCR). In vitro EC-iPSC-ECs maintained a higher CD31(+) population than FB-iPSC-ECs and CPC-iPSC-ECs with long-term culturing and passaging. These results indicate that cellular origin may influence lineage differentiation propensity of human iPSCs; hence, the somatic memory carried by early passage iPSCs should be carefully considered before clinical translation.

  3. Effects of cellular origin on differentiation of human induced pluripotent stem cell–derived endothelial cells

    Science.gov (United States)

    Zhao, Ming-Tao; Jahanbani, Fereshteh; Lee, Won Hee; Snyder, Michael P.

    2016-01-01

    Human induced pluripotent stem cells (iPSCs) can be derived from various types of somatic cells by transient overexpression of 4 Yamanaka factors (OCT4, SOX2, C-MYC, and KLF4). Patient-specific iPSC derivatives (e.g., neuronal, cardiac, hepatic, muscular, and endothelial cells [ECs]) hold great promise in drug discovery and regenerative medicine. In this study, we aimed to evaluate whether the cellular origin can affect the differentiation, in vivo behavior, and single-cell gene expression signatures of human iPSC–derived ECs. We derived human iPSCs from 3 types of somatic cells of the same individuals: fibroblasts (FB-iPSCs), ECs (EC-iPSCs), and cardiac progenitor cells (CPC-iPSCs). We then differentiated them into ECs by sequential administration of Activin, BMP4, bFGF, and VEGF. EC-iPSCs at early passage (10 < P < 20) showed higher EC differentiation propensity and gene expression of EC-specific markers (PECAM1 and NOS3) than FB-iPSCs and CPC-iPSCs. In vivo transplanted EC-iPSC–ECs were recovered with a higher percentage of CD31+ population and expressed higher EC-specific gene expression markers (PECAM1, KDR, and ICAM) as revealed by microfluidic single-cell quantitative PCR (qPCR). In vitro EC-iPSC–ECs maintained a higher CD31+ population than FB-iPSC–ECs and CPC-iPSC–ECs with long-term culturing and passaging. These results indicate that cellular origin may influence lineage differentiation propensity of human iPSCs; hence, the somatic memory carried by early passage iPSCs should be carefully considered before clinical translation. PMID:27398408

  4. Retinoic acid induces differentiation of buffalo (Bubalus bubalis) embryonic stem cells into germ cells.

    Science.gov (United States)

    Shah, Syed Mohmad; Singla, Suresh Kumar; Palta, Prabhat; Manik, Radhey Sham; Chauhan, Manmohan Singh

    2017-08-30

    Development of precise and reproducible culture system for in vitro differentiation of embryonic stem (ES) cells into germ cells counts as a major leap forward for understanding not only the remarkable process of gametogenesis, otherwise obscured by limited availability of precursor primordial germ cells (PGCs), but in finally treating the catastrophic infertility. Taking into account the significant role of retinoic acid (RA) during in vivo gametogenesis, we designed the present study to investigate the effects of its stimulation on directing the differentiation of ES cells into germ cells. The effects of RA were analyzed across dose-and-time upon various stages of gametogenesis like PGC induction, meiosis initiation and completion, haploid cell formation and development of the final gamete (oocyte and spermatozoa). Out of the series of RA doses (2, 4, 8, 16, 20 and 30μM), 16μM RA for 8day culture interval was found to induce highest expression of PGC- and meiosis-associated genes like DAZL, VASA, SYCP3, MLH1, TNP1/2 and PRM2, while mature germ cell genes like BOULE and TEKT1 (Spermatocyte markers), GDF9 and ZP2 (Oocyte markers) showed higher expression at 2μM RA dose, suggesting functional concentration-gradient of RA activity. Immunocytochemistry revealed expression of germ lineage-specific markers like: c-KIT, DAZL and VASA (PGC-markers); SYCP3, MLH1 and PROTAMINE1 (Meiotic-markers); ACROSIN and HAPRIN (Spermatocyte-markers); and GDF9 and ZP4 (Oocyte-markers) in optimally differentiated embryoid bodies (EBs) and adherent cultures. We observed significantly reduced (pcell population, indicating completion of meiosis. Oocyte-like structures (OLS) were obtained in adherent differentiated cultures. They had a big nucleus and a zona pellucida (ZP4) coat. They showed progression through 2-cell, 4-cell, 8-cell, morula and blastocyst-like structures upon extended culture beyond 14days. Copyright © 2017. Published by Elsevier B.V.

  5. Extended passaging increases the efficiency of neural differentiation from induced pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Koehler Karl R

    2011-08-01

    Full Text Available Abstract Background The use of induced pluripotent stem cells (iPSCs for the functional replacement of damaged neurons and in vitro disease modeling is of great clinical relevance. Unfortunately, the capacity of iPSC lines to differentiate into neurons is highly variable, prompting the need for a reliable means of assessing the differentiation capacity of newly derived iPSC cell lines. Extended passaging is emerging as a method of ensuring faithful reprogramming. We adapted an established and efficient embryonic stem cell (ESC neural induction protocol to test whether iPSCs (1 have the competence to give rise to functional neurons with similar efficiency as ESCs and (2 whether the extent of neural differentiation could be altered or enhanced by increased passaging. Results Our gene expression and morphological analyses revealed that neural conversion was temporally delayed in iPSC lines and some iPSC lines did not properly form embryoid bodies during the first stage of differentiation. Notably, these deficits were corrected by continual passaging in an iPSC clone. iPSCs with greater than 20 passages (late-passage iPSCs expressed higher expression levels of pluripotency markers and formed larger embryoid bodies than iPSCs with fewer than 10 passages (early-passage iPSCs. Moreover, late-passage iPSCs started to express neural marker genes sooner than early-passage iPSCs after the initiation of neural induction. Furthermore, late-passage iPSC-derived neurons exhibited notably greater excitability and larger voltage-gated currents than early-passage iPSC-derived neurons, although these cells were morphologically indistinguishable. Conclusions These findings strongly suggest that the efficiency neuronal conversion depends on the complete reprogramming of iPSCs via extensive passaging.

  6. Involvement of microRNAs in regulation of osteoblastic differentiation in mouse induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Hideharu Okamoto

    Full Text Available UNLABELLED: BACKGOROUND: MicroRNAs (miRNAs, which regulate biological processes by annealing to the 3'-untranslated region (3'-UTR of mRNAs to reduce protein synthesis, have been the subject of recent attention as a key regulatory factor in cell differentiation. The effects of some miRNAs during osteoblastic differentiation have been investigated in mesenchymal stem cells, however they still remains to be determined in pluripotent stem cells. METHODOLOGY/PRINCIPAL FINDINGS: Bone morphogenic proteins (BMPs are potent activators of osteoblastic differentiation. In the present study, we profiled miRNAs during osteoblastic differentiation of mouse induced pluripotent stem (iPS cells by BMP-4, in which expression of important osteoblastic markers such as Rux2, osterix, osteopontin, osteocalcin, PTHR1 and RANKL were significantly increased. A miRNA array analysis revealed that six miRNAs including miR-10a, miR-10b, miR-19b, miR-9-3p, miR-124a and miR-181a were significantly downregulated. Interestingly, miR-124a and miR-181a directly target the transcription factors Dlx5 and Msx2, both of which were increased by about 80-and 30-fold, respectively. In addition, transfection of miR-124a and miR-181a into mouse osteo-progenitor MC3T3-E1 cells significantly reduced expression of Dlx5, Runx2, osteocalcin and ALP, and Msx2 and osteocalcin, respectively. Finally, transfection of the anti-miRNAs of these six miRNAs, which are predicted to target Dlx5 and Msx2, into mouse iPS cells resulted in a significant increase in several osteoblastic differentiation markers such as Rux2, Msx2 and osteopontin. CONCLUSIONS/SIGNIFICANCE: In the present study, we demonstrate that six miRNAs including miR-10a, miR-10b, miR-19b, miR-9-3p, miR-124a and miR-181a miRNAs, especially miR-124a and miR-181a, are important regulatory factors in osteoblastic differentiation of mouse iPS cells.

  7. Preliminary Study on in Vitro Induced Differentiation of Embryonic Stem Cells into Neurons

    Institute of Scientific and Technical Information of China (English)

    JianGe; ShunongLi; 等

    2002-01-01

    Purpose;To study preliminarily in vitro induced differentiation of embryonic stem cells into neurons for further investigation of an alternative for the treatment of glaumatous neuropathy.Materials and methods:Supernatant of cultured Buffalo rat liver cells (buffalorat liver cell-conditioned medium,BRL-CM)was used for culturing embryonic stem cells(ES-D3 cell line)..Morphological features of undifferentiated ES cells were studied by HE staining and electron microscopy.Based on the methods used by Bain et al,we modified the methods and used retinoic,acid(RA) as an inducer to differentiate ES-D3 cells and cytosine arabinoside(Ara-C) as inhibitor of proliferative cells.The growth of the cells was observed under phase contrast microscope.Reuslts:ES-D3 cells cultured by BRL-CM grew in aggregates and remained undifferentiated.Electromicroscopy showed large nucleus and a large amount of mitochondria in undifferentiated ES cells and many processes on the surfaces.In the first day after the adding of retinoic acid,some neuron-like cells with one,two or more processes were present.In the second day after adding RA and the first day after the plus of 10μm Ara-C,a large amount of neuron-like cells appeared,with the formation of neuron-like networks.Conclusions:Combined use of RA and Ara-C can induce ES cells to different into neuron-like cells.Our present preliminary study might provide insights into an alternative for the treatment of glaucomatous neuropathy by the transplantation of embryonic stem cells.Eye Science 2000;16:1-6.

  8. Ascorbic acid inhibits TPA-induced HL-60 cell differentiation by decreasing cellular H₂O₂ and ERK phosphorylation.

    Science.gov (United States)

    Yiang, Giou-Teng; Chen, Jen-Ni; Wu, Tsai-Kun; Wang, Hsueh-Fang; Hung, Yu-Ting; Chang, Wei-Jung; Chen, Chinshuh; Wei, Chyou-Wei; Yu, Yung-Luen

    2015-10-01

    Retinoic acid (RA), vitamin D and 12-O‑tetradecanoyl phorbol-13-acetate (TPA) can induce HL-60 cells to differentiate into granulocytes, monocytes and macrophages, respectively. Similar to RA and vitamin D, ascorbic acid also belongs to the vitamin family. High‑dose ascorbic acid (>100 µM) induces HL‑60 cell apoptosis and induces a small fraction of HL‑60 cells to express the granulocyte marker, CD66b. In addition, ascorbic acid exerts an anti‑oxidative stress function. Oxidative stress is required for HL‑60 cell differentiation following treatment with TPA, however, the effect of ascorbic acid on HL‑60 cell differentiation in combination with TPA treatment remains to be fully elucidated. The aim of the present study was to investigate the cellular effects of ascorbic acid treatment on TPA-differentiated HL-60 cells. TPA-differentiated HL-60 cells were used for this investigation, this study and the levels of cellular hydrogen peroxide (H2O2), caspase activity and ERK phosphorylation were determined following combined treatment with TPA and ascorbic acid. The results demonstrated that low‑dose ascorbic acid (5 µM) reduced the cellular levels of H2O2 and inhibited the differentiation of HL‑60 cells into macrophages following treatment with TPA. In addition, the results of the present study further demonstrated that low‑dose ascorbic acid inactivates the ERK phosphorylation pathway, which inhibited HL‑60 cell differentiation following treatment with TPA.

  9. ERK2-Pyruvate Kinase Axis Permits Phorbol 12-Myristate 13-Acetate-induced Megakaryocyte Differentiation in K562 Cells.

    Science.gov (United States)

    Chaman, Noor; Iqbal, Mohammad Askandar; Siddiqui, Farid Ahmad; Gopinath, Prakasam; Bamezai, Rameshwar N K

    2015-09-25

    Metabolic changes that contribute to differentiation are not well understood. Overwhelming evidence shows the critical role of glycolytic enzyme pyruvate kinase (PK) in directing metabolism of proliferating cells. However, its role in metabolism of differentiating cells is unclear. Here we studied the role of PK in phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation in human leukemia K562 cells. We observed that PMA treatment decreased cancer-type anabolic metabolism but increased ATP production, along with up-regulated expression of two PK isoforms (PKM2 and PKR) in an ERK2-dependent manner. Interestingly, silencing of PK (PKM2 and PKR) inhibited PMA-induced megakaryocytic differentiation, as revealed by decreased expression of megakaryocytic differentiation marker CD61 and cell cycle behavior. Further, PMA-induced ATP production reduced greatly upon PK silencing, suggesting that PK is required for ATP synthesis. In addition to metabolic effects, PMA treatment also translocated PKM2, but not PKR, into nucleus. ERK1/2 knockdowns independently and together suggested the role of ERK2 in the up-regulation of both the isoforms of PK, proposing a role of ERK2-PK isoform axis in differentiation. Collectively, our findings unravel ERK2 guided PK-dependent metabolic changes during PMA induction, which are important in megakaryocytic differentiation.

  10. ERK2-Pyruvate Kinase Axis Permits Phorbol 12-Myristate 13-Acetate-induced Megakaryocyte Differentiation in K562 Cells*

    Science.gov (United States)

    Chaman, Noor; Iqbal, Mohammad Askandar; Siddiqui, Farid Ahmad; Gopinath, Prakasam; Bamezai, Rameshwar N. K.

    2015-01-01

    Metabolic changes that contribute to differentiation are not well understood. Overwhelming evidence shows the critical role of glycolytic enzyme pyruvate kinase (PK) in directing metabolism of proliferating cells. However, its role in metabolism of differentiating cells is unclear. Here we studied the role of PK in phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation in human leukemia K562 cells. We observed that PMA treatment decreased cancer-type anabolic metabolism but increased ATP production, along with up-regulated expression of two PK isoforms (PKM2 and PKR) in an ERK2-dependent manner. Interestingly, silencing of PK (PKM2 and PKR) inhibited PMA-induced megakaryocytic differentiation, as revealed by decreased expression of megakaryocytic differentiation marker CD61 and cell cycle behavior. Further, PMA-induced ATP production reduced greatly upon PK silencing, suggesting that PK is required for ATP synthesis. In addition to metabolic effects, PMA treatment also translocated PKM2, but not PKR, into nucleus. ERK1/2 knockdowns independently and together suggested the role of ERK2 in the up-regulation of both the isoforms of PK, proposing a role of ERK2-PK isoform axis in differentiation. Collectively, our findings unravel ERK2 guided PK-dependent metabolic changes during PMA induction, which are important in megakaryocytic differentiation. PMID:26269597

  11. Ascorbic acid delivered by mesoporous silica nanoparticles induces the differentiation of human embryonic stem cells into cardiomyocytes.

    Science.gov (United States)

    Ren, Mingming; Han, Zhen; Li, Jinglai; Feng, Gang; Ouyang, Shuyuan

    2015-11-01

    Embryonic stem (ES) cells offer the potential to generate all cell types in the body, which provide a promising approach to repair tissue damage or dysfunction. In the past decade, great efforts have been made to induce the differentiation of ES cells into numerous types of cells, such as adipocytes, neurocytes and cardiomyocytes. However, the low differentiated efficiency and successful rate limit the development of induction of the differentiation of stem cells for tissue engineering. Here, we utilize ascorbic acid (AA)-loaded fluorescent TRITC-mesoporous silica nanoparticles (TMSN-AA) as a potential tool to induce the differentiation of human ES cells into cardiomyocytes. The treatment of human ES cells by TMSN-AA nanoplex arrests cell cycle at G1 phase and decreases the expression of stemness genes octamer-binding transcription factor 4 (OCT4) and sex determining region Y-box 2 (SOX2), which exhibits more significant induction efficiency of stem cell differentiation than the treatment by AA alone. Furthermore, we have tested the myocardial marker genes cardiac Troponin I (cTnI) and fetal liver kinase 1 (FLK-1), and found these genes are up-regulated by TMSN-AA nanoplex. Importantly, this work demonstrates the more efficient induction efficiency of human ES cells differentiation by the nanoparticle-drug formulation. Our studies reveal a novel approach based on MSNs as nanocarriers to induce the differentiation of human ES cells into cardiomyocytes efficiently and feasibly, and offer the potential perspectives for tissue engineering, eventually in clinical applications.

  12. Increased cyclooxygenase-2 and thromboxane synthase expression is implicated in diosgenin-induced megakaryocytic differentiation in human erythroleukemia cells.

    Science.gov (United States)

    Cailleteau, C; Liagre, B; Battu, S; Jayat-Vignoles, C; Beneytout, J L

    2008-09-01

    Differentiation induction as a therapeutic strategy has, so far, the greatest impact in hematopoietic malignancies, most notably leukemia. Diosgenin is a very interesting natural product because, depending on the specific dose used, its biological effect is very different in HEL (human erythroleukemia) cells. For example, at 10 microM, diosgenin induced megakaryocytic differentiation, in contrast to 40 microM diosgenin, which induced apoptosis in HEL cells previously demonstrated using sedimentation field-flow fractionation (SdFFF). The goal of this work focused on the correlation between cyclooxygenase-2 (COX-2) and thromboxane synthase (TxS) and megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, the technique of SdFFF, having been validated in our models, was used in this new study as an analytical tool that provided us with more or less enriched differentiated cell fractions that could then be used for further analyses of enzyme protein expression and activity for the first time. In our study, we showed the implication of COX-2 and TxS in diosgenin-induced megakaryocytic differentiation in HEL cells. Furthermore, we showed that the analytical technique of SdFFF may be used as a tool to confirm our results as a function of the degree of cell differentiation.

  13. Simvastatin induces differentiation of rabbit articular chondrocytes via the ERK-1/2 and p38 kinase pathways.

    Science.gov (United States)

    Han, Yohan; Kim, Song Ja

    2016-08-15

    Statins are competitive inhibitors of hydroxy-methyl-glutaryl Coenzyme A (HMG-CoA) reductase, a key enzyme involved in the conversion of HMG-CoA to the cholesterol precursor mevalonate. Some statins, such as simvastatin (simvastatin), have been shown to have anti-cancer and anti-inflammatory effects, reducing cartilage degradation in osteoarthritic rabbits in vivo. However, the regulatory mechanisms undergirding simvastatin mediated chondrocyte differentiation have not been well elucidated. Thus, we investigated the action and mechanism of simvastatin on differentiation of rabbit articular chondrocytes through western blot analyses, RT-PCR, and immunohistochemical (IHC) and immunofluorescence (IF) staining. Simvastatin treatment was found to induce type II collagen expression and sulfated-proteoglycan synthesis in a dose- and time-dependent manner. Indeed, RT-PCR revealed increased expression of type II collagen on treatment with simvastatin. Both IHC and IF staining indicated differentiation of chondrocytes. Simvastatin treatment reduced activation of ERK-1/2 and stimulated activation of p38 kinase. Inhibition of ERK-1/2 with PD98059 enhanced simvastatin induced differentiation, whereas inhibition of p38 kinase with SB203580 inhibited simvastatin induced differentiation. Simvastatin treatment also inhibits loss of type II collagen in serial monolayer culture. Collectively, our results indicate that ERK-1/2 and p38 kinase regulate simvastatin-induced differentiation of chondrocytes in opposing manners. Thus, these findings suggest that simvastatin may be a potential therapeutic drug for osteoarthritis.

  14. Isoliquiritigenin-induced effects on Nrf2 mediated antioxidant defence in the HL-60 cell monocytic differentiation.

    Science.gov (United States)

    Chen, Hongmei; Zhang, Bo; Yuan, Xuan; Yao, Ying; Zhao, Hong; Sun, Xiling; Zheng, Quisheng

    2013-11-01

    To evaluate the role of redox homeostasis in differentiation in human promyelocytic leukemia cells (HL-60) induced by isoliquiritigenin (ISL) through modulation of the nuclear erythroid-related factor 2/antioxidant responsive element (Nrf2/ARE) pathway. Morphological changes, cell surface markers CD11b/CD14, and nitroblue tetrazolium (NBT)-reducing ability were used to determine the differentiation of HL-60, and 2,7-dichlorofluorescein was used to detect the level of intracellular reactive oxygen species (ROS). Thiobarbituric acid test was utilised to determine the levels of malondialdehyde production in ISL-treated HL-60. The study determines and presents the redox state of the ratio of reduced/oxidised glutathione as a consequence of progression from differentiation in HL-60. Expression levels of the Nrf2/ARE downstream target genes were determined by quantitative polymerase chain reaction. Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) inhibitors, apocynin (APO), and diphenyleneiodonium (DPI) were used for the preliminary study to determine the potential downstream targets regulated by NADPH oxidase in ISL-induced HL-60 differentiation. The data showed a strong dose-response relationship between ISL exposure and the characteristics of HL-60 differentiation, namely, morphology changes, NBT reductive activities, and expression levels of surface antigens CD11b/CD14. Intercellular redox homeostasis changes toward oxidation during drug exposure are necessary to support ISL-induced differentiation. The unique expression levels of the Nrf2/ARE downstream target genes in the differentiation of HL-60 recorded a statistically significant and dose-dependent increase (P < 0.05), which were suppressed by NADPH oxidase inhibitor, APO, and DPI. ISL as a differentiation-inducing agent with mechanisms involved in the Nrf2/ARE pathway to modulate intercellular redox homeostasis, and thus, facilitate differentiation.

  15. NGF induces adult stem Leydig cells to proliferate and differentiate during Leydig cell regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Lei [Department of Cell Biology, College of Life Science and Technology, Jinan University, 510632 Guangzhou (China); Wang, Huaxi [Southern Medical University, 510515 Guangzhou (China); Yang, Yan [College of Pharmacy, Jinan University, 510632 Guangzhou (China); Liu, Hui [Department of Cell Biology, College of Life Science and Technology, Jinan University, 510632 Guangzhou (China); Zhang, Qihao; Xiang, Qi [Department of Cell Biology, College of Life Science and Technology, Jinan University, 510632 Guangzhou (China); National Engineering Research Center of Genetic Medicine, 510632 Guangzhou (China); Ge, Renshan [Population Council, Rockefeller University, 10065 New York (United States); Su, Zhijian, E-mail: tjnuszj@jnu.edu.cn [Department of Cell Biology, College of Life Science and Technology, Jinan University, 510632 Guangzhou (China); Huang, Yadong, E-mail: tydhuang@jnu.edu.cn [Department of Cell Biology, College of Life Science and Technology, Jinan University, 510632 Guangzhou (China); National Engineering Research Center of Genetic Medicine, 510632 Guangzhou (China)

    2013-06-28

    Highlights: •Nerve growth factor has shown significant changes on mRNA levels during Adult Leydig cells regeneration. •We established the organ culture model of rat seminiferous tubules with ethane dimethyl sulphonate (EDS) treatment. •Nerve growth factor has shown proliferation and differentiation-promoting effects on Adult stem Leydig cells. •Nerve growth factor induces progenitor Leydig cells to proliferate and differentiate and immature Leydig cells to proliferate. -- Abstract: Nerve growth factor (NGF) has been reported to be involved in male reproductive physiology. However, few reports have described the activity of NGF during Leydig cell development. The objective of the present study was to examine the role of NGF during stem-Leydig-cell (SLC) regeneration. We investigated the effects of NGF on Leydig-cell (LC) regeneration by measuring mRNA levels in the adult rat testis after ethane dimethanesulfonate (EDS) treatment. Furthermore, we used the established organ culture model of rat seminiferous tubules to examine the regulation of NGF during SLC proliferation and differentiation using EdU staining, real-time PCR and western blotting. Progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs) were also used to investigate the effects of NGF on LCs at different developmental stages. NGF mRNA levels changed significantly during Leydig-cell regeneration in vivo. In vitro, NGF significantly promoted the proliferation of stem Leydig cells and also induced steroidogenic enzyme gene expression and 3β-HSD protein expression. The data from PLCs and ILCs showed that NGF could increase Cyclin D1 and Hsd 17b3 mRNA levels in PLCs and Cyclin D1 mRNA levels in ILCs. These results indicate that NGF may play an important role during LC regeneration by regulating the proliferation and differentiation of LCs at different developmental stages, from SLCs to PLCs and from PLCs to ILCs. The discovery of this effect of NGF on Leydig cells will provide useful

  16. Retinoic acid alleviates Con A-induced hepatitis and differentially regulates effector production in NKT cells.

    Science.gov (United States)

    Lee, Kyoo-A; Song, You Chan; Kim, Ga-Young; Choi, Gyeyoung; Lee, Yoon-Sook; Lee, Jung-Mi; Kang, Chang-Yuil

    2012-07-01

    Retinoic acid (RA) is a diverse regulator of immune responses. Although RA promotes natural killer T (NKT) cell activation in vitro by increasing CD1d expression on antigen-presenting cells (APCs), the direct effects of RA on NKT-cell responses in vivo are not known. In the present study, we demonstrated the effect of RA on the severity of Con A-induced hepatitis and molecular changes of NKT cells. First, we demonstrated that Con A-induced liver damage was ameliorated by RA. In correlation with cytokine levels in serum, RA regulated the production of IFN-γ and IL-4 but not TNF-α by NKT cells without influencing the NKT-cell activation status. However, RA did not alleviate α-GalCer-induced liver injury, even though it reduced IFN-γ and IL-4 but not TNF-α levels in serum. This regulation was also detected when liver mononuclear cells (MNCs) or NKT hybridoma cells were treated with RA in vitro. The regulatory effect of RA on NKT cells was mediated by RAR-α, and RA reduced the phosphorylation of MAPK. These results suggest that RA differentially modulates the production of effector cytokines by NKT cells in hepatitis, and the suppressive effect of RA on hepatitis varies with the pathogenic mechanism of liver injury.

  17. MicroRNA-1 effectively induces differentiation of myocardial cells from mouse bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Zhao, Xiao-Ling; Yang, Bo; Ma, Li-Na; Dong, Yan-Hua

    2016-11-01

    In this research, bone marrow mesenchymal stem cells (BMSCs) were isolated from mouse, and induced differentiation into myocardial cells in vitro after overexpression of miR-1a. The results showed that the BMSCs could induce differentiation into myocardial cells under the special condition medium, but when the miR-1a was over-expressed in BMSCs, the differentiation efficiency and induction time of myocardial cells from BMSCs could be promoted. This reason was demonstrated that Delta-like 1 (Dll-1) was a transcriptional repressor of myocardium gene expression during myocardium differentiation, miR-1a reduced Dll-1 levels, leading to the accumulation of myocardium gene mRNA and a dramatic increase in myocardium gene protein.

  18. Interaction with RXR is necessary for NPM-RAR-induced myeloid differentiation blockade.

    Science.gov (United States)

    Rush, Elizabeth A; Pollock, Sheri L; Abecassis, Irina; Redner, Robert L

    2013-12-01

    The t(5;17)(q35;q21) APL variant results in expression of a fusion protein linking the N-terminus of nucleophosmin (NPM) to the C-terminus of the retinoic acid receptor alpha (RAR). We have previously shown that NPM-RAR is capable of binding to DNA either as a homodimer or heterodimer with RXR. To determine the biological significance of NPM-RAR/RXR interaction, we developed two mutants of NPM-RAR that showed markedly diminished ability to bind RXR. U937 subclones expressing the NPM-RAR mutants showed significantly less inhibition of vitamin D3/TGFbeta-induced differentiation, compared with NPM-RAR. These results support the hypothesis that RXR interaction is necessary for NPM-RAR-mediated myeloid maturation arrest.

  19. On the q-deformed differential operators and induced su(n)-Toda field theory

    CERN Document Server

    Hssaini, M; Maroufi, B; Sedra, M B

    2000-01-01

    We build in this paper the algebra of q-deformed pseudo-differential operators shown to be an essential step towards setting a q-deformed integrability program. In fact, using the results of this q-deformed algebra, we derive the q-analogues of the generalised KdV hierarchy. We focus in particular the first leading orders of this q-deformed hierarchy namely the q-KdV and q- Boussinesq integrable systems. We present also the q-generalisation of the conformal transformations of the currents and discuss the primarity condition of the fields by using the Volterra gauge group transformations for the q-covariant Lax operators. An induced su(n)-Toda(su(2)-Liouville) field theory construction is discussed and other important features are presented.

  20. Development of a system of measuring double-differential cross sections for proton-induced reactions

    Energy Technology Data Exchange (ETDEWEB)

    Harada, M.; Watanabe, Y.; Sato, K. [Kyushu Univ., Fukuoka (Japan); Meigo, S.

    1997-03-01

    We report the present status of a counter telescope and a data acquisition system which are being developed for the measurement of double-differential cross sections of all light-charged particles emitted from proton-induced reactions on {sup 12}C at incident energies less than 90 MeV. The counter telescope consists of an active collimator made of a plastic scintillator, two thin silicon {Delta}E-detectors and a CsI(Tl) E-detectors with photo-diode readout. Signals from each detector are processed using the data acquisition system consisting of the front-end electronics (CAMAC) and two computers connected with the ethernet LAN: a personal computer as the data collector and server, and a UNIX workstation as the monitor and analyzer. (author)

  1. Rehmannia glutinosa oligosaccharide induces differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells.

    Science.gov (United States)

    Wang, X H; Du, H W; Guo, X H; Wang, S W; Zhou, R B; Li, Y; Li, Z B; Zhao, Y S; Zhu, Q L

    2016-10-17

    The aim of this study was to observe the effect of Rehmannia glutinosa oligosaccharide (RGO) on differentiation of bone marrow mesenchymal stem cells (MSCs) into cardiomyocyte-like cells . Rat MSCs were isolated, treated, and grouped as follows: RGO treatment group, 5-azacytidine (5-aza) treatment group, RGO + 5-aza treatment group, and control group. Following a four-week induction period, cardiac troponin I (cTnI) levels in MSCs were quantified by chemiluminescence, and the levels of myocardial enzymes creatine kinase (CK) and creatine kinase isoenzyme-MB (CK-MB) were measured using a dry chemistry analyzer. The cTnI- and connexin 43 (Cx43)-positive MSC population was identified by immunofluorescence, and expression levels of cTnI and Cx43 were analyzed by western blots. Following induction, cTnI, CK, and CK-MB levels were significantly higher in the RGO + 5-aza group as compared with the RGO and 5-aza groups (P < 0.05). In addition, fluorescence intensity of cTnI and Cx43 was higher in the RGO + 5-aza group as compared with the RGO and 5-aza groups. No cTnI- or Cx43-positive cells were detected in the control group. Western blot analysis further confirmed that cTnI and Cx43 were not expressed in the control group, while cTnI and Cx43 was higher in the RGO + 5-aza group than in the RGO and 5-aza groups. These results suggest that MSCs can be induced by RGO to differentiate into cardiomyocyte-like cells in vitro, and that RGO in combination with 5-aza enhance differentiation of MSCs.

  2. Osteogenic Differentiation of Adipose-Derived Stem Cells Is Hypoxia-Inducible Factor-1 Independent

    Science.gov (United States)

    Sahai, Suchit; Williams, Amanda; Skiles, Matthew L.

    2013-01-01

    Tissue engineering is a promising approach to repair critical-size defects in bone. Damage to vasculature at the defect site can create a lower O2 environment compared with healthy bone. Local O2 levels influence stem cell behavior, as O2 is not only a nutrient, but also a signaling molecule. The hypoxia-inducible factor-1 (HIF-1) is a transcription factor that regulates a wide range of O2-related genes and its contribution in bone repair/formation is an important area that can be exploited. In this study, we examined the effect of low O2 environments (1% and 2% O2) on the osteogenic differentiation of adipose-derived stem cells in both two-dimensional (2-D) and three-dimensional (3-D) culture systems. To determine the role of HIF-1 in the differentiation process, an inhibitor was used to block the HIF-1 activity. The samples were examined for osteogenesis markers as measured by quantification of the alkaline phosphatase (ALP) activity, mineral deposition, and expression of osteonectin (ON) and osteopontin (OPN). Results show a downregulation of the osteogenic markers (ALP activity, mineralization, ON, OPN) in both 1% and 2% O2 when compared to 20% O2 in both 2-D and 3-D culture. Vascular endothelial growth factor secretion over 28 days was significantly higher in low O2 environments and HIF-1 inhibition reduced this effect. The inhibition of the HIF-1 activity did not have a significant impact on the expression of the osteogenic markers, suggesting HIF-1-independent inhibition of osteogenic differentiation in hypoxic conditions. PMID:23394201

  3. Nat1 promotes translation of specific proteins that induce differentiation of mouse embryonic stem cells

    Science.gov (United States)

    Sugiyama, Hayami; Takahashi, Kazutoshi; Yamamoto, Takuya; Iwasaki, Mio; Narita, Megumi; Nakamura, Masahiro; Rand, Tim A.; Nakagawa, Masato; Watanabe, Akira; Yamanaka, Shinya

    2017-01-01

    Novel APOBEC1 target 1 (Nat1) (also known as “p97,” “Dap5,” and “Eif4g2”) is a ubiquitously expressed cytoplasmic protein that is homologous to the C-terminal two thirds of eukaryotic translation initiation factor 4G (Eif4g1). We previously showed that Nat1-null mouse embryonic stem cells (mES cells) are resistant to differentiation. In the current study, we found that NAT1 and eIF4G1 share many binding proteins, such as the eukaryotic translation initiation factors eIF3 and eIF4A and ribosomal proteins. However, NAT1 did not bind to eIF4E or poly(A)-binding proteins, which are critical for cap-dependent translation initiation. In contrast, compared with eIF4G1, NAT1 preferentially interacted with eIF2, fragile X mental retardation proteins (FMR), and related proteins and especially with members of the proline-rich and coiled-coil–containing protein 2 (PRRC2) family. We also found that Nat1-null mES cells possess a transcriptional profile similar, although not identical, to the ground state, which is established in wild-type mES cells when treated with inhibitors of the ERK and glycogen synthase kinase 3 (GSK3) signaling pathways. In Nat1-null mES cells, the ERK pathway is suppressed even without inhibitors. Ribosome profiling revealed that translation of mitogen-activated protein kinase kinase kinase 3 (Map3k3) and son of sevenless homolog 1 (Sos1) is suppressed in the absence of Nat1. Forced expression of Map3k3 induced differentiation of Nat1-null mES cells. These data collectively show that Nat1 is involved in the translation of proteins that are required for cell differentiation. PMID:28003464

  4. Multilineage-differentiating stress-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts

    OpenAIRE

    Wakao, Shohei; Kitada, Masaaki; Kuroda, Yasumasa; Shigemoto, Taeko; Matsuse, Dai; Akashi, Hideo; Tanimura, Yukihiro; Tsuchiyama, Kenichiro; Kikuchi, Tomohiko; Goda, Makoto; Nakahata, Tatsutoshi; Fujiyoshi, Yoshinori; Dezawa, Mari

    2011-01-01

    The stochastic and elite models have been proposed for the mechanism of induced pluripotent stem (iPS) cell generation. In this study we report a system that supports the elite model. We previously identified multilineage-differentiating stress-enduring (Muse) cells in human dermal fibroblasts that are characterized by stress tolerance, expression of pluripotency markers, self-renewal, and the ability to differentiate into endodermal-, mesodermal-, and ectodermal-lineage cells from a single c...

  5. Potential Role of Dentin Sialoprotein by Inducing Dental Pulp Mesenchymal Stem Cell Differentiation and Mineralization for Dental Tissue Repair

    OpenAIRE

    Zhi Chen; Shuo Chen; Li-An Wu; Guo-Hua Yuan; Guo-Bin Yang

    2010-01-01

    Introduction: Dentin sialoprotein (DSP) is a dentin extracellular matrix protein, a unique marker of dentinogenesis and plays a vital role in odontoblast differentiation and dentin mineralization. Recently, studies have shown that DSP induces differentiation and mineralization of periodontal ligament stem cells and dental papilla mesenchymal cells in vitro and rescues dentin deficiency and increases enamel mineralization in animal models.The hypothesis: DSP as a nature therapeutic agent stimu...

  6. Differential inhibition of oxidized LDL-induced apoptosis in human endothelial cells treated with different flavonoids.

    Science.gov (United States)

    Jeong, Yu-Jin; Choi, Yean-Jung; Kwon, Hyang-Mi; Kang, Sang-Wook; Park, Hyoung-Sook; Lee, Myungsook; Kang, Young-Hee

    2005-05-01

    High plasma level of cholesterol is a well-known risk factor for atherosclerotic diseases. Oxidized LDL induces cellular and nuclear damage that leads to apoptotic cell death. We tested the hypothesis that flavonoids may function as antioxidants with regard to LDL incubated with 5 microm-Cu(2+) alone or in combination with human umbilical vein endothelial cells (HUVEC). Cytotoxicity and formation of thiobarbituric acid-reactive substances induced by Cu(2+)-oxidized LDL were examined in the presence of various subtypes of flavonoid. Flavanols, flavonols and flavanones at a non-toxic dose of 50 microm markedly inhibited LDL oxidation by inhibiting the formation of peroxidative products. In contrast, the flavones luteolin and apigenin had no such effect, with >30 % of cells killed after exposure to 0.1 mg LDL/ml. Protective flavonoids, especially (-)-epigallocatechin gallate, quercetin, rutin and hesperetin, inhibited HUVEC nuclear condensation and fragmentation induced by Cu(2+)-oxidized LDL. In addition, immunochemical staining and Western blot analysis revealed that anti-apoptotic Bcl-2 expression was enhanced following treatment with these protective flavonoids. However, Bax expression and caspase-3 cleavage stimulated by 18 h incubation with oxidized LDL were reduced following treatment with these protective flavonoids. The down-regulation of Bcl-2 and up-regulation of caspase-3 activation were reversed by the cytoprotective flavonoids, (-)-epigallocatechin gallate, quercetin and hesperetin, at >/=10 microm. These results suggest that flavonoids may differentially prevent Cu(2+)-oxidized LDL-induced apoptosis and promote cell survival as potent antioxidants. Survival potentials of certain flavonoids against cytotoxic oxidized LDL appeared to stem from their disparate chemical structure. Furthermore, dietary flavonoids may have therapeutic potential for protecting the endothelium from oxidative stress and oxidized LDL-triggered atherogenesis.

  7. Differential effects of stress-induced cortisol responses on recollection and familiarity-based recognition memory.

    Science.gov (United States)

    McCullough, Andrew M; Ritchey, Maureen; Ranganath, Charan; Yonelinas, Andrew

    2015-09-01

    Stress-induced changes in cortisol can impact memory in various ways. However, the precise relationship between cortisol and recognition memory is still poorly understood. For instance, there is reason to believe that stress could differentially affect recollection-based memory, which depends on the hippocampus, and familiarity-based recognition, which can be supported by neocortical areas alone. Accordingly, in the current study we examined the effects of stress-related changes in cortisol on the processes underlying recognition memory. Stress was induced with a cold-pressor test after incidental encoding of emotional and neutral pictures, and recollection and familiarity-based recognition memory were measured one day later. The relationship between stress-induced cortisol responses and recollection was non-monotonic, such that subjects with moderate stress-related increases in cortisol had the highest levels of recollection. In contrast, stress-related cortisol responses were linearly related to increases in familiarity. In addition, measures of cortisol taken at the onset of the experiment showed that individuals with higher levels of pre-learning cortisol had lower levels of both recollection and familiarity. The results are consistent with the proposition that hippocampal-dependent memory processes such as recollection function optimally under moderate levels of stress, whereas more cortically-based processes such as familiarity are enhanced even with higher levels of stress. These results indicate that whether post-encoding stress improves or disrupts recognition memory depends on the specific memory process examined as well as the magnitude of the stress-induced cortisol response.

  8. Oligonucleotide microarray identifies genes differentially expressed during tumorigenesis of DMBA-induced pancreatic cancer in rats.

    Directory of Open Access Journals (Sweden)

    Jun-Chao Guo

    Full Text Available The extremely dismal prognosis of pancreatic cancer (PC is attributed, at least in part, to lack of early diagnosis. Therefore, identifying differentially expressed genes in multiple steps of tumorigenesis of PC is of great interest. In the present study, a 7,12-dimethylbenzanthraene (DMBA-induced PC model was established in male Sprague-Dawley rats. The gene expression profile was screened using an oligonucleotide microarray, followed by real-time quantitative polymerase chain reaction (qRT-PCR and immunohistochemical staining validation. A total of 661 differentially expressed genes were identified in stages of pancreatic carcinogenesis. According to GO classification, these genes were involved in multiple molecular pathways. Using two-way hierarchical clustering analysis, normal pancreas, acute and chronic pancreatitis, PanIN, early and advanced pancreatic cancer were completely discriminated. Furthermore, 11 upregulated and 142 downregulated genes (probes were found by Mann-Kendall trend Monotone test, indicating homologous genes of rat and human. The qRT-PCR and immunohistochemistry analysis of CXCR7 and UBe2c, two of the identified genes, confirmed the microarray results. In human PC cell lines, knockdown of CXCR7 resulted in decreased migration and invasion. Collectively, our data identified several promising markers and therapeutic targets of PC based on a comprehensive screening and systemic validation.

  9. Human macrophage colony-stimulating factor induces the differentiation of trophoblast.

    Science.gov (United States)

    Saito, S; Saito, M; Enomoto, M; Ito, A; Motoyoshi, K; Nakagawa, T; Ichijo, M

    1993-01-01

    When human cytotrophoblastic cells in the early stage of pregnancy were cultured in a serum-free medium in the presence of human macrophage colony-stimulating factor (M-CSF), the cytotrophoblastic cells fused and formed a typical syncytiotrophoblast which had a dense distribution of microvilli revealed under an electron microscope. On the other hand, cytotrophoblasts incubated with anti-M-CSF antibody showed hardly any syncytiotrophoblast formation. Following this finding, we studied the differentiation of chorionic cells from the viewpoint of hormone secretion. When cytotrophoblasts were incubated in the presence of M-CSF, the supernatant of the culture showed an increase in human chorionic gonadotropin and human placental lactogen levels in proportion to the concentration of M-CSF added. When cytotrophoblasts were incubated in the presence of anti-M-CSF antibody or anti-fms antibody, human chorionic gonadotropin and human placental lactogen secretion were suppressed. Thus, M-CSF was morphologically and endocrinologically found to induce the differentiation of chorionic cells.

  10. D609 induces vascular endothelial cells and marrow stromal cells differentiation into neuron-like cells

    Institute of Scientific and Technical Information of China (English)

    Nan WANG; Chun-qing DU; Shao-shan WANG; Kun XIE; Shang-li ZHANG; Jun-ying MIAO

    2004-01-01

    AIM: To investigate the effect of tricyclodecane-9-yl-xanthogenate (D609) on cell differentiation in vascular endothelial cells (VECs) and marrow stromal cells (MSCs). METHODS: Morphological changes were observed under phase contrast microscope. Electron microscope and immunostaining were used for VECs identification. The expressions of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were examined by immunohistochemistry. RESULTS: After 6 h of induction with D609, some VECs showed morphological changes characteristic of neurones. 9 h later, more VECs became neuron-like cells. About 30.8 % of VECs displayed positive NSE (P<0.01), while the expression of GFAP was negative. When MSCs were exposed to D609, the cells displayed neuronal morphologies, such as pyramidal cell bodies and processes formed extensive networks at 3 h. 6 h later, almost all of the cells exhibited a typical neuronal appearance, and 85.6 % of MSCs displayed intensive positive NSE, but GFAP did not express. CONCLUSION: D609 induces VECs and MSCs differentiation into neuron-like cells.

  11. In vitro induced dopaminergic differentiation of expanded rat mesencephalic neural stem cell

    Institute of Scientific and Technical Information of China (English)

    ZHENG Min; WANG Dongmei; JIAO Wenchang; LI Haiming; ZHAO Lianxu; BAI Chixian; WANG Yaping; PEI Xuetao

    2003-01-01

    Neural stem cell (NSC) is the progenitor of the neural system with the character of self-renew and having the potential to differentiate into all the phenotypes in the central nervous system (CNS). NSC may serve as a source of cell transplantation for the treatment of neurodegenerative diseases to replace degenerative neurons. In this study, NSCs derived from E12.5 rat mesencephalon were maintained and expanded using a serum-free defined medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). While proliferating, the cells were immunoreactive for nestin and remained multipotent to generate neurons, astrocytes, and oligodendrocytes. After 15 times passage the total number of the cell expanded about 2.4×104 fold. Compared with untreated cultures, ascorbic acid (AA) treatment led to more dopaminergic (DAergic) differentitiation as indicated by the expression of tyrosine hydroxylase (TH). With the concentration increasing, more TH+ neurons were obtained. 100 μmol/L AA could lead to a increase more than 20-fold, and a concentration of 10 μmol/L could lead to nearly 5-fold increase in TH+ cells. However, the ratio of TH+ cells was not improved any longer with the AA increasing above the concentration of 100 μmol/L. The results demonstrate that expanded NSCs can be induced to differentiate into dopamine neurons in vitro, which can provide enough cell population for the cell transplantation, as a main intervention for the neurodegenerative diseases such as Parkinson's disease.

  12. Cutting Edge: Helicobacter pylori Induces Nuclear Hypersegmentation and Subtype Differentiation of Human Neutrophils In Vitro

    Science.gov (United States)

    Whitmore, Laura C.; Weems, Megan N.

    2017-01-01

    Helicobacter pylori infects the human stomach and causes a spectrum of disease that includes gastritis, peptic ulcers, and gastric adenocarcinoma. A chronic, neutrophil-rich inflammatory response characterizes this infection. It is established that H. pylori stimulates neutrophil chemotaxis and a robust respiratory burst, but other aspects of this interaction are incompletely defined. We demonstrate here that H. pylori induces N1-like subtype differentiation of human neutrophils as indicated by profound nuclear hypersegmentation, a CD62Ldim, CD16bright, CD11bbright, CD66bbright, CD63bright surface phenotype, proinflammatory cytokine secretion, and cytotoxicity. Hypersegmentation requires direct neutrophil–H. pylori contact as well as transcription and both host and bacterial protein synthesis, but not urease, NapA, VacA, CagA, or CagT. The concept of neutrophil plasticity is new and, to our knowledge, these data are the first evidence that neutrophils can undergo subtype differentiation in vitro in response to bacterial pathogen infection. We hypothesize that these changes favor H. pylori persistence and disease. PMID:28148734

  13. Oligonucleotide microarray identifies genes differentially expressed during tumorigenesis of DMBA-induced pancreatic cancer in rats.

    Science.gov (United States)

    Guo, Jun-Chao; Li, Jian; Yang, Ying-Chi; Zhou, Li; Zhang, Tai-Ping; Zhao, Yu-Pei

    2013-01-01

    The extremely dismal prognosis of pancreatic cancer (PC) is attributed, at least in part, to lack of early diagnosis. Therefore, identifying differentially expressed genes in multiple steps of tumorigenesis of PC is of great interest. In the present study, a 7,12-dimethylbenzanthraene (DMBA)-induced PC model was established in male Sprague-Dawley rats. The gene expression profile was screened using an oligonucleotide microarray, followed by real-time quantitative polymerase chain reaction (qRT-PCR) and immunohistochemical staining validation. A total of 661 differentially expressed genes were identified in stages of pancreatic carcinogenesis. According to GO classification, these genes were involved in multiple molecular pathways. Using two-way hierarchical clustering analysis, normal pancreas, acute and chronic pancreatitis, PanIN, early and advanced pancreatic cancer were completely discriminated. Furthermore, 11 upregulated and 142 downregulated genes (probes) were found by Mann-Kendall trend Monotone test, indicating homologous genes of rat and human. The qRT-PCR and immunohistochemistry analysis of CXCR7 and UBe2c, two of the identified genes, confirmed the microarray results. In human PC cell lines, knockdown of CXCR7 resulted in decreased migration and invasion. Collectively, our data identified several promising markers and therapeutic targets of PC based on a comprehensive screening and systemic validation.

  14. Expandable and Rapidly Differentiating Human Induced Neural Stem Cell Lines for Multiple Tissue Engineering Applications

    Directory of Open Access Journals (Sweden)

    Dana M. Cairns

    2016-09-01

    Full Text Available Limited availability of human neurons poses a significant barrier to progress in biological and preclinical studies of the human nervous system. Current stem cell-based approaches of neuron generation are still hindered by prolonged culture requirements, protocol complexity, and variability in neuronal differentiation. Here we establish stable human induced neural stem cell (hiNSC lines through the direct reprogramming of neonatal fibroblasts and adult adipose-derived stem cells. These hiNSCs can be passaged indefinitely and cryopreserved as colonies. Independently of media composition, hiNSCs robustly differentiate into TUJ1-positive neurons within 4 days, making them ideal for innervated co-cultures. In vivo, hiNSCs migrate, engraft, and contribute to both central and peripheral nervous systems. Lastly, we demonstrate utility of hiNSCs in a 3D human brain model. This method provides a valuable interdisciplinary tool that could be used to develop drug screening applications as well as patient-specific disease models related to disorders of innervation and the brain.

  15. The receptor for advanced glycation end products (RAGE affects T cell differentiation in OVA induced asthma.

    Directory of Open Access Journals (Sweden)

    Eitan M Akirav

    Full Text Available The receptor for glycation end products (RAGE has been previously implicated in shaping the adaptive immune response. RAGE is expressed in T cells after activation and constitutively in T cells from patients with diabetes. The effects of RAGE on adaptive immune responses are not clear: Previous reports show that RAGE blockade affects Th1 responses. To clarify the role of RAGE in adaptive immune responses and the mechanisms of its effects, we examined whether RAGE plays a role in T cell activation in a Th2 response involving ovalbumin (OVA-induced asthma in mice. WT and RAGE deficient wild-type and OT-II mice, expressing a T cell receptor specific for OVA, were immunized intranasally with OVA. Lung cellular infiltration and T cell responses were analyzed by immunostaining, FACS, and multiplex bead analyses for cytokines. RAGE deficient mice showed reduced cellular infiltration in the bronchial alveolar lavage fluid and impaired T cell activation in the mediastinal lymph nodes when compared with WT mice. In addition, RAGE deficiency resulted in reduced OT-II T cell infiltration of the lung and impaired IFNγ and IL-5 production when compared with WT mice and reduced infiltration when transferred into WT hosts. When cultured under conditions favoring the differentiation of T cells subsets, RAGE deficient T cells showed reduced production of IFNγ but increased production of IL-17. Our data show a stimulatory role for RAGE in T activation in OVA-induced asthma. This role is largely mediated by the effects of RAGE on T cell proliferation and differentiation. These findings suggest that RAGE may play a regulatory role in T cell responses following immune activation.

  16. Transforming growth factor-β1 induces intestinal myofibroblast differentiation and modulates their migration

    Institute of Scientific and Technical Information of China (English)

    Julia Brenmoehl; Sandra Nicole Miller; Claudia Hofmann; Daniela Vogl; Werner Falk; Jürgen Scholmerich; Gerhard Rogler

    2009-01-01

    AIM:To investigate the effects of transforming growth factor β1 (TGF-β1) on the differentiation of colonic lamina propria fibroblasts (CLPF) into myofibroblasts in vitro. METHODS:Primary CLPF cultures were incubated with TGF-β1 and analyzed for production of α-smooth muscle actin (α-SMA), fibronectin (FN) and FN isoforms. Migration assays were performed in a modified 48-well Boyden chamber. Levels of total and phosphorylated focal adhesion kinase (FAK) in CLPF were analyzed after induction of migration. RESULTS:Incubation of CLPF with TGF-β1 for 2 d did not change α-SMA levels, while TGF-β1 treatment for 6 d significantly increased α-SMA production. Short term incubation (6 h) with TGF-β1 enhanced CLPF migration, while long term treatment (6 d) of CLPF with TGF-β1 reduced migration to 15%-37% compared to untreated cells. FN and FN isoform mRNA expression were increased after short term incubation with TGF-β1 (2 d) in contrast to long term incubation with TGF-β1 for 6 d. After induction of migration, TGF-β1-preincubated CLPF showed higher amounts of FN and its isoforms and lower levels of total and phosphorylated FAK than untreated cells. CONCLUSION:Long term incubation of CLPF with TGF-β1 induced differentiation into myofibroblasts with enhanced α-SMA, reduced migratory potential and FAK phosphorylation, and increased FN production. In contrast, short term contact (6 h) of fibroblasts with TGF-β1 induced a dose-dependent increase of cell migration and FAK phosphorylation without induction of α-SMA production.

  17. Notch signaling modulates hypoxia-induced neuroendocrine differentiation of human prostate cancer cells.

    Science.gov (United States)

    Danza, Giovanna; Di Serio, Claudia; Rosati, Fabiana; Lonetto, Giuseppe; Sturli, Niccolò; Kacer, Doreen; Pennella, Antonio; Ventimiglia, Giuseppina; Barucci, Riccardo; Piscazzi, Annamaria; Prudovsky, Igor; Landriscina, Matteo; Marchionni, Niccolò; Tarantini, Francesca

    2012-02-01

    Prostate carcinoma is among the most common causes of cancer-related death in men, representing 15% of all male malignancies in developed countries. Neuroendocrine differentiation (NED) has been associated with tumor progression, poor prognosis, and with the androgen-independent status. Currently, no successful therapy exists for advanced, castration-resistant disease. Because hypoxia has been linked to prostate cancer progression and unfavorable outcome, we sought to determine whether hypoxia would impact the degree of neuroendocrine differentiation of prostate cancer cells in vitro. Exposure of LNCaP cells to low oxygen tension induced a neuroendocrine phenotype, associated with an increased expression of the transcription factor neurogenin3 and neuroendocrine markers, such as neuron-specific enolase, chromogranin A, and β3-tubulin. Moreover, hypoxia triggered a significant decrease of Notch 1 and Notch 2 mRNA and protein expression, with subsequent downregulation of Notch-mediated signaling, as shown by reduced levels of the Notch target genes, Hes1 and Hey1. NED was promoted by attenuation of Hes1 transcription, as cells expressing a dominant-negative form of Hes1 displayed increased levels of neuroendocrine markers under normoxic conditions. Although hypoxia downregulated Notch 1 and Notch 2 mRNA transcription and receptor activation also in the androgen-independent cell lines, PC-3 and Du145, it did not change the extent of NED in these cultures, suggesting that androgen sensitivity may be required for transdifferentiation to occur. Hypoxia induces NED of LNCaP cells in vitro, which seems to be driven by the inhibition of Notch signaling with subsequent downregulation of Hes1 transcription. ©2011 AACR.

  18. The receptor for advanced glycation end products (RAGE) affects T cell differentiation in OVA induced asthma.

    Science.gov (United States)

    Akirav, Eitan M; Henegariu, Octavian; Preston-Hurlburt, Paula; Schmidt, Ann Marie; Clynes, Raphael; Herold, Kevan C

    2014-01-01

    The receptor for glycation end products (RAGE) has been previously implicated in shaping the adaptive immune response. RAGE is expressed in T cells after activation and constitutively in T cells from patients with diabetes. The effects of RAGE on adaptive immune responses are not clear: Previous reports show that RAGE blockade affects Th1 responses. To clarify the role of RAGE in adaptive immune responses and the mechanisms of its effects, we examined whether RAGE plays a role in T cell activation in a Th2 response involving ovalbumin (OVA)-induced asthma in mice. WT and RAGE deficient wild-type and OT-II mice, expressing a T cell receptor specific for OVA, were immunized intranasally with OVA. Lung cellular infiltration and T cell responses were analyzed by immunostaining, FACS, and multiplex bead analyses for cytokines. RAGE deficient mice showed reduced cellular infiltration in the bronchial alveolar lavage fluid and impaired T cell activation in the mediastinal lymph nodes when compared with WT mice. In addition, RAGE deficiency resulted in reduced OT-II T cell infiltration of the lung and impaired IFNγ and IL-5 production when compared with WT mice and reduced infiltration when transferred into WT hosts. When cultured under conditions favoring the differentiation of T cells subsets, RAGE deficient T cells showed reduced production of IFNγ but increased production of IL-17. Our data show a stimulatory role for RAGE in T activation in OVA-induced asthma. This role is largely mediated by the effects of RAGE on T cell proliferation and differentiation. These findings suggest that RAGE may play a regulatory role in T cell responses following immune activation.

  19. Kaempferol as a flavonoid induces osteoblastic differentiation via estrogen receptor signaling

    Directory of Open Access Journals (Sweden)

    Guo Ava

    2012-04-01

    Full Text Available Abstract Background Flavonoids, a group of compounds mainly derived from vegetables and herbal medicines, chemically resemble estrogen and some have been used as estrogen substitutes. Kaempferol, a flavonol derived from the rhizome of Kaempferia galanga L., is a well-known phytoestrogen possessing osteogenic effects that is also found in a large number of plant foods. The herb K. galanga is a popular traditional aromatic medicinal plant that is widely used as food spice and in medicinal industries. In the present study, both the estrogenic and osteogenic properties of kaempferol are evaluated. Methods Kaempferol was first evaluated for its estrogenic properties, including its effects on estrogen receptors. The osteogenic properties of kaempferol were further determined its induction effects on specific osteogenic enzymes and genes as well as the mineralization process in cultured rat osteoblasts. Results Kaempferol activated the transcriptional activity of pERE-Luc (3.98 ± 0.31 folds at 50 μM and induced estrogen receptor α (ERα phosphorylation in cultured rat osteoblasts, and this ER activation was correlated with induction and associated with osteoblast differentiation biomarkers, including alkaline phosphatase activity and transcription of osteoblastic genes, e.g., type I collagen, osteonectin, osteocalcin, Runx2 and osterix. Kaempferol also promoted the mineralization process of osteoblasts (4.02 ± 0.41 folds at 50 μM. ER mediation of the kaempferol-induced effects was confirmed by pretreatment of the osteoblasts with an ER antagonist, ICI 182,780, which fully blocked the induction effect. Conclusion Our results showed that kaempferol stimulates osteogenic differentiation of cultured osteoblasts by acting through the estrogen receptor signaling.

  20. Exocrine pancreas ER stress is differentially induced by different fatty acids.

    Science.gov (United States)

    Danino, Hila; Ben-Dror, Karin; Birk, Ruth

    2015-12-10

    Exocrine pancreas acinar cells have a highly developed endoplasmic reticulum (ER), accommodating their high protein production rate. Overload of dietary fat (typical to obesity) is a recognized risk factor in pancreatitis and pancreatic cancer. Dietary fat, especially saturated fat, has been suggested by others and us to induce an acinar lipotoxic effect. The effect of different dietary fatty acids on the ER stress response is unknown. We studied the effect of acute (24h) challenge with different fatty acids (saturated, mono and poly-unsaturated) at different concentrations (between 200 and 500µM, typical to normal and obese states, respectively), testing fat accumulation, ER stress indicators, X-box binding protein 1 (Xbp1) splicing and nuclear translocation, as well as unfolded protein response (UPR) transcripts and protein levels using exocrine pancreas acinar AR42J and primary cells. Acute exposure of AR42J cells to different fatty acids caused increased accumulation of triglycerides, dependent on the type of fat. Different FAs had different effects on ER stress: most notably, saturated palmitic acid significantly affected the UPR response, as demonstrated by altered Xbp1 splicing, elevation in transcript levels of UPR (Xbp, CHOP, Bip) and immune factors (Tnfα, Tgfβ), and enhanced Xbp1 protein levels and Xbp1 time-dependent nuclear translocation. Poly-unsaturated FAs caused milder elevation of ER stress markers, while mono-unsaturated oleic acid attenuated the ER stress response. Thus, various fatty acids differentially affect acinar cell fat accumulation and, apart from oleic acid, induce ER stress. The differential effect of the various fatty acids could have potential nutritional and therapeutic implications.

  1. Neuronal differentiation of human iPS cells induced by baicalin via regulation of bHLH gene expression.

    Science.gov (United States)

    Morita, Akihiro; Soga, Kohei; Nakayama, Hironobu; Ishida, Torao; Kawanishi, Shosuke; Sato, Eisuke F

    2015-09-25

    Efficient differentiation is important for regenerative medicine based on pluripotent stem cells, including treatment of neurodegenerative disorders and trauma. Baicalin promotes neuronal differentiation of neural stem/progenitor cells of rats and mice. To evaluate the suitability of baicalin for neuronal differentiation of human iPS cells, we investigated whether it promotes neuronal differentiation in human iPS cells and monitored basic helix-loop-helix (bHLH) gene expression during neuronal differentiation. Baicalin promoted neuronal differentiation and inhibited glial differentiation, suggesting that baicalin can influence the neuronal fate decision in human iPS cells. Notch signaling, which is upstream of bHLH proteins, was not involved in baicalin-induced neuronal differentiation. Baicalin treatment did not down-regulate Hes1 gene expression, but it reduced Hes1 protein levels and up-regulated Ascl1 gene expression. Thus, baicalin promoted neuronal differentiation via modulation of bHLH transcriptional factors. Therefore, baicalin has potential to be used as a small-molecule drug for regenerative treatment of neurodegenerative disorders.

  2. Differentiation of HL-60 promyelocytes to granulocytes induced via the activation of protein kinase-C by benzene

    Energy Technology Data Exchange (ETDEWEB)

    Carlson, C.; O' Connor, A.; Kalf, G. (Rutgers-the State Univ., Piscataway, NJ (United States) Thomas Jefferson Univ., Philadelphia, PA (United States))

    1991-03-15

    Benzene is a hematotoxin which affects the development of bone marrow progenitor cells and a leukemogen which causes acute myelogenous leukemia. The authors studied the effect of benzene on the differentiation of progenitors of the myeloid lineage, using HL-60 promyelocytic leukemia cells which can be induced to differentiate to granulocytes via the activation of protein kinase-C (PKC) by DMSO and retinoic acid. Exposure of HL-60 cells to 5 mM benzene for 5 min. results in the activation of PKC as measured by an increases in the phosphorylation of cellular proteins in a whole cell assay including proteins pp17 and pp27 reported by Feuerstein and Cooper to be involved in HL-60 cell differentiation. The increase in protein phosphorylation observed with benzene was equally as great as that observed with 100 ng/mL PMA, used as a control. Under the same conditions, benzene induces differentiation of the promyelocytes into granulocytes as measured by the acquisition of superoxide production and granulocyte morphology. Preincubation with 40 {mu}M sphinganine, a PKC inhibitor, prevents the benzene-induced increase in cellular protein phosphorylation and the differentiation to granulocytes. These results indicate that benzene, by activation of PKC, can affect myeloid differentiation which may play a role in the ability of benzene to cause acute myelogenous leukemia.

  3. Differentiation of bone marrow derived Thy-1+β2M-cells into hepatocytes induced by coculture with transgenic CFSCs

    Institute of Scientific and Technical Information of China (English)

    WANG Yunfang; NAN Xue; ZHANG Rui; LI Yanhua; YUE Wen; YAN Fang; PEI Xuetao

    2004-01-01

    Studies of transplantation in vivo indicted that bone marrow derived stem cells had a potential to differentiate into mature hepatocytes. However, there are lots of doubts and uncertainties in the influencing factors and control agents of effectively inducing stem cell differentiation in vitro, the efficiency of stem cells' differentiation into hepatocytes and differentiated cells' life-span and functional state,etc. In this study, rat bone marrow derived Thy-1+β2M- cells (BDTCs) were induced to differentiate into hepatocytes by co-culturing with CFSC/HGF feeder layers which expressed hHGF efficiently and stably. RT-PCR and immunofluorescent texts proved induced BDTCs expressed infant and adult hepatocyte specific genes. Further more, these cells displayed functions of indocyanine green (ICG) uptake, ammonium metabolism and albumin production. It was shown that growth factors together with hepatic nonparenchyma cells provided a feasible microenvironment for differentiation of bone marrow stem cells into hepatocytes. The studies not only provided a significant biological model for going deep into the mechanism of stem cell plasticity, but also offered a theoretical and technical foundation of gene and stem cell engineering-based regenerative medicine for end-stage liver diseases.

  4. Bilobalide induces neuronal differentiation of P19 embryonic carcinoma cells via activating Wnt/β-catenin pathway.

    Science.gov (United States)

    Liu, Mei; Guo, Jingjing; Wang, Juan; Zhang, Luyong; Pang, Tao; Liao, Hong

    2014-08-01

    Bilobalide, a natural product extracted from Ginkgo biloba leaf, is known to exhibit a number of pharmacological activities. So far, whether it could affect embryonic stem cell differentiation is still unknown. The main aim of this study was to investigate the effect of bilobalide on P19 embryonic carcinoma cells differentiation and the underlying mechanisms. Our results showed that bilobalide induced P19 cells differentiation into neurons in a concentration- and time-dependent manner. We also found that bilobalide promoted neuronal differentiation through activation of Wnt/β-catenin signaling pathway. Exposure to bilobalide increased inactive GSK-3β phosphorylation, further induced the nuclear accumulation of β-catenin, and also up-regulated the expression of Wnt ligands Wnt1 and Wnt7a. Neuronal differentiation induced by bilobalide was totally abolished by XAV939, an inhibitor of Wnt/β-catenin pathway. These results revealed a novel role of bilobalide in neuronal differentiation from P19 embryonic cells acting through Wnt/β-catenin signaling pathway, which would provide a better insight into the beneficial effects of bilobalide in brain diseases.

  5. Mitochondrial reactive oxygen species regulate adipocyte differentiation of mesenchymal stem cells in hematopoietic stress induced by arabinosylcytosine.

    Directory of Open Access Journals (Sweden)

    Weimin Wang

    Full Text Available The increase in adipocytes induced by chemotherapeutic drugs may play a negative role in hematopoietic recovery. However, the mechanism underlying adipocyte differentiation of mesenchymal stem cells (MSCs in hematopoietic stress is still unknown. Hence, the involvement of reactive oxygen species (ROS in adipocyte differentiation under hematopoietic stress was investigated in vitro and in vivo.The roles of cellular ROS in adipogenesis were investigated in vivo through an adipocyte hyperplasia marrow model under hematopoietic stress induced by arabinosylcytosine (Ara-C and in vitro via adipocyte differentiation of human MSCs. ROS levels were detected using the CM-H2DCFDA probe and Mito-SOX dye. Adipogenesis was evaluated by histopathology and oil red O staining, whereas detection of mRNA levels of antioxidant enzymes and adipogenesis markers was performed using quantitative real-time polymerase chain reaction analysis.ROS were found to play an important role in regulating adipocyte differentiation of MSCs by activating peroxisome proliferator-activated receptor gamma (PPARγ, while the antioxidant N-acetyl-L-cysteine acts through ROS to inhibit adipocyte differentiation. The elevated ROS levels induced by Ara-C were caused by both over-generation of mitochondrial ROS and reduction of antioxidant enzymes (Cu/Zn Superoxide dismutase and catalase. Our findings suggest that a mitochondrial-targeted antioxidant could diminish adipocyte differentiation.

  6. Elevated TrkA receptor expression is associated with all-trans retinoic acid-induced neuroblastoma differentiation.

    Science.gov (United States)

    Gao, Q; Chen, C F; Dong, Q; Hou, L; Chen, X; Zhi, Y L; Li, X; Lu, H T; Zhang, H Y

    2015-10-27

    Neuroblastoma is the most common and one of the deadliest among pediatric tumors; however, a subset of infants with neuroblastoma display spontaneous regression. The mechanism of spontaneous regression remains to be elucidated. TrkA plays an essential role in the differentiation and functionality of neurons; abundant TrkA expression is associated with favorable prognosis of neuroblastoma. All-trans retinoic acid (ATRA), a first-line drug for acute promyelocytic leukemia (APL) treatment, has been shown to induce differentiation and inhibit cell growth. Neuroblastoma tissues in our hospital inpatient were collected, primary cell culture was performed, and the cells were separated and purified to be cell line. Trypan blue exclusion was used to count the numbers of cells alive, morphological changes were observed under the phase-contrast microscope. RT-PCR was used to determine the expression level of TrkA. In this study, a human neuroblastoma cell line was successfully established; in addition, we demonstrated that ATRA induces growth arrest and promotes the differentiation of neuroblastoma cells. In addition, ATRA was shown to significantly increase the levels of TrkA mRNA expression. Therefore, we concluded that the elevated expression of the TrkA receptor is associated with ATRA-induced growth arrest and differentiation o neuroblastoma cells. The results of this study provide a theoretical basis for the clinical application of differentiation-inducing ATRA for neuroblastoma therapy.

  7. An ester extract of Cochinchina momordica seeds induces differentiation of melanoma B16 F1 cells via MAPKs signaling.

    Science.gov (United States)

    Zhao, Lian-Mei; Han, Li-Na; Ren, Feng-Zhi; Chen, Shu-Hong; Liu, Li-Hua; Wang, Ming-Xia; Sang, Mei-Xiang; Shan, Bao-En

    2012-01-01

    Cochinchina momordica seeds (CMS) have been widely used due to antitumor activity by Mongolian tribes of China. However, the details of the underlying mechanisms remain unknown. In the present study, we found that an EtOAc (ethyl ester) extract of CMS (CMSEE) induced differentiation and caused growth inhibition of melanoma B16 F1 cells. CMSEE at the concentration of 5-200 μg/ml exhibited strongest anti-proliferative effects on B16 F1 cells among other CMS fractions (water or petroleum ether). Moreover, CMSEE induced melanoma B16 F1 cell differentiation, characterized by dendrite-like outgrowth, increasing melanogenesis production, as well as enhancing tyrosinase activity. Western blot analysis showed that sustained phosphorylation of p38 MAP accompanied by decrease in ERK1/2 and JNK dephosphorylation were involved in CMSEE-induced B16 F1 cell differentiation. Notably, 6 compounds that were isolated and identified may be responsible for inducing differentiation of CMSEE. These results indicated that CMSEE contributes to the differentiation of B16 F1 cells through modulating MAPKs activity, which may throw some light on the development of potentially therapeutic strategies for melanoma treatment.

  8. p53 is required for brown adipogenic differentiation and has a protective role against diet-induced obesity

    DEFF Research Database (Denmark)

    Molchadsky, A; Ezra, O; Amendola, P G

    2013-01-01

    that p53 is implicated in protection against diet-induced obesity. In striking contrast, our data shows that p53 exerts a positive regulatory effect on brown adipocyte differentiation. Abrogation of p53 function in skeletal muscle committed cells reduced their capacity to differentiate into brown...... adipocytes and histological analysis of brown adipose tissue revealed an impaired morphology in both embryonic and adult p53-null mice. Thus, depending on the specific adipogenic differentiation program, p53 may exert a positive or a negative effect. This cell type dependent regulation reflects an additional...

  9. Soluble Jagged 1/Fc chimera protein induces the differentiation and maturation of bone marrow-derived dendritic cells

    Institute of Scientific and Technical Information of China (English)

    XING FeiYue; LIU Jing; YU Zhe; JI YuHua

    2008-01-01

    A soluble Jagged 1/Fc chimera protein (Jagged 1/Fc) was directly used to induce differentiation and maturation of bone marrow-derived dendritic cells (DCs) in mice in vitro. A model of inducing and am-plifying DCs in vitro was established. The effect of Jagged 1/Fc on morphology of DCs induced by both rmGM-CSF and rmlL-4 was observed under a confocal microscope. A fluorescein-labeled monoclonal antibody staining combined with flow cytometry was applied to detect the effect of Jagged 1/Fc on the expression of CD11c, MHC-Ⅱ, CD86, CD80 and CD40 molecules on the surface of DCs. The results showed that Jagged 1/Fc did not affect the morphological properties of DC differentiation induced by both rmGM-CSF and rmlL-4. But it could promote the differentiation and maturation of DCs induced by both. The effect of it was strikingly different in the expression profile of co-stimulating molecules and the morphologic properties of DCs from lipopolysaccharide (LPS). The levels of MHC-Ⅱ and CD40 molecule expression on the surface of DCs stimulated by Jagged 1/Fc were significantly lower than those stimulated by LPS, and the level of CD80 expression on the surface of DCs induced by Jagged 1/Fc was near to that induced by LPS. Jagged 1/Fc had no influence on the expression of CD86 mole-cule on the surface of DCs. Jagged 1/Fc when used alone could not maintain the growth, differentiation and maturation of DCs. All the findings indicate that Jagged 1/Fc influences the differentiation and maturation of DCs, which is not markedly similar to LPS, providing important evidence for its devel-opment and application as a novel immunosuppressant.

  10. Lipopolysaccharide-activated microglial-induced neuroglial cell differentiation in bone marrow mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Xiaoguang Luo; Chunlin Ge; Yan Ren; Hongmei Yu; Zhe Wu; Qiushuang Wang; Chaodong Zhang

    2008-01-01

    BACKGROUND: Microglia are very sensitive to environmental changes, often becoming activated by pathological conditions. Activated microglia can exert a dual role in injury and repair in various diseases of the central nervous system, including cerebral ischemia, Parkinson's disease, and Alzheimer's disease. OBJECTIVE: An immortal microglial cell line, BV2, was treated with varying concentrations of lipopolysaccharide (LPS) to induce a pathological situation. Supernatant was harvested and incubated with bone marrow mesenchymal stem cells and, concomitantly, bone marrow mesenchymal stem cell differentiation was observed. DESIGN: A controlled observation, in vitro experiment. SETTING: Department of Neurology, First Affiliated Hospital of China Medical University. MATERIALS: Five male 2-3-week-old Sprague Dawley rats were purchased from Animal Laboratory Center of China Medical University and included in this study. The protocol was performed in accordance with ethical guidelines for the use and care of animals. The microglial cell line BV2 was produced by Cell Research Institute of Chinese Academy of Sciences. LPS was produced by Sigma Company, USA. METHODS: This study was performed in the Central Laboratory of China Medical University from September 2006 to March 2007. Rat femoral and tibial bone marrow was collected for separation and primary culture of bone marrow mesenchymal stem cells. Bone marrow mesenchymal stem cell cultures were divided into 5 groups: control group, non-activated group, as well as low-, medium-, and high-dose LPS groups. In the control group, bone marrow mesenchymal stem cells were cultured with Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovine serum (volume fraction 0.1). In the non-activated group, bone marrow mesenchymal stem cells were incubated with non-activated BV2 supernatant. In the low-, medium-, and high-dose LPS groups, bone marrow mesenchymal stem cells were incubated with LPS (0.01, 0.1 and 1

  11. Fluoxetine induces proliferation and inhibits differentiation of hypothalamic neuroprogenitor cells in vitro.

    Directory of Open Access Journals (Sweden)

    Lígia Sousa-Ferreira

    Full Text Available A significant number of children undergo maternal exposure to antidepressants and they often present low birth weight. Therefore, it is important to understand how selective serotonin reuptake inhibitors (SSRIs affect the development of the hypothalamus, the key center for metabolism regulation. In this study we investigated the proliferative actions of fluoxetine in fetal hypothalamic neuroprogenitor cells and demonstrate that fluoxetine induces the proliferation of these cells, as shown by increased neurospheres size and number of proliferative cells (Ki-67+ cells. Moreover, fluoxetine inhibits the differentiation of hypothalamic neuroprogenitor cells, as demonstrated by decreased number of mature neurons (Neu-N+ cells and increased number of undifferentiated cells (SOX-2+ cells. Additionally, fluoxetine-induced proliferation and maintenance of hypothalamic neuroprogenitor cells leads to changes in the mRNA levels of appetite regulator neuropeptides, including Neuropeptide Y (NPY and Cocaine-and-Amphetamine-Regulated-Transcript (CART. This study provides the first evidence that SSRIs affect the development of hypothalamic neuroprogenitor cells in vitro with consequent alterations on appetite neuropeptides.

  12. Nanopit-induced osteoprogenitor cell differentiation: The effect of nanopit depth

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    Martin J Davison

    2016-05-01

    Full Text Available We aimed to assess osteogenesis in osteoprogenitor cells by nanopits and to assess optimal feature depth. Topographies of depth 80, 220 and 333 nm were embossed onto polycaprolactone discs. Bone marrow–derived mesenchymal stromal cells were seeded onto polycaprolactone discs, suspended in media and incubated. Samples were fixed after 3 and 28 days. Cells were stained for the adhesion molecule vinculin and the osteogenic transcription factor RUNX2 after 3 days. Adhesion was lowest on planar controls and it was the shallowest, and 80-nm-deep pits supported optimal adhesion formation. Deep pits (80 and 220 nm induced most RUNX2 accumulation. After 28 days, osteocalcin and osteopontin expression were used as markers of osteoblastic differentiation. Deep pits (220 nm produced cells with the highest concentrations of osteopontin and osteocalcin. All topographies induced higher expression levels than controls. We demonstrated stimulation of osteogenesis in a heterogeneous population of mesenchymal stromal cells. All nanopit depths gave promising results with an optimum depth of 220 nm after 28 days. Nanoscale modification of implant surfaces could optimise fracture union or osteointegration.

  13. Half-filled energy bands induced negative differential resistance in nitrogen-doped graphene.

    Science.gov (United States)

    Li, Xiao-Fei; Lian, Ke-Yan; Qiu, Qi; Luo, Yi

    2015-03-07

    Nitrogen-doping brings novel properties and promising applications into graphene, but the underlying mechanism is still in debate. To determine the key factor in motivating the negative differential resistance (NDR) behaviour of nitrogen-doped graphene, the electronic structure and transport properties of an 11-dimer wide nitrogen-doped armchair graphene nanoribbon (N-AGNR) were systematically studied by first principles calculations. Both the effect of interaction between N-dopants and the effect of doping-sublattice on the NDR were examined for the first time. Taking into account the two effects, N-AGNR becomes metallic or semiconducting depending on the doping configuration, and its Fermi level varies in a large range. NDR was firmly verified not to be intrinsic for N-AGNRs. However, it is totally determined by whether nitrogen-doping induces half-filled energy bands (HFEBs) because it is HFEBs that cross the Fermi level and determine the transport properties of N-AGNR under low biases. With the bias increasing, the transmission spectrum near the Fermi level showed a flag shape, and therefore, the corresponding transport channel is totally suppressed at a certain bias, resulting in the NDR behaviour with a configuration-dependent peak-to-valley current ratio (PVCR) up to 10(4). Our findings give new insights into the microscopic mechanism of chemical doping induced NDR behaviour and will be useful in building NDR-based nanodevices in the future.

  14. Antidepressant imipramine induces human astrocytes to differentiate into cells with neuronal phenotype.

    Science.gov (United States)

    Cabras, Stefano; Saba, Francesca; Reali, Camilla; Scorciapino, Maria Laura; Sirigu, Annarita; Talani, Giuseppe; Biggio, Giovanni; Sogos, Valeria

    2010-06-01

    Several recent studies have expanded our conception of the role of astrocytes in neurogenesis, proposing that these cells may contribute to this phenomenon not only as a source of trophic substances, but also as stem cells themselves. We recently observed in vitro that human mature astrocytes can be induced to differentiate into cells with a neuronal phenotype. Antidepressant drugs have been shown to increase neurogenesis in the adult rodent hippocampus. In order to better understand the role of astroglia in antidepressant-induced neurogenesis, primary astrocyte cultures were treated with the antidepressant imipramine. Cell morphology was rapidly modified by treatment. In fact, whereas untreated astrocytes showed large, flat morphology, after a few hours of treatment cells exhibited a round-shaped cell body with long, thin processes. The expression of neuronal markers was analysed by immunocytochemistry, Western Blot and RT-PCR at different treatment times. Results showed an increase in neuronal markers such as neurofilament and neuron-specific enolase (NSE), whereas glial fibrillary acidic protein (GFAP) and nestin expression were not significantly modified by treatment. Similar results were obtained with fluoxetine and venlafaxine. Hes1 mRNA significantly increased after 2 h of treatment, suggesting involvement of this transcription factor in this process. These results confirm the role of astrocytes in neurogenesis and suggest that these cells may represent one of the targets of antidepressants.

  15. Differential expression of ETS family transcription factors in NCCIT human embryonic carcinoma cells upon retinoic acid-induced differentiation.

    Science.gov (United States)

    Park, Sung-Won; Do, Hyun-Jin; Ha, Woo Tae; Han, Mi-Hee; Song, Hyuk; Uhm, Sang-Jun; Chung, Hak-Jae; Kim, Jae-Hwan

    2014-01-01

    E26 transformation-specific (ETS) transcription factors play important roles in normal and tumorigenic processes during development, differentiation, homeostasis, proliferation, and apoptosis. To identify critical ETS factor(s) in germ cell-derived cancer cells, we examined the expression patterns of the 27 ETS transcription factors in naive and differentiated NCCIT human embryonic carcinoma cells, which exhibit both pluripotent and tumorigenic characteristics. Overall, expression of ETS factors was relatively low in NCCIT cells. Among the 27 ETS factors, polyomavirus enhancer activator 3 (PEA3) and epithelium-specific ETS transcription factor-1 (ESE-1) exhibited the most significant changes in their expression levels. Western blot analysis confirmed these patterns, revealing reduced levels of PEA3 protein and elevated levels of ESE-1 protein in differentiated cells. PEA3 increased the proportion of cells in S-phase and promoted cell growth, whereas ESE-1 reduced proliferation potential. These data suggest that PEA3 and ESE-1 may play important roles in pluripotent and tumorigenic embryonic carcinoma cells. These findings contribute to our understanding of the functions of oncogenic ETS factors in germ cell-derived stem cells during processes related to tumorigenesis and pluripotency.

  16. Enteroendocrine cells, stem cells and differentiation progenitors in rats with TNBS-induced colitis.

    Science.gov (United States)

    El-Salhy, Magdy; Mazzawi, Tarek; Umezawa, Kazuo; Gilja, Odd Helge

    2016-12-01

    Patients with inflammatory bowel disease (IBD), as well as animal models of human IBD have abnormal enteroendocrine cells. The present study aimed to identify the possible mechanisms underlying these abnormalities. For this purpose, 40 male Wistar rats were divided into 4 groups as follows: the control group, the group with trinitrobenzene sulfonic acid (TNBS)-induced colitis with no treatment (TNBS group), the group with TNBS-induced colitis treated with 3-[(dodecylthiocarbonyl)-methyl]-glutarimide (DTCM-G; an activator protein-1 inhibitor) (DTCM-G group), and the group with TNBS-induced colitis treated with dehydroxymethylepoxyquinomicin (DHMEQ; a nuclear factor-κB inhibitor) treatment (DHMEQ group). Three days following the administration of TNBS, the rats were treated as follows: those in the control and TNBS groups received 0.5 ml of the vehicle [0.5% carboxymethyl cellulose (CMC)], those in the DTCM-G group received DTCM-G at 20 mg/kg body weight in 0.5% CMC, and those in the DHMEQ group received DHMEQ at 15 mg/kg body weight in 0.5% CMC. All injections were administered intraperitoneally twice daily for 5 days. The rats were then sacrificed, and tissue samples were taken from the colon. The tissue sections were stained with hemotoxylin-eosin and immunostained for chromogranin A (CgA), serotonin, peptide YY (PYY), oxyntomodulin, pancreatic polypeptide (PP), somatostatin, Musashi1 (Msi1), Math1, Neurogenin3 (Neurog3) and NeuroD1. The staining was quantified using image analysis software. The densities of CgA-, PYY-, PP-, Msi1-, Neurog3- and NeuroD1-positive cells were significantly lower in the TNBS group than those in the control group, while those of serotonin-, oxyntomodulin- and somatostatin-positive cells were significantly higher in the TNBS group than those in the control group. Treatment with either DTCM-G or DHMEQ restored the densities of enteroendocrine cells, stem cells and their progenitors to normal levels. It was thus

  17. The differentiation inducer, dimethyl sulfoxide, transiently increases the intracellular calcium ion concentration in various cell types.

    Science.gov (United States)

    Morley, P; Whitfield, J F

    1993-08-01

    Dimethyl sulfoxide (DMSO) initiates a coordinated differentiation program in various cell types but the mechanism(s) by which DMSO does this is not understood. In this study, the effect of DMSO on intracellular calcium ion concentration ([Ca2+]i) was determined in primary cultures of chicken ovarian granulosa cells from the two largest preovulatory follicles of laying hens, and in three cell lines: undifferentiated P19 embryonal carcinoma cells, 3T3-L1 fibroblasts, and Friend murine erythroleukemia (MEL) cells. [Ca2+]i was measured in cells loaded with the Ca(2+)-specific fluoroprobe Fura-2. There was an immediate (i.e., within 5 sec), transient, two to sixfold increase in [Ca2+]i after exposing all cell types to 1% DMSO. DMSO was effective between 0.2 and 1%. The prompt DMSO-induced [Ca2+]i spike in all of the cell types was not prevented by incubating the cells in Ca(2+)-free medium containing 2 mM EGTA or by pretreating them with the Ca(2+)-channel blockers methoxyverapamil (D600; 100 microM), nifedipine (20 microM), or cobalt (5 mM). However, when granulosa cells, 3T3-L1 cells, or MEL cells were pretreated with lanthanum (La3+; 1 mM), which blocks both Ca2+ channels and membrane Ca2+ pumps, there was a sustained increase in [Ca2+]i in response to 1% DMSO. By contrast, pretreating P19 cells with La3+ (1 mM) did not prolong the DMSO-triggered [Ca2+]i transient. In all cases, the DMSO-induced [Ca2+]i surge was unaffected by pretreating the cells with the inhibitors of inositol phospholipid hydrolysis, neomycin (1.5 mM) or U-73, 122 (2.5 microM). These results suggest that DMSO almost instantaneously triggers the release of Ca2+ from intracellular stores through a common mechanism in cells in primary cultures and in cells of a variety of established lines, but this release is not mediated through phosphoinositide breakdown. This large, DMSO-induced Ca2+ spike may play a role in the induction of cell differentiation by DMSO.

  18. Lapatinib induces autophagic cell death and differentiation in acute myeloblastic leukemia

    Directory of Open Access Journals (Sweden)

    Chen YJ

    2016-07-01

    Full Text Available Yu-Jen Chen,1–4 Li-Wen Fang,5 Wen-Chi Su,6,7 Wen-Yi Hsu,1 Kai-Chien Yang,1 Huey-Lan Huang8 1Department of Medical Research, 2Department of Radiation Oncology, Mackay Memorial Hospital, 3Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, 4Institute of Pharmacology, Taipei Medical University, Taipei, 5Department of Nutrition, I-Shou University, Kaohsiung, 6Research Center for Emerging Viruses, China Medical University Hospital, 7Graduate Institute of Clinical Medical Science, China Medical University, Taichung, 8Department of Bioscience Technology, College of Health Science, Chang Jung Christian University, Tainan, Taiwan, Republic of China Abstract: Lapatinib is an oral-form dual tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR or ErbB/Her superfamily members with anticancer activity. In this study, we examined the effects and mechanism of action of lapatinib on several human leukemia cells lines, including acute myeloid leukemia (AML, chronic myeloid leukemia (CML, and acute lymphoblastic leukemia (ALL cells. We found that lapatinib inhibited the growth of human AML U937, HL-60, NB4, CML KU812, MEG-01, and ALL Jurkat T cells. Among these leukemia cell lines, lapatinib induced apoptosis in HL-60, NB4, and Jurkat cells, but induced nonapoptotic cell death in U937, K562, and MEG-01 cells. Moreover, lapatinib treatment caused autophagic cell death as shown by positive acridine orange staining, the massive formation of vacuoles as seen by electronic microscopy, and the upregulation of LC3-II, ATG5, and ATG7 in AML U937 cells. Furthermore, autophagy inhibitor 3-methyladenine and knockdown of ATG5, ATG7, and Beclin-1 using short hairpin RNA (shRNA partially rescued lapatinib-induced cell death. In addition, the induction of phagocytosis and ROS production as well as the upregulation of surface markers CD14 and CD68 was detected in lapatinib-treated U937 cells, suggesting the induction of

  19. Rhipicephalus microplus salivary gland molecules induce differential CD86 expression in murine macrophages

    Directory of Open Access Journals (Sweden)

    Tidwell Jason P

    2010-11-01

    Full Text Available Abstract Background Tick parasitism is a major impediment for cattle production in many parts of the world. The southern cattle tick, Rhipicephalus (Boophilus microplus, is an obligate hematophagous parasite of domestic and wild animals that serves as vector of infectious agents lethal to cattle. Tick saliva contains molecules evolved to modulate host innate and adaptive immune responses which facilitates blood feeding and pathogen transmission. Tick feeding promotes CD4 T cell polarization to a Th2 profile usually accompanied by down-regulation of Th1 cytokines through as yet undefined mechanisms. Co-stimulatory molecules on antigen presenting cells are central to development of T cell responses including Th1 and Th2 responses. Tick induced changes to antigen presenting cell signal transduction pathways are largely unknown. Here we document the ability of R. microplus salivary gland extracts (SGE to effect differential CD86 expression. Results We examined changes in co-stimulatory molecule expression in murine RAW 264.7 cells in response to R. microplus SGE exposure in the presence of the toll-like receptor 4 (TLR4 ligand, LPS. After 24 hrs, CD86, but not CD80, was preferentially up-regulated on mouse macrophage RAW 264.7 cells when treated with SGE and then LPS, but not SGE alone. CD80 and CD40 expression was increased with LPS, but the addition of SGE did not alter expression. Higher concentrations of SGE were less effective at increasing CD86 RNA expression. The addition of mitogen or extracellular kinase (MEK inhibitor, PD98059, significantly reduced the ability for SGE to induce CD86 expression, indicating activation of MEK is necessary for SGE induced up-regulation. Conclusions Molecules in SGE of R. microplus have a concentration-dependent effect on differential up-regulation of CD86 in a macrophage cell line activated by the TLR4 ligand, LPS. This CD86 up-regulation is at least partially dependent on the ERK1/2 pathway and may serve to

  20. Effect of pirfenidone on proliferation, TGF-β-induced myofibroblast differentiation and fibrogenic activity of primary human lung fibroblasts.

    Science.gov (United States)

    Conte, Enrico; Gili, Elisa; Fagone, Evelina; Fruciano, Mary; Iemmolo, Maria; Vancheri, Carlo

    2014-07-16

    Pirfenidone is an orally active small molecule that has been shown to inhibit the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis. Although pirfenidone exhibits well documented antifibrotic and antiinflammatory activities, in vitro and in vivo, its molecular targets and mechanisms of action have not been elucidated. In this study, we investigated the effects of pirfenidone on proliferation, TGF-β-induced differentiation and fibrogenic activity of primary human lung fibroblasts (HLFs). Pirfenidone reduced fibroblast proliferation and attenuated TGF-β-induced α-smooth muscle actin (SMA) and pro-collagen (Col)-I mRNA and protein levels. Importantly, pirfenidone inhibited TGF-β-induced phosphorylation of Smad3, p38, and Akt, key factors in the TGF-β pathway. Together, these results demonstrate that pirfenidone modulates HLF proliferation and TGF-β-mediated differentiation into myofibroblasts by attenuating key TGF-β-induced signaling pathways.

  1. Ascorbic acid enhances the cardiac differentiation of induced pluripotent stem cells through promoting the proliferation of cardiac progenitor cells

    Institute of Scientific and Technical Information of China (English)

    Nan Cao; Bin Wei; Liu Wang; Ying Jin; Huang-Tian Yang; Zumei Liu; Zhongyan Chen; Jia Wang; Taotao Chen; Xiaoyang Zhao; Yu Ma; Lianju Qin; Jiuhong Kang

    2012-01-01

    Generation of induced pluripotent stem cells (iPSCs) has opened new avenues for the investigation of heart diseases,drug screening and potential autologous cardiac regeneration.However,their application is hampered by inefficient cardiac differentiation,high interline variability,and poor maturation of iPSC-derived cardiomyoeytes (iPS-CMs).To identify efficient inducers for cardiac differentiation and maturation of iPSCs and elucidate the mechanisms,we systematically screened sixteen cardiomyocyte inducers on various murine (m) iPSCs and found that only ascorbic acid (AA) consistently and robustly enhanced the cardiac differentiation of eleven lines including eight without spontaneous cardiogenic potential.We then optimized the treatment conditions and demonstrated that differentiation day 2-6,a period for the specification of cardiac progenitor cells (CPCs),was a critical time for AA to take effect.This was further confirmed by the fact that AA increased the expression of cardiovascular but not mesodermal markers.Noteworthily,AA treatment led to approximately 7.3-fold (miPSCs) and 30.2-fold (human iPSCs) augment in the yield of iPS-CMs.Such effect was attributed to a specific increase in the proliferation of CPCs via the MEK-ERK1/2 pathway by promoting collagen synthesis.In addition,AA-induced cardiomyocytes showed better sareomerie organization and enhanced responses of action potentials and calcium transients to β-adrenergic and muscarinic stimulations.These findings demonstrate that AA is a suitable cardiomyocyte inducer for iPSCs to improve cardiac differentiation and maturation simply,universally,and efficiently.These findings also highlight the importance of stimulating CPC proliferation by manipulating extracellular microenvironment in guiding cardiac differentiation of the pluripotent stem cells.

  2. Estrogen deficiency induces the differentiation of IL-17 secreting Th17 cells: a new candidate in the pathogenesis of osteoporosis.

    Directory of Open Access Journals (Sweden)

    Abdul M Tyagi

    Full Text Available Th17 cells produce IL-17, and the latter promotes bone loss in collagen-induced arthritis in mice. Blocking IL-17 action in mouse model of rheumatoid arthritis reduces disease symptoms. These observations suggest that Th17 cells may be involved in the pathogenesis of bone loss. However, the role of Th17 cell in estrogen (E2 deficiency-induced bone loss is still not very clear. We investigated the effect of E2 on Th17 differentiation in vivo and IL-17 mediated regulation of osteoclast and osteoblast differentiation. Additionally, effect of IL-17 functional block under E2 deficiency-induced bone loss was studied. In murine bone marrow cells, E2 suppressed IL-17 mediated osteoclast differentiation. IL-17 inhibited formation of mineralized nodules in osteoblasts and this effect was suppressed by E2. E2 treatment to mouse calvarial osteoblasts inhibited the IL-17-induced production of osteoclastogenic cytokines and NF-kB translocation. In ovariectomized mice, there was increase in the number of Th17 cells, transcription factors promoting Th17 cell differentiation and circulating IL-17 levels. These effects were reversed by E2 supplementation. Treatment of neutralizing IL-17 monoclonal antibody to Ovx mice mitigated the E2 deficiency-induced trabecular bone loss and reversed the decreased osteoprotegerin-to-receptor activator of nuclear factor kappa B ligand (RANKL transcript levels in long bones, increased osteoclast differentiation from the bone marrow precursor cells and decreased osteoblast differentiation from the bone marrow stromal cells. Our findings indicate that E2 deficiency leads to increased differentiation of Th17 cells with attendant up regulation of STAT3, ROR-γt and ROR-α and downregulation of Foxp3 which antagonizes Th17 cell differentiation. Increased IL-17 production in turn induces bone loss by increasing pro-osteoclastogenic cytokines including TNF-α, IL-6 and RANKL from osteoblasts and functional block of IL-17 prevents bone

  3. ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Xinhua [Department of Spine Surgery, Xiangya Hospital of Central South University, Changsha (China); Wang, Xiaoyuan [Department of Nephrology, Xi An Honghui Hospital, Xi an (China); Hu, Xiongke; Chen, Yong; Zeng, Kefeng [Department of Spine Surgery, Xiangya Hospital of Central South University, Changsha (China); Zhang, Hongqi, E-mail: zhq9699@126.com [Department of Spine Surgery, Xiangya Hospital of Central South University, Changsha (China)

    2015-07-01

    Although 17β-estradial (E2) is known to stimulate bone formation, the underlying mechanisms are not fully understood. Recent studies have implicated the Wnt/β-catenin pathway as a major signaling cascade in bone biology. The interactions between Wnt/β-catenin signaling pathway and estrogen signaling pathways have been reported in many tissues. In this study, E2 significantly increased the expression of β-catenin by inducing phosphorylations of GSK3β at serine 9. ERβ siRNAs were transfected into MC3T3-E1 cells and revealed that ERβ involved E2-induced osteoblasts proliferation and differentiation via Wnt/β-catenin signaling. The osteoblast differentiation genes (BGP, ALP and OPN) and proliferation related gene (cyclin D1) expression were significantly induced by E2-mediated ERβ. Furthermore immunofluorescence and immunoprecipitation analysis demonstrated that E2 induced the accumulation of β-catenin protein in the nucleus which leads to interaction with T-cell-specific transcription factor/lymphoid enhancer binding factor (TCF/LEF) transcription factors. Taken together, these findings suggest that E2 promotes osteoblastic proliferation and differentiation by inducing proliferation-related and differentiation-related gene expression via ERβ/GSK-3β-dependent Wnt/β-catenin signaling pathway. Our findings provide novel insights into the mechanisms of action of E2 in osteoblastogenesis. - Highlights: • 17β-estradial (E2) promotes GSK3-β phosphorylation. • E2 activates the Wnt/β-catenin signaling pathway. • The Wnt/β-catenin signaling pathway interacts with estrogen signaling pathways. • E2-mediated ER induced osteoblast differentiation and proliferation related genes expression.

  4. Taurine Protected Against the Impairments of Neural Stem Cell Differentiated Neurons Induced by Oxygen-Glucose Deprivation.

    Science.gov (United States)

    Xiao, Bo; Liu, Huazhen; Gu, Zeyun; Liu, Sining; Ji, Cheng

    2015-11-01

    Cell transplantation of neural stem cells (NSCs) is a promising approach for neurological recovery both structurally and functionally. However, one big obstacle is to promote differentiation of NSCs into neurons and the followed maturation. In the present study, we aimed to investigate the protective effect of taurine on the differentiation of NSCs and subsequent maturation of their neuronal lineage, when exposed to oxygen-glucose deprivation (OGD). The results suggested that taurine (5-20 mM) promoted the viability and proliferation of NSCs, and it protected against 8 h of OGD induced impairments. Furthermore, 20 mM taurine promoted NSCs to differentiate into neurons after 7 days of culture, and it also protected against the suppressive impairments of 8 h of OGD. Consistently, taurine (20 mM) promoted the neurite sprouting and outgrowth of the NSC differentiated neurons after 14 days of differentiation, which were significantly inhibited by OGD (8 h). At D21, the mushroom spines and spine density were promoted or restored by 20 mM taurine. Taken together, the enhanced viability and proliferation of NSCs, more differentiated neurons and the promoted maturation of neurons by 20 mM taurine support its therapeutic application during stem cell therapy to enhance neurological recovery. Moreover, it protected against the impairments induced by OGD, which may highlight its role for a more direct therapeutic application especially in an ischemic stroke environment.

  5. Development of a rapid culture method to induce adipocyte differentiation of human bone marrow-derived mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Ninomiya, Yuichi [Translational Research Center, Saitama International Medical, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298 (Japan); Sugahara-Yamashita, Yzumi; Nakachi, Yutaka; Tokuzawa, Yoshimi; Okazaki, Yasushi [Division of Functional Genomics and Systems Medicine, Research Center for Genomic Medicine, Saitama Medical University, Saitama 350-1241 (Japan); Nishiyama, Masahiko, E-mail: yamacho@saitama-med.ac.jp [Translational Research Center, Saitama International Medical, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298 (Japan)

    2010-04-02

    Human mesenchymal stem cells (hMSCs) derived from bone marrow are multipotent stem cells that can regenerate mesenchymal tissues such as adipose, bone or muscle. It is thought that hMSCs can be utilized as a cell resource for tissue engineering and as human models to study cell differentiation mechanisms, such as adipogenesis, osteoblastogenesis and so on. Since it takes 2-3 weeks for hMSCs to differentiate into adipocytes using conventional culture methods, the development of methods to induce faster differentiation into adipocytes is required. In this study we optimized the culture conditions for adipocyte induction to achieve a shorter cultivation time for the induction of adipocyte differentiation in bone marrow-derived hMSCs. Briefly, we used a cocktail of dexamethasone, insulin, methylisobutylxanthine (DIM) plus a peroxisome proliferator-activated receptor {gamma} agonist, rosiglitazone (DIMRo) as a new adipogenic differentiation medium. We successfully shortened the period of cultivation to 7-8 days from 2-3 weeks. We also found that rosiglitazone alone was unable to induce adipocyte differentiation from hMSCs in vitro. However, rosiglitazone appears to enhance hMSC adipogenesis in the presence of other hormones and/or compounds, such as DIM. Furthermore, the inhibitory activity of TGF-{beta}1 on adipogenesis could be investigated using DIMRo-treated hMSCs. We conclude that our rapid new culture method is very useful in measuring the effect of molecules that affect adipogenesis in hMSCs.

  6. Sensitivity of nuclear c-myc levels and induction to differentiation-inducing agents in human colon tumor cell lines.

    Science.gov (United States)

    Taylor, C W; Kim, Y S; Childress-Fields, K E; Yeoman, L C

    1992-02-29

    Six human colon tumor cell lines were analyzed for their constitutive levels of the c-myc protein. The nuclear proto-oncogene, c-myc, was detected as an expressed product in all of the human colon tumor cell lines analyzed. The poorly differentiated cell lines HCT116, RKO and C showed c-myc levels that averaged 2-fold greater than their well-differentiated counterparts, i.e., GEO, CBS and FET. When c-myc levels and responses to serum induction were analyzed in the presence of inducers of differentiation, i.e., dimethylformamide, retinoic acid, sodium butyrate and TGF-beta, distinct patterns of sensitivity and resistance emerged. Nuclear c-myc levels were reduced in all the colon cell phenotypes treated with dimethylformamide or sodium butyrate. Only the well-differentiated human colon tumor cell lines were responsive to transforming growth factor-beta. Only one of the human colon tumor cell lines (GEO) responded to retinoic acid. Increased levels of c-myc protein were found to correlate well with greater growth rates and with poor differentiation class. Similarly, a parallel sensitivity to down-regulation of c-myc levels and attenuation of c-myc induction curves for inducers of differentiation were observed in growth sensitive human colon tumor cell lines.

  7. Inhibitory effect of leptin on rosiglitazone-induced differentiation of primary adipocytes prepared from TallyHO/Jng mice

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ki Young; Kim, Joo Young; Sung, Yoon-Young; Jung, Won Hoon; Kim, Hee-Youn; Park, Ji Seon; Cheon, Hyae Gyeong [Medicinal Science Division, Korea Research Institute of Chemical Technology, 100 Jang-dong, Yuseong, 305-600 Daejon (Korea, Republic of); Rhee, Sang Dal, E-mail: sdrhee@krict.re.kr [Medicinal Science Division, Korea Research Institute of Chemical Technology, 100 Jang-dong, Yuseong, 305-600 Daejon (Korea, Republic of)

    2011-03-25

    Research highlights: {yields} In this study, we investigated the effects of leptin on adipocyte differentiation prepared from subcutaneous fat of TallyHo mice. {yields} Leptin inhibited the adipocytes differentiation at physiological concentration via inhibition of PPAR{gamma} expression. {yields} Inhibitors of ERK and STAT1 restored the leptin's inhibitory activity both in vitro and in vivo. -- Abstract: The effects of leptin on rosiglitazone-induced adipocyte differentiation were investigated in the primary adipocytes prepared from subcutaneous fat of TallyHO/Jng (TallyHO) mouse, a recently developed model animal for type 2 diabetes mellitus (T2DM). The treatment of leptin inhibited the rosiglitazone-induced adipocyte differentiation with a decreased expression of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) a key adipogenic transcription factor, both in mRNA and protein levels. Leptin (10 nM) was sufficient to inhibit the adipocyte differentiation, which seemed to come from increased expression of leptin receptor genes in the fat of TallyHO mice. The inhibition of adipogenesis by leptin was restored by the treatment of inhibitors for extracellular-signal-regulated kinase (ERK) (PD98059) and signal transducer and activator of transcription-1 (STAT1) (fludarabine). Furthermore, in vivo intraperitoneal administration of PD98059 and fludarabine increased the PPAR{gamma} expression in the subcutaneous fat of TallyHO mice. These data suggest that leptin could inhibit the PPAR{gamma} expression and adipocyte differentiation in its physiological concentration in TallyHO mice.

  8. Social defeat during adolescence and adulthood differentially induce BDNF-regulated immediate early genes

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    Caroline M. Coppens

    2011-11-01

    Full Text Available Stressful life events generally enhance the vulnerability for the development of human psychopathologies such as anxiety disorders and depression. The incidence rates of adult mental disorders steeply rises during adolescence in parallel with a structural and functional reorganization of the neural circuitry underlying stress reactivity. However, the mechanisms underlying susceptibility to stress and manifestation of mental disorders during adolescence are little understood. We hypothesized that heightened sensitivity to stress during adolescence reflects age-dependent differences in the expression of activity-dependent genes involved in synaptic plasticity. Therefore, we compared the effect of social stress during adolescence with social stress in adulthood on the expression of a panel of genes linked to induction of long-term potentiation (LTP and brain-derived neurotrophic factor (BDNF signaling. We show that social defeat during adolescence and adulthood differentially regulates expression of the immediate early genes BDNF, Arc, Carp, and Tieg1, as measured by qPCR in tissue lysates from prefrontal cortex, nucleus accumbens, and hippocampus. In the hippocampus, mRNA levels for all four genes were robustly elevated following social defeat in adolescence, whereas none were induced by defeat in adulthood. The relationship to coping style was also examined using adult reactive and proactive coping rats. Gene expression levels of reactive and proactive animals were similar in the prefrontal cortex and hippocampus. However, a trend toward a differential expression of BDNF and Arc mRNA in the nucleus accumbens was detected. BDNF mRNA was increased in the nucleus accumbens of proactive defeated animals, whereas the expression level in reactive defeated animals was comparable to control animals. The results demonstrate striking differences in immediate early gene expression in response to social defeat in adolescent and adult rats.

  9. Different types of exercise induce differential effects on neuronal adaptations and memory performance.

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    Lin, Tzu-Wei; Chen, Shean-Jen; Huang, Tung-Yi; Chang, Chia-Yuan; Chuang, Jih-Ing; Wu, Fong-Sen; Kuo, Yu-Min; Jen, Chauying J

    2012-01-01

    Different exercise paradigms show differential effects on various forms of memory. We hypothesize that the differential effects of exercises on memory performance are caused by different neuroplasticity changes in relevant brain regions in response to different exercise trainings. We examined the effects of treadmill running (TR) and wheel running (WR) on the Pavlovian fear conditioning task that assesses learning and memory performance associated with the amygdala (cued conditioning) and both the amygdala and hippocampus (contextual conditioning). The skeletal muscle citrate synthase activity, an indicator of aerobic capacity, was elevated in rats received 4 w of TR, but not WR. While both TR and WR elevated the contextual conditional response, only TR facilitated the cued conditional response. Using a single-neuron labeling technique, we found that while both TR and MR enlarged the dendritic field and increased the spine density in hippocampal CA3 neurons, only TR showed these effects in basolateral amygdalar neurons. Moreover, both types of exercise upregulated synaptic proteins (i.e., TrkB and SNAP-25) in the hippocampus; however only TR showed similar effects in the amygdala. Injection of K252a, a TrkB kinase inhibitor, in the dorsal hippocampus or basolateral amygdala abolished the exercise-facilitated contextual or cued fear learning and memory performance, respectively, regardless of the types of exercise. In summary, our results supported that different types of exercise affect the performance of learning and memory via BDNF-TrkB signaling and neuroplasticity in specific brain regions. The brain region-specific neuronal adaptations are possibly induced by various levels of intensity/stress elicited by different types of exercise. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Vorinostat induces apoptosis and differentiation in myeloid malignancies: genetic and molecular mechanisms.

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    Gabriela Silva

    Full Text Available BACKGROUND: Aberrant epigenetic patterns are central in the pathogenesis of haematopoietic diseases such as myelodysplastic syndromes (MDS and acute myeloid leukaemia (AML. Vorinostat is a HDACi which has produced responses in these disorders. The purpose of this study was to address the functional effects of vorinostat in leukemic cell lines and primary AML and MDS myeloid cells and to dissect the genetic and molecular mechanisms by which it exerts its action. METHODOLOGY/PRINCIPAL FINDINGS: Functional assays showed vorinostat promoted cell cycle arrest, inhibited growth, and induced apoptosis and differentiation of K562, HL60 and THP-1 and of CD33(+ cells from AML and MDS patients. To explore the genetic mechanism for these effects, we quantified gene expression modulation by vorinostat in these cells. Vorinostat increased expression of genes down-regulated in MDS and/or AML (cFOS, COX2, IER3, p15, RAI3 and suppressed expression of genes over-expressed in these malignancies (AXL, c-MYC, Cyclin D1 and modulated cell cycle and apoptosis genes in a manner which would favor cell cycle arrest, differentiation, and apoptosis of neoplastic cells, consistent with the functional assays. Reporter assays showed transcriptional effect of vorinostat on some of these genes was mediated by proximal promoter elements in GC-rich regions. Vorinostat-modulated expression of some genes was potentiated by mithramycin A, a compound that interferes with SP1 binding to GC-rich DNA sequences, and siRNA-mediated SP1 reduction. ChIP assays revealed vorinostat inhibited DNA binding of SP1 to the proximal promoter regions of these genes. These results suggest vorinostat transcriptional action in some genes is regulated by proximal promoter GC-rich DNA sequences and by SP1. CONCLUSION: This study sheds light on the effects of vorinostat in AML and MDS and supports the implementation of clinical trials to explore the use of vorinostat in the treatment of these diseases.

  11. Differential analysis of muscle fatigue induced elbow and wrist tremor in controlled laparoscopic manoeuvring.

    Science.gov (United States)

    Chandra, Sourav; Hayashibe, Mitsuhiro; Thondiyath, Asokan; Ramalingam, Manickam

    2017-09-01

    Fatigue induced hand tremor (FIT) is a primary limiting concern for the prolonged surgical intervention in minimally invasive surgery (MIS) and robot-assisted-minimally invasive surgery (RAMIS). A thorough analysis is necessary to understand the FIT characteristics in laparoscopic tool movement. The primary aim of this study is to perform a differential analysis of the elbow and wrist tremor due to muscle fatigue in laparoscopic manoeuvring. We have introduced a joint angle based tremor analysis method, which enables us to perform a differential study of FIT characteristics at the individual joint. Experimental data was acquired from a group of subjects during static and dynamic laparoscopic movement in an imitative RAMIS master manipulation scenario. A repetitive task was performed with a total span of 1 h for observing the effect of muscle fatigue. Along with the joint angle variation, surface electromyography (sEMG) signal was also studied in the analysis. The wrist tremor is more predominant than tremor generated at the elbow, especially in highly fatigued condition. The high-frequency tremor (>4 Hz) is contributed by the wrist joint. Moreover, the variation of the wrist and elbow tremor ratio was found to be dependent upon the experience of the surgeons. In this work, we have investigated the attribution of elbow and wrist joints in FIT during laparoscopic tool manipulation. The outcomes may be useful for the design of robot-assisted surgical manipulator, and can be used for quality assessment of surgical training as well. Copyright © 2016 John Wiley & Sons, Ltd.

  12. 25-Hydroxyvitamin D3 induces osteogenic differentiation of human mesenchymal stem cells

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    Lou, Yan-Ru; Toh, Tai Chong; Tee, Yee Han; Yu, Hanry

    2017-01-01

    25-Hydroxyvitamin D3 [25(OH)D3] has recently been found to be an active hormone. Its biological actions are demonstrated in various cell types. 25(OH)D3 deficiency results in failure in bone formation and skeletal deformation. Here, we investigated the effect of 25(OH)D3 on osteogenic differentiation of human mesenchymal stem cells (hMSCs). We also studied the effect of 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3], a metabolite of 25(OH)D3. One of the vitamin D responsive genes, 25(OH)D3-24-hydroxylase (cytochrome P450 family 24 subfamily A member 1) mRNA expression is up-regulated by 25(OH)D3 at 250–500 nM and by 1α,25-(OH)2D3 at 1–10 nM. 25(OH)D3 and 1α,25-(OH)2D3 at a time-dependent manner alter cell morphology towards osteoblast-associated characteristics. The osteogenic markers, alkaline phosphatase, secreted phosphoprotein 1 (osteopontin), and bone gamma-carboxyglutamate protein (osteocalcin) are increased by 25(OH)D3 and 1α,25-(OH)2D3 in a dose-dependent manner. Finally, mineralisation is significantly increased by 25(OH)D3 but not by 1α,25-(OH)2D3. Moreover, we found that hMSCs express very low level of 25(OH)D3-1α-hydroxylase (cytochrome P450 family 27 subfamily B member 1), and there is no detectable 1α,25-(OH)2D3 product. Taken together, our findings provide evidence that 25(OH)D3 at 250–500 nM can induce osteogenic differentiation and that 25(OH)D3 has great potential for cell-based bone tissue engineering. PMID:28211493

  13. Regional differentiation of retinoic acid-induced human pluripotent embryonic carcinoma stem cell neurons.

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    Dennis E Coyle

    Full Text Available The NTERA2 cl D1 (NT2 cell line, derived from human teratocarcinoma, exhibits similar properties as embryonic stem (ES cells or very early neuroepithelial progenitors. NT2 cells can be induced to become postmitotic central nervous system neurons (NT2N with retinoic acid. Although neurons derived from pluripotent cells, such as NT2N, have been characterized for their neurotransmitter phenotypes, their potential suitability as a donor source for neural transplantation also depends on their ability to respond to localized environmental cues from a specific region of the CNS. Therefore, our study aimed to characterize the regional transcription factors that define the rostocaudal and dorsoventral identity of NT2N derived from a monolayer differentiation paradigm using quantitative PCR (qPCR. Purified NT2N mainly expressed both GABAergic and glutamatergic phenotypes and were electrically active but did not form functional synapses. The presence of immature astrocytes and possible radial glial cells was noted. The NT2N expressed a regional transcription factor code consistent with forebrain, hindbrain and spinal cord neural progenitors but showed minimal expression of midbrain phenotypes. In the dorsoventral plane NT2N expressed both dorsal and ventral neural progenitors. Of major interest was that even under the influence of retinoic acid, a known caudalization factor, the NT2N population maintained a rostral phenotype subpopulation which expressed cortical regional transcription factors. It is proposed that understanding the regional differentiation bias of neurons derived from pluripotent stem cells will facilitate their successful integration into existing neuronal networks within the CNS.

  14. NGF induces adult stem Leydig cells to proliferate and differentiate during Leydig cell regeneration.

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    Zhang, Lei; Wang, Huaxi; Yang, Yan; Liu, Hui; Zhang, Qihao; Xiang, Qi; Ge, Renshan; Su, Zhijian; Huang, Yadong

    2013-06-28

    Nerve growth factor (NGF) has been reported to be involved in male reproductive physiology. However, few reports have described the activity of NGF during Leydig cell development. The objective of the present study was to examine the role of NGF during stem-Leydig-cell (SLC) regeneration. We investigated the effects of NGF on Leydig-cell (LC) regeneration by measuring mRNA levels in the adult rat testis after ethane dimethanesulfonate (EDS) treatment. Furthermore, we used the established organ culture model of rat seminiferous tubules to examine the regulation of NGF during SLC proliferation and differentiation using EdU staining, real-time PCR and western blotting. Progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs) were also used to investigate the effects of NGF on LCs at different developmental stages. NGF mRNA levels changed significantly during Leydig-cell regeneration in vivo. In vitro, NGF significantly promoted the proliferation of stem Leydig cells and also induced steroidogenic enzyme gene expression and 3β-HSD protein expression. The data from PLCs and ILCs showed that NGF could increase Cyclin D1 and Hsd 17b3 mRNA levels in PLCs and Cyclin D1 mRNA levels in ILCs. These results indicate that NGF may play an important role during LC regeneration by regulating the proliferation and differentiation of LCs at different developmental stages, from SLCs to PLCs and from PLCs to ILCs. The discovery of this effect of NGF on Leydig cells will provide useful information for developing new potential therapies for PADAM (Partial Androgen Deficiency in the Aging Male). Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Effect of angiotensin II on proliferation and differentiation of mouse induced pluripotent stem cells into mesodermal progenitor cells

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    Ishizuka, Toshiaki, E-mail: tishizu@ndmc.ac.jp [Department of Pharmacology, National Defense Medical College, Tokorozawa, Saitama 359-8513 (Japan); Goshima, Hazuki; Ozawa, Ayako; Watanabe, Yasuhiro [Department of Pharmacology, National Defense Medical College, Tokorozawa, Saitama 359-8513 (Japan)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Treatment with angiotensin II enhanced LIF-induced DNA synthesis of mouse iPS cells. Black-Right-Pointing-Pointer Angiotensin II may enhance the DNA synthesis via induction of superoxide. Black-Right-Pointing-Pointer Treatment with angiotensin II significantly increased JAK/STAT3 phosphorylation. Black-Right-Pointing-Pointer Angiotensin II enhanced differentiation into mesodermal progenitor cells. Black-Right-Pointing-Pointer Angiotensin II may enhance the differentiation via activation of p38 MAPK. -- Abstract: Previous studies suggest that angiotensin receptor stimulation may enhance not only proliferation but also differentiation of undifferentiated stem/progenitor cells. Therefore, in the present study, we determined the involvement of the angiotensin receptor in the proliferation and differentiation of mouse induced pluripotent stem (iPS) cells. Stimulation with angiotensin II (Ang II) significantly increased DNA synthesis in mouse iPS cells cultured in a medium with leukemia inhibitory factor (LIF). Pretreatment of the cells with either candesartan (a selective Ang II type 1 receptor [AT{sub 1}R] antagonist) or Tempol (a cell-permeable superoxide scavenger) significantly inhibited Ang II-induced DNA synthesis. Treatment with Ang II significantly increased JAK/STAT3 phosphorylation. Pretreatment with candesartan significantly inhibited Ang II- induced JAK/STAT3 phosphorylation. In contrast, induction of mouse iPS cell differentiation into Flk-1-positive mesodermal progenitor cells was performed in type IV collagen (Col IV)- coated dishes in a differentiation medium without LIF. When Col IV-exposed iPS cells were treated with Ang II for 5 days, the expression of Flk-1 was significantly increased compared with that in the cells treated with the vehicle alone. Pretreatment of the cells with both candesartan and SB203580 (a p38 MAPK inhibitor) significantly inhibited the Ang II- induced increase in Flk-1 expression

  16. Shockwaves induce osteogenic differentiation of human mesenchymal stem cells through ATP release and activation of P2X7 receptors.

    Science.gov (United States)

    Sun, Dahui; Junger, Wolfgang G; Yuan, Changji; Zhang, Wenyan; Bao, Yi; Qin, Daming; Wang, Chengxue; Tan, Lei; Qi, Baochang; Zhu, Dong; Zhang, Xizheng; Yu, Tiecheng

    2013-06-01

    Shockwave treatment promotes bone healing of nonunion fractures. In this study, we investigated whether this effect could be due to adenosine 5'-triphosphate (ATP) release-induced differentiation of human mesenchymal stem cells (hMSCs) into osteoprogenitor cells. Cultured bone marrow-derived hMSCs were subjected to shockwave treatment and ATP release was assessed. Osteogenic differentiation and mineralization of hMSCs were evaluated by examining alkaline phosphatase activity, osteocalcin production, and calcium nodule formation. Expression of P2X7 receptors and c-fos and c-jun mRNA was determined with real-time reverse transcription polymerase chain reaction and Western blotting. P2X7-siRNA, apyrase, P2 receptor antagonists, and p38 MAPK inhibitors were used to evaluate the roles of ATP release, P2X7 receptors, and p38 MAPK signaling in shockwave-induced osteogenic hMSCs differentiation. Shockwave treatment released significant amounts (≈ 7 μM) of ATP from hMSCs. Shockwaves and exogenous ATP induced c-fos and c-jun mRNA transcription, p38 MAPK activation, and hMSC differentiation. Removal of ATP with apyrase, targeting of P2X7 receptors with P2X7-siRNA or selective antagonists, or blockade of p38 MAPK with SB203580 prevented osteogenic differentiation of hMSCs. Our findings indicate that shockwaves release cellular ATP that activates P2X7 receptors and downstream signaling events that caused osteogenic differentiation of hMSCs. We conclude that shockwave therapy promotes bone healing through P2X7 receptor signaling, which contributes to hMSC differentiation.

  17. A PU.1 suppressive target gene, metallothionein 1G, inhibits retinoic acid-induced NB4 cell differentiation.

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    Naomi Hirako

    Full Text Available We recently revealed that myeloid master regulator SPI1/PU.1 directly represses metallothionein (MT 1G through its epigenetic activity of PU.1, but the functions of MT1G in myeloid differentiation remain unknown. To clarify this, we established MT1G-overexpressing acute promyelocytic leukemia NB4 (NB4MTOE cells, and investigated whether MT1G functionally contributes to all-trans retinoic acid (ATRA-induced NB4 cell differentiation. Real-time PCR analyses demonstrated that the inductions of CD11b and CD11c and reductions in myeloperoxidase and c-myc by ATRA were significantly attenuated in NB4MTOE cells. Morphological examination revealed that the percentages of differentiated cells induced by ATRA were reduced in NB4MTOE cells. Since G1 arrest is a hallmark of ATRA-induced NB4 cell differentiation, we observed a decrease in G1 accumulation, as well as decreases in p21WAF1/CIP1 and cyclin D1 inductions, by ATRA in NB4MTOE cells. Nitroblue tetrazolium (NBT reduction assays revealed that the proportions of NBT-positive cells were decreased in NB4MTOE cells in the presence of ATRA. Microarray analyses showed that the changes in expression of several myeloid differentiation-related genes (GATA2, azurocidin 1, pyrroline-5-carboxylate reductase 1, matrix metallopeptidase -8, S100 calcium-binding protein A12, neutrophil cytosolic factor 2 and oncostatin M induced by ATRA were disturbed in NB4MTOE cells. Collectively, overexpression of MT1G inhibits the proper differentiation of myeloid cells.

  18. Sound Waves Induce Neural Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells via Ryanodine Receptor-Induced Calcium Release and Pyk2 Activation.

    Science.gov (United States)

    Choi, Yura; Park, Jeong-Eun; Jeong, Jong Seob; Park, Jung-Keug; Kim, Jongpil; Jeon, Songhee

    2016-10-01

    Mesenchymal stem cells (MSCs) have shown considerable promise as an adaptable cell source for use in tissue engineering and other therapeutic applications. The aims of this study were to develop methods to test the hypothesis that human MSCs could be differentiated using sound wave stimulation alone and to find the underlying mechanism. Human bone marrow (hBM)-MSCs were stimulated with sound waves (1 kHz, 81 dB) for 7 days and the expression of neural markers were analyzed. Sound waves induced neural differentiation of hBM-MSC at 1 kHz and 81 dB but not at 1 kHz and 100 dB. To determine the signaling pathways involved in the neural differentiation of hBM-MSCs by sound wave stimulation, we examined the Pyk2 and CREB phosphorylation. Sound wave induced an increase in the phosphorylation of Pyk2 and CREB at 45 min and 90 min, respectively, in hBM-MSCs. To find out the upstream activator of Pyk2, we examined the intracellular calcium source that was released by sound wave stimulation. When we used ryanodine as a ryanodine receptor antagonist, sound wave-induced calcium release was suppressed. Moreover, pre-treatment with a Pyk2 inhibitor, PF431396, prevented the phosphorylation of Pyk2 and suppressed sound wave-induced neural differentiation in hBM-MSCs. These results suggest that specific sound wave stimulation could be used as a neural differentiation inducer of hBM-MSCs.

  19. Effect of IFN-γ and dexamethasone on TGF-β1-induced human fetal lung fibroblast-myofibroblast differentiation

    Institute of Scientific and Technical Information of China (English)

    LiGU; Yuan-jueZHU; Zi-jianGUO; Xing-xiangXU; Wen-bingXU

    2004-01-01

    AIM: To study whether Smads signaling pathway was involved in human fetal lung fibroblast-myofibroblast differentiation induced by transforming growth factor (TGF)-β1 and the role of interferon (IFN)-γ, dexamethasone (DEX) in the fibroblast-myofibroblast differentiation. METHODS: α-Smooth muscle actin (α-SMA), Smad2/3, and Smad7 protein were assessed by Western blot. Collagen protein was analyzed by measuring hydroxyproline, α-SMA and collagen III mRNA were assessed by RT-PCR. Myofibroblasts morphology and Smad2/3 nuclear translocation were assessed by immunohistochemistry. The overexpression of Smad7, a negative mediator of Smads signaling pathway, was acquired by transfection of Smad7 vector. RESULTS: During fibroblast-myofibroblast differentiation induced by TGF-β1, IFN-γ 200 μg/L markedly blocked TGF-β1-induced α-SMA protein expression (PO.05) and collagen protein (P>0.05) and mRNA expression (P>0.05) and did not change myofibroblasts morphology. Transient transfection of Smad7 vector resulted in significant inhibition of TGF-β1-induced α-SMA expression (P<0.01). IFN-γ 200 μg/L did not block TGF-β1-stimulated Smad2/3 phosphorylation and their nuclear translocation. CONCLUSION: TGF-131 induced fibroblastmyofibroblast differentiation in a Smad proteins-dependent manner. IFN-γ could block this process but it was not mediated by interrupting smad2/3 phosphorylation and their nuclear translocation and DEX played a synergism withTGF-β1. Differentiated myofibroblasts, however, were resistant to both IFN-γ and DEX.

  20. Proteomic identification of novel differentiation plasma protein markers in hypobaric hypoxia-induced rat model.

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    Yasmin Ahmad

    Full Text Available BACKGROUND: Hypobaric hypoxia causes complex changes in the expression of genes, including stress related genes and corresponding proteins that are necessary to maintain homeostasis. Whereas most prior studies focused on single proteins, newer methods allowing the simultaneous study of many proteins could lead to a better understanding of complex and dynamic changes that occur during the hypobaric hypoxia. METHODS: In this study we investigated the temporal plasma protein alterations of rat induced by hypobaric hypoxia at a simulated altitude of 7620 m (25,000 ft, 282 mm Hg in a hypobaric chamber. Total plasma proteins collected at different time points (0, 6, 12 and 24 h, separated by two-dimensional electrophoresis (2-DE and identified using matrix assisted laser desorption ionization time of flight (MALDI-TOF/TOF. Biological processes that were enriched in the plasma proteins during hypobaric hypoxia were identified using Gene Ontology (GO analysis. According to their properties and obvious alterations during hypobaric hypoxia, changes of plasma concentrations of Ttr, Prdx-2, Gpx -3, Apo A-I, Hp, Apo-E, Fetub and Nme were selected to be validated by Western blot analysis. RESULTS: Bioinformatics analysis of 25 differentially expressed proteins showed that 23 had corresponding candidates in the database. The expression patterns of the eight selected proteins observed by Western blot were in agreement with 2-DE results, thus confirming the reliability of the proteomic analysis. Most of the proteins identified are related to cellular defense mechanisms involving anti-inflammatory and antioxidant activity. Their presence reflects the consequence of serial cascades initiated by hypobaric hypoxia. CONCLUSION/SIGNIFICANCE: This study provides information about the plasma proteome changes induced in response to hypobaric hypoxia and thus identification of the candidate proteins which can act as novel biomarkers.

  1. Marek’s disease virus infection induces widespread differential chromatin marks in inbred chicken lines

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    Mitra Apratim

    2012-10-01

    Full Text Available Abstract Background Marek’s disease (MD is a neoplastic disease in chickens caused by the MD virus (MDV. Successful vaccine development against MD has resulted in increased virulence of MDV and the understanding of genetic resistance to the disease is, therefore, crucial to long-term control strategies. Also, epigenetic factors are believed to be one of the major determinants of disease response. Results Here, we carried out comprehensive analyses of the epigenetic landscape induced by MDV, utilizing genome-wide histone H3 lysine 4 and lysine 27 trimethylation maps from chicken lines with varying resistance to MD. Differential chromatin marks were observed on genes previously implicated in the disease such as MX1 and CTLA-4 and also on genes reported in other cancers including IGF2BP1 and GAL. We detected bivalent domains on immune-related transcriptional regulators BCL6, CITED2 and EGR1, which underwent dynamic changes in both lines as a result of MDV infection. In addition, putative roles for GAL in the mechanism of MD progression were revealed. Conclusion Our results confirm the presence of widespread epigenetic differences induced by MD in chicken lines with different levels of genetic resistance. A majority of observed epigenetic changes were indicative of increased levels of viral infection in the susceptible line symptomatic of lowered immunocompetence in these birds caused by early cytolytic infection. The GAL system that has known anti-proliferative effects in other cancers is also revealed to be potentially involved in MD progression. Our study provides further insight into the mechanisms of MD progression while revealing a complex landscape of epigenetic regulatory mechanisms that varies depending on host factors.

  2. Varicella-Zoster Virus glycoprotein expression differentially induces the unfolded protein response in infected cells.

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    John Earl Carpenter

    2014-07-01

    Full Text Available Varicella-zoster virus (VZV is a human herpesvirus that spreads to children as varicella or chicken pox. The virus then establishes latency in the nervous system and re-emerges, typically decades later, as zoster or shingles. We have reported previously that VZV induces autophagy in infected cells as well as exhibiting evidence of the Unfolded Protein Response (UPR: XBP1 splicing, a greatly expanded Endoplasmic Reticulum (ER and CHOP expression. Herein we report the results of a UPR specific PCR array that measures the levels of mRNA of 84 different components of the UPR in VZV infected cells as compared to tunicamycin treated cells as a positive control and uninfected, untreated cells as a negative control. Tunicamycin is a mixture of chemicals that inhibits N-linked glycosylation in the ER with resultant protein misfolding and the UPR. We found that VZV differentially induces the UPR when compared to tunicamycin treatment. For example, tunicamycin treatment moderately increased (8 fold roughly half of the array elements while downregulating only three (one ERAD and two FOLD components. VZV infection on the other hand upregulated 33 components including a little described stress sensor CREB-H (64 fold as well as ER membrane components INSIG and gp78, which modulate cholesterol synthesis while downregulating over 20 components mostly associated with ERAD and FOLD. We hypothesize that this expression pattern is associated with an expanding ER with downregulation of active degradation by ERAD and apoptosis as the cell attempts to handle abundant viral glycoprotein synthesis.

  3. Acute ozone-induced differential gene expression profiles in rat lung.

    Science.gov (United States)

    Nadadur, Srikanth S; Costa, Daniel L; Slade, Ralph; Silbjoris, Robert; Hatch, Gary E

    2005-12-01

    Ozone is an oxidant gas that can directly induce lung injury. Knowledge of the initial molecular events of the acute O3 response would be useful in developing biomarkers of exposure or response. Toward this goal, we exposed rats to toxic concentrations of O3 (2 and 5 ppm) for 2 hr and the molecular changes were assessed in lung tissue 2 hr postexposure using a rat cDNA expression array containing 588 characterized genes. Gene array analysis indicated differential expression in almost equal numbers of genes for the two exposure groups: 62 at 2 ppm and 57 at 5 ppm. Most of these genes were common to both exposure groups, suggesting common roles in the initial toxicity response. However, we also identified the induction of nine genes specific to 2-ppm (thyroid hormone-beta receptor c-erb-A-beta; and glutathione reductase) or 5-ppm exposure groups (c-jun, induced nitric oxide synthase, macrophage inflammatory protein-2, and heat shock protein 27). Injury markers in bronchoalveolar lavage fluid (BALF) were used to assess immediate toxicity and inflammation in rats similarly exposed. At 2 ppm, injury was marked by significant increases in BALF total protein, N-acetylglucosaminidase, and lavageable ciliated cells. Because infiltration of neutrophils was observed only at the higher 5 ppm concentration, the distinctive genes suggested a potential amplification role for inflammation in the gene profile. Although the specific gene interactions remain unclear, this is the first report indicating a dose-dependent direct and immediate induction of gene expression that may be separate from those genes involved in inflammation after acute O3 exposure.

  4. Prenatal diagnosis by isoenzymic differentiation of Treacher Collins' syndrome induced by retinoids in rats

    DEFF Research Database (Denmark)

    Granström, G; Kirkeby, S

    1990-01-01

    ear ossicles, short cochleas, defectively differentiated Meckel's cartilages, micrognathia, rudimentary malar bones, lateral facial clefts, fistulas and skin tags, all of which were similar to Treacher Collins' syndrome in man. The defects were accompanied by a pathological differentiation pattern...

  5. Use of high pressure liquid chromatography to study chemically induced alterations in the pattern of benzo(a)pyrene metabolism. [Rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Freudenthal, R.I.; Leber, A.P.; Emmerling, D.; Clarke, P.

    1975-11-01

    The metabolism of radiolabeled benzo(a)pyrene (BP) by control, 3-methylcholanthrene (3-MC) induced and 1,1,1-trichloropropene-2,3-oxide (TCPO)-inhibited rat liver microsomes was measured using fluorescence, radiometric, and high-pressure liquid chromatographic (HPLC) assays. Significant differences in the total measurable metabolism of BP by the three microsomal enzyme incubations resulted from the use of the three assay procedures. Appreciable differences in the concentration of the metabolite fractions after 3-MC induction and TCPO inhibition are clearly demonstrated. NMR analysis revealed that while the 3-hydroxy-BP fraction is greater than 90 percent pure, the 9-hydroxy fraction contains a number of metabolites having essentially identical retention times.

  6. A Case of Habitual Neck Compression Induced Electroencephalogram Abnormalities: Differentiating from Epileptic Seizures Using a Tc-99m HMPAO SPECT

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    Choi, Hongyoon; Seo, Minseok; Lee, Hoyoung; Kim, Youngsoo; Yun, Changho; Kim, Sangeun; Park, Sungho [Seoul National Univ. Bundang Hospital, Seongnam (Korea, Republic of)

    2014-06-15

    Self-induced hypoxia has been reported particularly in adolescents, and it can result in neurological injury. Here, we present a case of electroencephalogram (EEG) abnormalities induced by habitual neck compression differentiated from epileptic seizures by Tc-99m HMPAO SPECT. A 19-year-old male was admitted for evaluation of recurrent generalized tonic-clonic seizures. No interictal EEG abnormality was detected; however, abnormal slow delta waves were found immediately after habitual right neck compression. To differentiate EEG abnormalities due to a hemodynamic deficit induced by habitual neck compression from an epileptic seizure, Tc-99m HMPAO SPECT was performed immediately after right carotid artery compression. Abnormal delta waves were triggered, and cerebral hypoperfusion in the right internal carotid artery territory was detected on Tc-99m HMPAO SPECT. The slow delta wave detected on the EEG resulted from the cerebral hypoperfusion because of the habitual neck compression.

  7. Bone morphogenic protein-2 regulates the myogenic differentiation of PMVECs in CBDL rat serum-induced pulmonary microvascular remodeling

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Chang; Chen, Lin; Zeng, Jing; Cui, Jian; Ning, Jiao-nin [Department of Anesthesia, Southwest Hospital, The Third Military Medical University, Chongqing 400038 (China); Wang, Guan-song [Institute of Respiratory Disease, Xinqiao Hospital, The Third Military Medical University, Chongqing 400037 (China); Belguise, Karine; Wang, Xiaobo [Université P. Sabatier Toulouse III and CNRS, LBCMCP, 31062 Toulouse Cedex 9 (France); Qian, Gui-sheng [Institute of Respiratory Disease, Xinqiao Hospital, The Third Military Medical University, Chongqing 400037 (China); Lu, Kai-zhi [Department of Anesthesia, Southwest Hospital, The Third Military Medical University, Chongqing 400038 (China); Yi, Bin, E-mail: yibin1974@163.com [Department of Anesthesia, Southwest Hospital, The Third Military Medical University, Chongqing 400038 (China)

    2015-08-01

    Hepatopulmonary syndrome (HPS) is characterized by an arterial oxygenation defect induced by intrapulmonary vasodilation (IPVD) that increases morbidity and mortality. In our previous study, it was determined that both the proliferation and the myogenic differentiation of pulmonary microvascular endothelial cells (PMVECs) play a key role in the development of IPVD. However, the molecular mechanism underlying the relationship between IPVD and the myogenic differentiation of PMVECs remains unknown. Additionally, it has been shown that bone morphogenic protein-2 (BMP2), via the control of protein expression, may regulate cell differentiation including cardiomyocyte differentiation, neuronal differentiation and odontoblastic differentiation. In this study, we observed that common bile duct ligation (CBDL)-rat serum induced the upregulation of the expression of several myogenic proteins (SM-α-actin, calponin, SM-MHC) and enhanced the expression levels of BMP2 mRNA and protein in PMVECs. We also observed that both the expression levels of Smad1/5 and the activation of phosphorylated Smad1/5 were significantly elevated in PMVECs following exposure to CBDL-rat serum, which was accompanied by the down-regulation of Smurf1. The blockage of the BMP2/Smad signaling pathway with Noggin inhibited the myogenic differentiation of PMVECs, a process that was associated with relatively low expression levels of both SM-α-actin and calponin in the setting of CBDL-rat serum exposure, although SM-MHC expression was not affected. These findings suggested that the BMP2/Smad signaling pathway is involved in the myogenic differentiation of the PMVECs. In conclusion, our data highlight the pivotal role of BMP2 in the CBDL-rat serum-induced myogenic differentiation of PMVECs via the activation of both Smad1 and Smad5 and the down-regulation of Smurf1, which may represent a potential therapy for HPS-induced pulmonary vascular remodeling. - Highlights: • CBDL-rat serum promotes the myogenic

  8. Berberine Sulfate Attenuates Osteoclast Differentiation through RANKL Induced NF-κB and NFAT Pathways.

    Science.gov (United States)

    Zhou, Lin; Song, Fangming; Liu, Qian; Yang, Mingli; Zhao, Jinmin; Tan, Renxiang; Xu, Jun; Zhang, Ge; Quinn, Julian M W; Tickner, Jennifer; Xu, Jiake

    2015-11-13

    Osteoporosis, a metabolic bone disease, is characterized by an excessive formation and activation of osteoclasts. Anti-catabolic treatment using natural compounds has been proposed as a potential therapeutic strategy against the osteoclast related osteolytic diseases. In this study, the activity of berberine sulfate (an orally available form of berberine) on osteoclast differentiation and its underlying molecular mechanisms of action were investigated. Using bone marrow macrophages (BMMs) derived osteoclast culture system, we showed that berberine sulfate at the dose of 0.25, 0.5 and 1 μM significantly inhibited the formation of osteoclasts. Notably, berberine sulfate at these doses did not affect the BMM viability. In addition, we observed that berberine sulfate inhibited the expression of osteoclast marker genes, including cathepsin K (Ctsk), nuclear factor of activated T cells cytoplasmic 1 (NFATc1), tartrate resistant acid phosphatase (TRAcP, Acp5) and Vacuolar-type H+-ATPase V0 subunit D2 (V-ATPase d2). Luciferase reporter gene assay and Western blot analysis further revealed that berberine sulfate inhibits receptor for activation of nuclear factor ligand (RANKL)-induced NF-κB and NFAT activity. Taken together, our results suggest that berberine sulfate is a natural compound potentially useful for the treatment of osteoporosis.

  9. Gap state charge induced spin-dependent negative differential resistance in tunnel junctions

    Science.gov (United States)

    Jiang, Jun; Zhang, X.-G.; Han, X. F.

    2016-04-01

    We propose and demonstrate through first-principles calculation a new spin-dependent negative differential resistance (NDR) mechanism in magnetic tunnel junctions (MTJ) with cubic cation disordered crystals (CCDC) AlO x or Mg1-x Al x O as barrier materials. The CCDC is a class of insulators whose band gap can be changed by cation doping. The gap becomes arched in an ultrathin layer due to the space charge formed from metal-induced gap states. With an appropriate combination of an arched gap and a bias voltage, NDR can be produced in either spin channel. This mechanism is applicable to 2D and 3D ultrathin junctions with a sufficiently small band gap that forms a large space charge. It provides a new way of controlling the spin-dependent transport in spintronic devices by an electric field. A generalized Simmons formula for tunneling current through junction with an arched gap is derived to show the general conditions under which ultrathin junctions may exhibit NDR.

  10. Hepatic Differentiation of Murine Disease-Specific Induced Pluripotent Stem Cells Allows Disease Modelling In Vitro

    Directory of Open Access Journals (Sweden)

    Reto Eggenschwiler

    2011-01-01

    Full Text Available Direct reprogramming of somatic cells into pluripotent cells by retrovirus-mediated expression of OCT4, SOX2, KLF4, and C-MYC is a promising approach to derive disease-specific induced pluripotent stem cells (iPSCs. In this study, we focused on three murine models for metabolic liver disorders: the copper storage disorder Wilson's disease (toxic-milk mice, tyrosinemia type 1 (fumarylacetoacetate-hydrolase deficiency, FAH−/− mice, and alpha1-antitrypsin deficiency (PiZ mice. Colonies of iPSCs emerged 2-3 weeks after transduction of fibroblasts, prepared from each mouse strain, and were maintained as individual iPSC lines. RT-PCR and immunofluorescence analyses demonstrated the expression of endogenous pluripotency markers. Hepatic precursor cells could be derived from these disease-specific iPSCs applying an in vitro differentiation protocol and could be visualized after transduction of a lentiviral albumin-GFP reporter construct. Functional characterization of these cells allowed the recapitulation of the disease phenotype for further studies of underlying molecular mechanisms of the respective disease.

  11. Induced pluripotent stem cells from familial Alzheimer's disease patients differentiate into mature neurons with amyloidogenic properties.

    Science.gov (United States)

    Mahairaki, Vasiliki; Ryu, Jiwon; Peters, Ann; Chang, Qing; Li, Tong; Park, Tea Soon; Burridge, Paul W; Talbot, Conover C; Asnaghi, Laura; Martin, Lee J; Zambidis, Elias T; Koliatsos, Vassilis E

    2014-12-15

    Although the majority of Alzheimer's disease (AD) cases are sporadic, about 5% of cases are inherited in an autosomal dominant pattern as familial AD (FAD) and manifest at an early age. Mutations in the presenilin 1 (PSEN1) gene account for the majority of early-onset FAD. Here, we describe the generation of virus-free human induced pluripotent stem cells (hiPSCs) derived from fibroblasts of patients harboring the FAD PSEN1 mutation A246E and fibroblasts from healthy age-matched controls using nonintegrating episomal vectors. We have differentiated these hiPSC lines to the neuronal lineage and demonstrated that hiPSC-derived neurons have mature phenotypic and physiological properties. Neurons from mutant hiPSC lines express PSEN1-A246E mutations themselves and show AD-like biochemical features, that is, amyloidogenic processing of amyloid precursor protein (APP) indicated by an increase in β-amyloid (Aβ)42/Aβ40 ratio. FAD hiPSCs harboring disease properties can be used as humanized models to test novel diagnostic methods and therapies and explore novel hypotheses for AD pathogenesis.

  12. Gravimetric analysis and differential scanning calorimetric studies on glycerin-induced skin hydration.

    Science.gov (United States)

    Lee, Ae-Ri Cho; Moon, Hee Kyung

    2007-11-01

    A thermal gravimetric analysis (TGA) and a differential scanning calorimetry (DSC) were carried out to characterize the water property and an alteration of lipid phase transition of stratum corneum (SC) by glycerin. In addition, the relationship between steady state skin permeation rate and skin hydration in various concentrations of glycerin was investigated. Water vapor absorption-desorption was studied in the hairless mouse stratum corneum. Dry SC samples were exposed to different conc. of glycerin (0-50%) followed by exposure to dry air and the change in weight property was monitored over time by use of TGA. In DSC study, significant decrease in DeltaH of the lipid transition in 10% glycerin and water treated sample: the heat of lipid transition of normal, water, 10% glycerin treated SC were 6.058, 4.412 and 4.316 mJ/mg, respectively. In 10% glycerin treated SCs, the Tc of water shifts around 129 degrees C, corresponding to the weakly bound secondary water. In 40% glycerin treated SC, the Tc of water shifts to 144 degrees C corresponding to strongly bound primary water. There was a good correlation between the hydration property of the skin and the steady state skin flux with the correlation coefficient (r2=0.94). As the hydration increased, the steady state flux increased. As glycerin concentration increased, hydration property decreased. High diffusivity induced by the hydration effect of glycerin and water could be the major contributing factor for the enhanced skin permeation of nicotinic acid (NA).

  13. Light-induced negative differential resistance in graphene/Si-quantum-dot tunneling diodes.

    Science.gov (United States)

    Lee, Kyeong Won; Jang, Chan Wook; Shin, Dong Hee; Kim, Jong Min; Kang, Soo Seok; Lee, Dae Hun; Kim, Sung; Choi, Suk-Ho; Hwang, Euyheon

    2016-01-01

    One of the interesing tunneling phenomena is negative differential resistance (NDR), the basic principle of resonant-tunneling diodes. NDR has been utilized in various semiconductor devices such as frequency multipliers, oscillators, relfection amplifiers, logic switches, and memories. The NDR in graphene has been also reported theoretically as well as experimentally, but should be further studied to fully understand its mechanism, useful for practical device applications. Especially, there has been no observation about light-induced NDR (LNDR) in graphene-related structures despite very few reports on the LNDR in GaAs-based heterostructures. Here, we report first observation of LNDR in graphene/Si quantum dots-embedded SiO2 (SQDs:SiO2) multilayers (MLs) tunneling diodes. The LNDR strongly depends on temperature (T) as well as on SQD size, and the T dependence is consistent with photocurrent (PC)-decay behaviors. With increasing light power, the PC-voltage curves are more structured with peak-to-valley ratios over 2 at room temperature. The physical mechanism of the LNDR, governed by resonant tunneling of charge carriers through the minibands formed across the graphene/SQDs:SiO2 MLs and by their nonresonant phonon-assisted tunneling, is discussed based on theoretical considerations.

  14. Phytoestrogens induce differential estrogen receptor alpha- or Beta-mediated responses in transfected breast cancer cells.

    Science.gov (United States)

    Harris, D M; Besselink, E; Henning, S M; Go, V L W; Heber, D

    2005-09-01

    Increased intake of phytoestrogens may be associated with a lower risk of cancer in the breast and several other sites, although there is controversy surrounding this activity. One of the mechanisms proposed to explain the activity of phytoestrogens is their ability to bind and activate human estrogen receptor alpha (ERalpha) and human estrogen receptor beta (ERbeta). Nine phytoestrogens were tested for their ability to transactivate ERalpha or ERbeta at a range of doses. Mammary adenocarcinoma (MCF-7) cells were co-transfected with either ERalpha or ERbeta, and an estrogen-response element was linked to a luciferase reporter gene. Dose-dependent responses were compared with the endogenous ligand 17beta-estradiol. Purified genistein, daidzein, apigenin, and coumestrol showed differential and robust transactivation of ERalpha- and ERbeta-induced transcription, with an up to 100-fold stronger activation of ERbeta. Equol, naringenin, and kaempferol were weaker agonists. When activity was evaluated against a background of 0.5 nM 17beta-estradiol, the addition of genistein, daidzein, and resveratrol superstimulated the system, while kaempferol and quercetin were antagonists at the highest doses. This transfection assay provides an excellent model to evaluate the activation of ERalpha and ERbeta by different phytoestrogens in a breast cancer context and can be used as a screening bioassay tool to evaluate the estrogenic activity of extracts of herbs and foods.

  15. Microarray analysis of genes differentially expressed in placentas of pregnancy-induced hypertension patients

    Institute of Scientific and Technical Information of China (English)

    李东红; 黄飞; 郑维国; 姜锋; 高平

    2003-01-01

    Objective: To uncover new clue for the research of the etiology of pregnancy-induced hypertension (PIH) by testing the gene expression difference between preeclamptic placentas and normal ones. Methods: mRNA level of 4 PIH placentas were examined using 4000 feature cDNA microarray in comparison with the pooled control consisting of total RNA from 4 cases of PIH placentas after the control cDNA and experimental cDNA were labeled by cy3 and cy5 respectively. Results: Fifty-eight to 131 genes were found down or up-regulated in 4 runs of hybridization. Among the differentially expressed genes, 22 genes, including genes encoding secreted protein ADRP, CYR61, EPI and HIF2, had the concordance in at least 2 cases were up-regulated or down-regulated. Conclusion: cDNA microarray is a high throughput and time-saving method to monitor the altered gene expression and the result could provide interesting clue and strategy for the etiological research of PIH.

  16. Light-induced negative differential resistance in graphene/Si-quantum-dot tunneling diodes

    Science.gov (United States)

    Lee, Kyeong Won; Jang, Chan Wook; Shin, Dong Hee; Kim, Jong Min; Kang, Soo Seok; Lee, Dae Hun; Kim, Sung; Choi, Suk-Ho; Hwang, Euyheon

    2016-07-01

    One of the interesing tunneling phenomena is negative differential resistance (NDR), the basic principle of resonant-tunneling diodes. NDR has been utilized in various semiconductor devices such as frequency multipliers, oscillators, relfection amplifiers, logic switches, and memories. The NDR in graphene has been also reported theoretically as well as experimentally, but should be further studied to fully understand its mechanism, useful for practical device applications. Especially, there has been no observation about light-induced NDR (LNDR) in graphene-related structures despite very few reports on the LNDR in GaAs-based heterostructures. Here, we report first observation of LNDR in graphene/Si quantum dots-embedded SiO2 (SQDs:SiO2) multilayers (MLs) tunneling diodes. The LNDR strongly depends on temperature (T) as well as on SQD size, and the T dependence is consistent with photocurrent (PC)-decay behaviors. With increasing light power, the PC-voltage curves are more structured with peak-to-valley ratios over 2 at room temperature. The physical mechanism of the LNDR, governed by resonant tunneling of charge carriers through the minibands formed across the graphene/SQDs:SiO2 MLs and by their nonresonant phonon-assisted tunneling, is discussed based on theoretical considerations.

  17. Inhibition of GSK-3 induces differentiation and impaired glucose metabolism in renal cancer

    Science.gov (United States)

    Pal, Krishnendu; Cao, Ying; Gaisina, Irina N.; Bhattacharya, Santanu; Dutta, Shamit K.; Wang, Enfeng; Gunosewoyo, Hendra; Kozikowski, Alan P.; Billadeau, Daniel D.; Mukhopadhyay, Debabrata

    2014-01-01

    Glycogen synthase kinase-3 (GSK-3), a constitutively active serine/threonine kinase, is a key regulator of numerous cellular processes ranging from glycogen metabolism to cell cycle regulation and proliferation. Consistent with its involvement in many pathways, it has also been implicated in the pathogenesis of various human diseases including Type II diabetes, Alzheimer's disease, bipolar disorder, inflammation and cancer. Consequently it is recognized as an attractive target for the development of new drugs. In the present study, we investigated the effect of both pharmacological and genetic inhibition of GSK-3 in two different renal cancer cell lines. We have shown potent anti-proliferative activity of 9-ING-41, a maleimide-based GSK-3 inhibitor. The anti-proliferative activity is most likely caused by G0–G1 and G2-M phase arrest as evident from cell cycle analysis. We have established that inhibition of GSK-3 imparted a differentiated phenotype in renal cancer cells. We have also shown that GSK-3 inhibition induced autophagy, likely as a result of imbalanced energy homeostasis caused by impaired glucose metabolism. Additionally, we have demonstrated the antitumor activity of 9-ING-41 in two different subcutaneous xenograft RCC tumor models. To our knowledge, this is the first report describing autophagy induction due to GSK-3 inhibition in renal cancer cells. PMID:24327518

  18. Differential effects of aging and sex on stroke induced inflammation across the lifespan.

    Science.gov (United States)

    Manwani, Bharti; Liu, Fudong; Scranton, Victoria; Hammond, Matthew D; Sansing, Lauren H; McCullough, Louise D

    2013-11-01

    Aging and biological sex are critical determinants of stroke outcome. Post-ischemic inflammatory response strongly contributes to the extent of ischemic brain injury, but how this response changes with age and sex is unknown. We subjected young (5-6 months), middle aged (14-15 months) and aged (20-22 months), C57BL/6 male and female mice to transient middle cerebral artery occlusion (MCAO) and found that a significant age by sex interaction influenced histological stroke outcomes. Acute functional outcomes were worse with aging. Neutrophils, inflammatory macrophages, macrophages, dendritic cells (DCs) and microglia significantly increased in the brain post MCAO. Cycling females had higher Gr1(-) non-inflammatory macrophages and lower T cells in the brain after stroke and these correlated with serum estradiol levels. Estrogen loss in acyclic aged female mice exacerbated stroke induced splenic contraction. Advanced age increased T cells, DCs and microglia at the site of injury, which may be responsible for the exacerbated behavioral deficits in the aged. We conclude that aging and sex have differential effects on the post stroke inflammatory milieu. Putative immunomodulatory therapies need to account for this heterogeneity.

  19. Electrospun tilapia collagen nanofibers accelerating wound healing via inducing keratinocytes proliferation and differentiation.

    Science.gov (United States)

    Zhou, Tian; Wang, Nanping; Xue, Yang; Ding, Tingting; Liu, Xin; Mo, Xiumei; Sun, Jiao

    2016-07-01

    The development of biomaterials with the ability to induce skin wound healing is a great challenge in biomedicine. In this study, tilapia skin collagen sponge and electrospun nanofibers were developed for wound dressing. The collagen sponge was composed of at least two α-peptides. It did not change the number of spleen-derived lymphocytes in BALB/c mice, the ratio of CD4(+)/CD8(+) lymphocytes, and the level of IgG or IgM in Sprague-Dawley rats. The tensile strength and contact angle of collagen nanofibers were 6.72±0.44MPa and 26.71±4.88°, respectively. They also had good thermal stability and swelling property. Furthermore, the nanofibers could significantly promote the proliferation of human keratinocytes (HaCaTs) and stimulate epidermal differentiation through the up-regulated gene expression of involucrin, filaggrin, and type I transglutaminase in HaCaTs. The collagen nanofibers could also facilitate rat skin regeneration. In the present study, electrospun biomimetic tilapia skin collagen nanofibers were succesfully prepared, were proved to have good bioactivity and could accelerate rat wound healing rapidly and effectively. These biological effects might be attributed to the biomimic extracellular matrix structure and the multiple amino acids of the collagen nanofibers. Therefore, the cost-efficient tilapia collagen nanofibers could be used as novel wound dressing, meanwhile effectively avoiding the risk of transmitting animal disease in the future clinical apllication.

  20. Glycolipid dynamics in generation and differentiation of induced pluripotent stem cells.

    Science.gov (United States)

    Ojima, Takuma; Shibata, Eri; Saito, Shiho; Toyoda, Masashi; Nakajima, Hideki; Yamazaki-Inoue, Mayu; Miyagawa, Yoshitaka; Kiyokawa, Nobutaka; Fujimoto, Jun-ichiro; Sato, Toshinori; Umezawa, Akihiro

    2015-10-19

    Glycosphingolipids (GSLs) are glycoconjugates that function as mediators of cell adhesion and modulators of signal transduction. Some well-defined markers of undifferentiated human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are glycoconjugates, such as SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. However, Comprehensive GSL profiles of hiPSCs have not yet been elucidated. The global images of GSLs from the parental cells, hiPSCs, and differentiated cells revealed that there are parental cell-independent specific glycolipids, including Globo H (fucosyl-Gb5Cer) and H type1 antigen (fucosyl-Lc4Cer) that are novel markers for undifferentiated hiPSCs. Interestingly, undifferentiated hiPSCs expressed H type 1 antigen, specific for blood type O, regardless of the cells' genotypes. Thus, in this study, we defined the dynamics of GSL remodeling during reprogramming from parental cell sets to iPSC sets and thence to iPSC-neural cells.

  1. Curl flux induced drift in stochastic differential equations in the zero-mass limit

    Science.gov (United States)

    Wang, Jinhua; Yuan, Bo

    2016-11-01

    We consider the nonlinear stochastic dynamics of dissipative Hamiltonian systems with state-dependent friction and diffusion connected by the fluctuation-dissipation relation in high dimensions. The system under study has a close connection to Ao's framework in constructing a dynamical potential for non-equilibrium processes without detailed balance. We study the limiting case where the mass approaches zero and give a new and complete derivation of effective stochastic differential equations. Using the Ito stochastic integral convention, we show that the limiting effective Langevin equations have a new drift term. This extra term happens to be identical to the corresponding anti-Ito (or isothermal) integral (requiring constant temperature) in one dimension. We, however, cannot obtain this additional drift term using conventional stochastic integrals in high dimension. It is interesting to note that in a high-dimensional system, a curl flux induced drift may appear even if the diffusion matrix is constant. Our findings are supported by numerical simulations. We further analyze and discuss the role of this new drift term in calculating the classic escape time. For the first time, to our knowledge, the relation between the escape rate and the anti-Ito integral is presented. We also demonstrate that the derived diffusion equations give a new sampling algorithm which can increase convergence speed in a simple two-dimensional example.

  2. Combinations of Ashwagandha leaf extracts protect brain-derived cells against oxidative stress and induce differentiation.

    Directory of Open Access Journals (Sweden)

    Navjot Shah

    Full Text Available Ashwagandha, a traditional Indian herb, has been known for its variety of therapeutic activities. We earlier demonstrated anticancer activities in the alcoholic and water extracts of the leaves that were mediated by activation of tumor suppressor functions and oxidative stress in cancer cells. Low doses of these extracts were shown to possess neuroprotective activities in vitro and in vivo assays.We used cultured glioblastoma and neuroblastoma cells to examine the effect of extracts (alcoholic and water as well as their bioactive components for neuroprotective activities against oxidative stress. Various biochemical and imaging assays on the marker proteins of glial and neuronal cells were performed along with their survival profiles in control, stressed and recovered conditions. We found that the extracts and one of the purified components, withanone, when used at a low dose, protected the glial and neuronal cells from oxidative as well as glutamate insult, and induced their differentiation per se. Furthermore, the combinations of extracts and active component were highly potent endorsing the therapeutic merit of the combinational approach.Ashwagandha leaf derived bioactive compounds have neuroprotective potential and may serve as supplement for brain health.

  3. Natural cerebrolysin induces neuronal differentiation in bone marrow mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Zhengzhi Wu; Yinghong Li; Andrew C. J. Huang O; Ming Li; Min Yang; Manyin Chen

    2009-01-01

    MEASURES: Morphology of MSCs and neurite outgrowth during differentiation was observed under inverted phase contrast microscope. Neurite-positive cells were classified by neurite length that was longer than 1.5x the cell body diameter. Immunocytochemistry was used to identify purity of MSCs following passage, as well as expression of nidogen, neuron-specific enolase, glial fibrillary acidic protein, and microtubule-associated protein 2 following treatment with natural cerebrolysin, mRNA expression of neuron-specific enolase and glial fibrillary acidic protein was detected using semi-quantitative RT-PCR.RESULTS: After MSCs were treated with natural cerebrolysin for 3-5 hours, the cell bodies were larger, and small neurites - similar to neuronal neurites - were observed. The number of neurite-positive cells significantly increased compared with the control group (P 0.05).CONCLUSION: Natural cerebrolysin promoted neurite outgrowth and induced neuronal-like differentiation of MSCs.

  4. Shear stress induced by an interstitial level of slow flow increases the osteogenic differentiation of mesenchymal stem cells through TAZ activation.

    Directory of Open Access Journals (Sweden)

    Kyung Min Kim

    Full Text Available Shear stress activates cellular signaling involved in cellular proliferation, differentiation, and migration. However, the mechanisms of mesenchymal stem cell (MSC differentiation under interstitial flow are not fully understood. Here, we show the increased osteogenic differentiation of MSCs under exposure to constant, extremely low shear stress created by osmotic pressure-induced flow in a microfluidic chip. The interstitial level of shear stress in the proposed microfluidic system stimulated nuclear localization of TAZ (transcriptional coactivator with PDZ-binding motif, a transcriptional modulator of MSCs, activated TAZ target genes such as CTGF and Cyr61, and induced osteogenic differentiation. TAZ-depleted cells showed defects in shear stress-induced osteogenic differentiation. In shear stress induced cellular signaling, Rho signaling pathway was important forthe nuclear localization of TAZ. Taken together, these results suggest that TAZ is an important mediator of interstitial flow-driven shear stress signaling in osteoblast differentiation of MSCs.

  5. Differential Expression of microRNAs in the Ovaries from Letrozole-Induced Rat Model of Polycystic Ovary Syndrome.

    Science.gov (United States)

    Li, Dandan; Li, Chunjin; Xu, Ying; Xu, Duo; Li, Hongjiao; Gao, Liwei; Chen, Shuxiong; Fu, Lulu; Xu, Xin; Liu, Yongzheng; Zhang, Xueying; Zhang, Jingshun; Ming, Hao; Zheng, Lianwen

    2016-04-01

    Polycystic ovary syndrome (PCOS) is a complex and heterogeneous endocrine disorder. To understand the pathogenesis of PCOS, we established rat models of PCOS induced by letrozole and employed deep sequencing to screen the differential expression of microRNAs (miRNAs) in PCOS rats and control rats. We observed vaginal smear and detected ovarian pathological alteration and hormone level changes in PCOS rats. Deep sequencing showed that a total of 129 miRNAs were differentially expressed in the ovaries from letrozole-induced rat model compared with the control, including 49 miRNAs upregulated and 80 miRNAs downregulated. Furthermore, the differential expression of miR-201-5p, miR-34b-5p, miR-141-3p, and miR-200a-3p were confirmed by real-time polymerase chain reaction. Bioinformatic analysis revealed that these four miRNAs were predicted to target a large set of genes with different functions. Pathway analysis supported that the miRNAs regulate oocyte meiosis, mitogen-activated protein kinase (MAPK) signaling, phosphoinositide 3-kinase/Akt (PI3K-Akt) signaling, Rap1 signaling, and Notch signaling. These data indicate that miRNAs are differentially expressed in rat PCOS model and the differentially expressed miRNA are involved in the etiology and pathophysiology of PCOS. Our findings will help identify miRNAs as novel diagnostic markers and therapeutic targets for PCOS.

  6. Modulation of fibroblast growth factor receptor expression and signalling during retinoic acid-induced differentiation of Tera-2 teratocarcinoma cells.

    Science.gov (United States)

    Pertovaara, L; Tienari, J; Vainikka, S; Partanen, J; Saksela, O; Lehtonen, E; Alitalo, K

    1993-02-26

    We have analyzed the regulation of fibroblast growth factor receptors (FGFRs) during retinoic acid (RA) induced differentiation of Tera-2 human embryonal carcinoma cells. Undifferentiated Tera-2 cells expressed mRNAs for all four known FGFRs. Their differentiation led to loss of FGFR-4 mRNA expression and mRNA levels for FGFR-2 and FGFR-3 were considerably downregulated, whereas the mRNA levels for FGFR-1 remained unaltered. A substantial decrease in binding of K-FGF was found to occur upon RA-induced differentiation of the cells. In undifferentiated Tera-2 cells FGF stimulation caused an increase of c-fos mRNA, and c-jun mRNAs, but no increase of junB mRNA, whereas in the differentiated cells, FGFs strongly stimulated the expression of all three genes. Thus differentiation of the Tera-2 cells leads to marked changes in FGFR gene expression as well as to complex alterations in their responses to exogenous FGFs.

  7. Gallium nitride induces neuronal differentiation markers in neural stem/precursor cells derived from rat cerebral cortex.

    Science.gov (United States)

    Chen, Chi-Ruei; Li, Yi-Chen; Young, Tai-Horng

    2009-09-01

    In the present study, gallium nitride (GaN) was used as a substrate to culture neural stem/precursor cells (NSPCs), isolated from embryonic rat cerebral cortex, to examine the effect of GaN on the behavior of NSPCs in the presence of basic fibroblast growth factor (bFGF) in serum-free medium. Morphological studies showed that neurospheres maintained their initial shape and formed many long and thick processes with the fasciculate feature on GaN. Immunocytochemical characterization showed that GaN could induce the differentiation of NSPCs into neurons and astrocytes. Compared to poly-d-lysine (PDL), the most common substrate used for culturing neurons, there was considerable expression of synapsin I for differentiated neurons on GaN, suggesting GaN could induce the differentiation of NSPCs towards the mature differentiated neurons. Western blot analysis showed that the suppression of glycogen synthase kinase-3beta (GSK-3beta) activity was one of the effects of GaN-promoted NSPC differentiation into neurons. Finally, compared to PDL, GaN could significantly improve cell survival to reduce cell death after long-term culture. These results suggest that GaN potentially has a combination of electric characteristics suitable for developing neuron and/or NSPC chip systems.

  8. Mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-induced Smad1/5/8 phosphorylation

    Science.gov (United States)

    Liu, Jia; Saito, Kan; Maruya, Yuriko; Nakamura, Takashi; Yamada, Aya; Fukumoto, Emiko; Ishikawa, Momoko; Iwamoto, Tsutomu; Miyazaki, Kanako; Yoshizaki, Keigo; Ge, Lihong; Fukumoto, Satoshi

    2016-01-01

    Bone morphogenetic proteins (BMPs) regulate hard tissue formation, including bone and tooth. Growth differentiation factor 5 (GDF5), a known BMP, is expressed in cartilage and regulates chondrogenesis, and mutations have been shown to cause osteoarthritis. Notably, GDF5 is also expressed in periodontal ligament tissue; however, its role during tooth development is unclear. Here, we used cell culture and in vivo analyses to determine the role of GDF5 during tooth development. GDF5 and its associated BMP receptors are expressed at the protein and mRNA levels during postnatal tooth development, particularly at a stage associated with enamel formation. Furthermore, whereas BMP2 was observed to induce evidently the differentiation of enamel-forming ameloblasts, excess GDF5 induce mildly this differentiation. A mouse model harbouring a mutation in GDF5 (W408R) showed enhanced enamel formation in both the incisors and molars, but not in the tooth roots. Overexpression of the W408R GDF5 mutant protein was shown to induce BMP2-mediated mRNA expression of enamel matrix proteins and downstream phosphorylation of Smad1/5/8. These results suggest that mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-signalling. PMID:27030100

  9. Impact of mGluR5 during amphetamine-induced hyperactivity and conditioned hyperactivity in differentially reared rats.

    Science.gov (United States)

    Gill, Margaret J; Arnold, Jennifer C; Cain, Mary E

    2012-05-01

    3-((2-Methyl-1,3-thiazol-4-yl)ethynyl)pyridine hydrochloride (MTEP) is a metabotropic glutamate receptor 5 (mGluR5) antagonist that may alter drug sensitivity in differentially reared rats due to its involvement in the psychostimulant reward pathway and plasticity. The purpose of this study was to assess the effects of MTEP on acute amphetamine-induced hyperactivity, conditioned hyperactivity, and sensitization. Rats were reared in an enriched (EC), isolated (IC), or standard (SC) condition after which rats were either administered MTEP (1.0 mg/kg, ip) or saline prior to an acute (0.5 or 1.0 mg/kg, sc) or repeated (0.3 mg/kg, sc) amphetamine exposure. Rats undergoing repeated amphetamine exposure were administered MTEP prior to conditioned hyperactivity and sensitization tests. EC and SC rats administered with MTEP prior to acute amphetamine demonstrated attenuated amphetamine-induced locomotor activity compared to controls, while IC rats administered MTEP following repeated amphetamine exposure demonstrated attenuated amphetamine-induced locomotor activity. Interestingly, MTEP treatment only altered conditioned hyperactivity in EC rats, as MTEP pretreatment resulted in conditioned hyperactivity in EC rats while conditioned hyperactivity was not observed in EC rats pretreated with saline. Glutamatergic pathways are altered during differential rearing, which differentially alters the role of mGluR5 in EC, IC, and SC rats when administered psychostimulant acutely versus repeatedly. These findings suggest that differential rearing alters glutamatergic function, which reduces sensitivity to psychostimulants.

  10. Interleukin-17A Differentially Induces Inflammatory and Metabolic Gene Expression in the Adipose Tissues of Lean and Obese Mice.

    Science.gov (United States)

    Qu, Yine; Zhang, Qiuyang; Ma, Siqi; Liu, Sen; Chen, Zhiquan; Mo, Zhongfu; You, Zongbing

    2016-04-07

    The functions of interleukin-17A (IL-17A) in adipose tissues and adipocytes have not been well understood. In the present study, male mice were fed with a regular diet (n = 6, lean mice) or a high-fat diet (n = 6, obese mice) for 30 weeks. Subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) were analyzed for IL-17A levels. SAT and VAT were treated with IL-17A and analyzed for inflammatory and metabolic gene expression. Mouse 3T3-L1 pre-adipocytes were differentiated into adipocytes, followed with IL-17A treatment and analysis for inflammatory and metabolic gene expression. We found that IL-17A levels were higher in obese SAT than lean SAT; the basal expression of inflammatory and metabolic genes was different between SAT and VAT and between lean and obese adipose tissues. IL-17A differentially induced expression of inflammatory and metabolic genes, such as tumor necrosis factor α, Il-6, Il-1β, leptin, and glucose transporter 4, in adipose tissues of lean and obese mice. IL-17A also differentially induced expression of inflammatory and metabolic genes in pre-adipocytes and adipocytes, and IL-17A selectively activated signaling pathways in adipose tissues and adipocytes. These findings suggest that IL-17A differentially induces inflammatory and metabolic gene expression in the adipose tissues of lean and obese mice.

  11. Phospholipase C-gamma1 is required for subculture-induced terminal differentiation of normal human oral keratinocytes.

    Science.gov (United States)

    Oh, Ju-Eun; Kook, Joong-Ki; Park, Kyung-Hee; Lee, Gene; Seo, Byoung-Moo; Min, Byung-Moo

    2003-04-01

    Serial subculture of primary normal human oral keratinocytes (NHOKs) to the post-mitotic stage induces terminal differentiation, which is in part linked to elevated levels of phospholipase C (PLC)-gamma1. Therefore, PLC-gamma1 may be involved in the signal transduction system that leads to the calcium regulation of subculture-induced keratinocyte differentiation. To test this hypothesis, the expression of PLC-gamma1 in primary NHOKs was blocked by transfecting cells with the antisense PLC-gamma1 cDNA construct. These cells demonstrated dramatic reductions in PLC-gamma1 protein and in the differentiation markers involucrin and transglutaminase following calcium exposure and an increase (15-20%) in in vitro life span versus empty vector-transfected cells. In addition, we established the ability of antisense PLC-gamma1 to block the serial subculture-induced rise in intracellular calcium. Similar observations were made following treatment with the specific PLC inhibitor U73122. These results indicate that the terminal differentiation of NHOKs by serial subculture is associated with PLC-gamma1, which mediates calcium regulation by mobilizing intracellular calcium.

  12. Interleukin-17A Differentially Induces Inflammatory and Metabolic Gene Expression in the Adipose Tissues of Lean and Obese Mice

    Directory of Open Access Journals (Sweden)

    Yine Qu

    2016-04-01

    Full Text Available The functions of interleukin-17A (IL-17A in adipose tissues and adipocytes have not been well understood. In the present study, male mice were fed with a regular diet (n = 6, lean mice or a high-fat diet (n = 6, obese mice for 30 weeks. Subcutaneous adipose tissue (SAT and visceral adipose tissue (VAT were analyzed for IL-17A levels. SAT and VAT were treated with IL-17A and analyzed for inflammatory and metabolic gene expression. Mouse 3T3-L1 pre-adipocytes were differentiated into adipocytes, followed with IL-17A treatment and analysis for inflammatory and metabolic gene expression. We found that IL-17A levels were higher in obese SAT than lean SAT; the basal expression of inflammatory and metabolic genes was different between SAT and VAT and between lean and obese adipose tissues. IL-17A differentially induced expression of inflammatory and metabolic genes, such as tumor necrosis factor α, Il-6, Il-1β, leptin, and glucose transporter 4, in adipose tissues of lean and obese mice. IL-17A also differentially induced expression of inflammatory and metabolic genes in pre-adipocytes and adipocytes, and IL-17A selectively activated signaling pathways in adipose tissues and adipocytes. These findings suggest that IL-17A differentially induces inflammatory and metabolic gene expression in the adipose tissues of lean and obese mice.

  13. Differentiation of Human Bone Marrow Stromal Cells into Neural-Like Cells Induced by Sodium Ferulate in vitro

    Institute of Scientific and Technical Information of China (English)

    Yang Wang; Zhifeng Deng; Xianliang Lai; Wei Tu

    2005-01-01

    Human marrow stromal cells (hMSCs) are multipotential stem cells, capable of differentiating into bone, cartilage,fat and muscle. Several recent reports demonstrated that hMSCs have been also differentiated into neural cells.However, only a few reported inducers are applicable for clinical use. This work is to explore the effects of sodium ferulate (SF) on differentiation of hMSCs into neural cells in vitro. We found that hMSCs could be induced to the cells with typical neural morphology when cultured with SF. The cells express neural proteins, such as nestin,neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP). About 30% of the hMSC-derived cells expressed nestin when cultured with SF for 3 h, but no expression was detected after 24 h. The percentages of positive cells for NSE or GFAP were about 67% and 39% separately at 6 h, and reached the plateau phage after treatment with SF for 3 days. The data suggest that SF can induce hMSCs to differentiate into neural-like cells in vitro.

  14. Mycoplasma hyopneumoniae and Mycoplasma flocculare differential domains from orthologous surface proteins induce distinct cellular immune responses in mice.

    Science.gov (United States)

    Leal, Fernanda Munhoz Dos Anjos; Virginio, Veridiana Gomes; Martello, Carolina Lumertz; Paes, Jéssica Andrade; Borges, Thiago J; Jaeger, Natália; Bonorino, Cristina; Ferreira, Henrique Bunselmeyer

    2016-07-15

    Mycoplasma hyopneumoniae and Mycoplasma flocculare are two genetically close species found in the swine respiratory tract. Despite their similarities, while M. hyopneumoniae is the causative agent of porcine enzootic pneumonia, M. flocculare is a commensal bacterium. Genomic and transcriptional comparative analyses so far failed to explain the difference in pathogenicity between these two species. We then hypothesized that such difference might be, at least in part, explained by amino acid sequence and immunological or functional differences between ortholog surface proteins. In line with that, it was verified that approximately 85% of the ortholog surface proteins from M. hyopneumoniae 7448 and M. flocculare present one or more differential domains. To experimentally assess possible immunological implications of this kind of difference, the extracellular differential domains from one pair of orthologous surface proteins (MHP7448_0612, from M. hyopneumoniae, and MF_00357, from M. flocculare) were expressed in E. coli and used to immunize mice. The recombinant polypeptides (rMHP61267-169 and rMF35767-196, respectively) induced distinct cellular immune responses. While, rMHP61267-169 induced both Th1 and Th2 responses, rMF35767-196 induced just an early pro-inflammatory response. These results indicate that immunological properties determined by differential domains in orthologous surface protein might play a role in pathogenicity, contributing to elicit specific and differential immune responses against each species.

  15. Induction and enhancement of cardiac cell differentiation from mouse and human induced pluripotent stem cells with cyclosporin-A.

    Directory of Open Access Journals (Sweden)

    Masataka Fujiwara

    Full Text Available Induced pluripotent stem cells (iPSCs are novel stem cells derived from adult mouse and human tissues by reprogramming. Elucidation of mechanisms and exploration of efficient methods for their differentiation to functional cardiomyocytes are essential for developing cardiac cell models and future regenerative therapies. We previously established a novel mouse embryonic stem cell (ESC and iPSC differentiation system in which cardiovascular cells can be systematically induced from Flk1(+ common progenitor cells, and identified highly cardiogenic progenitors as Flk1(+/CXCR4(+/VE-cadherin(- (FCV cells. We have also reported that cyclosporin-A (CSA drastically increases FCV progenitor and cardiomyocyte induction from mouse ESCs. Here, we combined these technologies and extended them to mouse and human iPSCs. Co-culture of purified mouse iPSC-derived Flk1(+ cells with OP9 stroma cells induced cardiomyocyte differentiation whilst addition of CSA to Flk1(+ cells dramatically increased both cardiomyocyte and FCV progenitor cell differentiation. Spontaneously beating colonies were obtained from human iPSCs by co-culture with END-2 visceral endoderm-like cells. Appearance of beating colonies from human iPSCs was increased approximately 4.3 times by addition of CSA at mesoderm stage. CSA-expanded human iPSC-derived cardiomyocytes showed various cardiac marker expressions, synchronized calcium transients, cardiomyocyte-like action potentials, pharmacological reactions, and ultra-structural features as cardiomyocytes. These results provide a technological basis to obtain functional cardiomyocytes from iPSCs.

  16. Human Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells in Phenotypic Screening: A Transforming Growth Factor-β Type 1 Receptor Kinase Inhibitor Induces Efficient Cardiac Differentiation.

    Science.gov (United States)

    Drowley, Lauren; Koonce, Chad; Peel, Samantha; Jonebring, Anna; Plowright, Alleyn T; Kattman, Steven J; Andersson, Henrik; Anson, Blake; Swanson, Bradley J; Wang, Qing-Dong; Brolen, Gabriella

    2016-02-01

    Several progenitor cell populations have been reported to exist in hearts that play a role in cardiac turnover and/or repair. Despite the presence of cardiac stem and progenitor cells within the myocardium, functional repair of the heart after injury is inadequate. Identification of the signaling pathways involved in the expansion and differentiation of cardiac progenitor cells (CPCs) will broaden insight into the fundamental mechanisms playing a role in cardiac homeostasis and disease and might provide strategies for in vivo regenerative therapies. To understand and exploit cardiac ontogeny for drug discovery efforts, we developed an in vitro human induced pluripotent stem cell-derived CPC model system using a highly enriched population of KDR(pos)/CKIT(neg)/NKX2.5(pos) CPCs. Using this model system, these CPCs were capable of generating highly enriched cultures of cardiomyocytes under directed differentiation conditions. In order to facilitate the identification of pathways and targets involved in proliferation and differentiation of resident CPCs, we developed phenotypic screening assays. Screening paradigms for therapeutic applications require a robust, scalable, and consistent methodology. In the present study, we have demonstrated the suitability of these cells for medium to high-throughput screens to assess both proliferation and multilineage differentiation. Using this CPC model system and a small directed compound set, we identified activin-like kinase 5 (transforming growth factor-β type 1 receptor kinase) inhibitors as novel and potent inducers of human CPC differentiation to cardiomyocytes. Significance: Cardiac disease is a leading cause of morbidity and mortality, with no treatment available that can result in functional repair. This study demonstrates how differentiation of induced pluripotent stem cells can be used to identify and isolate cell populations of interest that can translate to the adult human heart. Two separate examples of phenotypic

  17. Ectopic Myf5 or MyoD prevents the neuronal differentiation program in addition to inducing skeletal muscle differentiation, in the chick neural tube.

    Science.gov (United States)

    Delfini, Marie-Claire; Duprez, Delphine

    2004-02-01

    Forced expression of the bHLH myogenic factors, Myf5 and MyoD, in various mammalian cell lines induces the full program of myogenic differentiation. However, this property has not been extensively explored in vivo. We have taken advantage of the chick model to investigate the effect of electroporation of the mouse Myf5 and MyoD genes in the embryonic neural tube. We found that misexpression of either mouse Myf5 or MyoD in the chick neural tube leads to ectopic skeletal muscle differentiation, assayed by the expression of the myosin heavy chains in the neural tube and neural crest derivatives. We also showed that the endogenous neuronal differentiation program is inhibited under the influence of either ectopic mouse Myf5 or MyoD. We used this new system to analyse, in vivo, the transcriptional regulation between the myogenic factors. We found that MyoD and Myogenin expression can be activated by ectopic mouse Myf5 or MyoD, while Myf5 expression cannot be activated either by mouse MyoD or by itself. We also analysed the transcriptional regulation between the myogenic factors and the different genes involved in myogenesis, such as Mef2c, Pax3, Paraxis, Six1, Mox1, Mox2 and FgfR4. We established the existence of an unexpected regulatory loop between MyoD and FgfR4. The consequences for myogenesis are discussed.

  18. Electron- and positron-induced ionization of water molecules: Theory versus experiment at the triply differential scale

    Science.gov (United States)

    Singh, P.; Purohit, G.; Champion, C.; Patidar, V.

    2014-03-01

    Triply differential cross sections for electron- and positron-induced ionization of the 3a1 orbital of the water molecule are calculated within the second-order distorted-wave Born approximation. In this context, distorted-wave functions are numerically calculated by modeling both the initial and the final channels whereas single-center Slater-type wave functions are used for describing the molecular target. A good agreement with the existing experimental data is observed. Differences in the trends of differential cross sections are observed for the electron- and positron-impact ionization.

  19. Adipogenic human adenovirus Ad-36 induces commitment, differentiation, and lipid accumulation in human adipose-derived stem cells

    DEFF Research Database (Denmark)

    Pasarica, Magdalena; Mashtalir, Nazar; McAllister, Emily J

    2008-01-01

    , the effect of the virus on commitment, differentiation, and lipid accumulation was investigated in vitro in primary human adipose-derived stem/stromal cells (hASC). Ad-36 infected hASC in a time- and dose-dependent manner. Even in the presence of osteogenic media, Ad-36-infected hASC showed significantly...... and the accumulation of its extracellular fraction. hASC from subjects harboring Ad-36 DNA in their adipose tissue due to natural infection had significantly greater ability to differentiate compared with Ad-36 DNA-negative counterparts, which offers a proof of concept. Thus, Ad-36 has the potential to induce...

  20. Micro-RNA-21 regulates TGF-β-induced myofibroblast differentiation by targeting PDCD4 in tumor-stroma interaction.

    Science.gov (United States)

    Yao, Qin; Cao, Siyu; Li, Chun; Mengesha, Asferd; Kong, Beihua; Wei, Mingqian

    2011-04-15

    Transforming growth factor-β1 (TGF-β1) induces stromal fibroblast-to-myofibroblast transdifferentiation in the tumor-stroma interactive microenvironment via modulation of multiple phenotypic and functional genes, which plays a critical role in tumor progression. Up to now, the involvement of micro-RNAs (miRNAs) and their roles in TGF-β1-induced myofibroblast differentiation in tumor-stroma interaction are unclear. Using quantitative real-time RT-PCR, we demonstrated that the expression of micro-RNA-21 (miR-21) was upregulated in activated fibroblasts after treatment with TGF-β1 or conditioned medium from cancer cells. To determine the potential roles of miR-21 in TGF-β1-mediated gene regulation during myofibroblast conversion, we showed that miR-21 expression was downregulated by miR-21 inhibitor and upregulated by miR-21 mimic. Interestingly, downregulation of miR-21 with the inhibitor effectively inhibited TGF-β1-induced myofibroblast differentiation while upregulation of miR-21 with a mimic significantly promoted myofibroblast differentiation. We further demonstrated that MiR-21 directly targeted and downregulated programmed cell death 4 (PDCD4) gene, which in turn acted as a negative regulator of several phenotypic and functional genes of myofibroblasts. Taken together, these results suggested that miR-21 participated in TGF-β1-induced myofibroblast transdifferentiation in cancer stroma by targeting PDCD4. Copyright © 2010 UICC.

  1. Manipulation of radiation-induced bystander effect in prostate adenocarcinoma by dose and tumor differentiation grade: in vitro study.

    Science.gov (United States)

    Tubin, Slavisa; Valeriani, Maurizio; Salerno, Gerardo; Bracci, Stefano; Stoppacciaro, Antonella; Cardelli, Patrizia; Osti, Mattia Falchetto; De Sanctis, Vitaliana; Minniti, Giuseppe; Maurizi Enrici, Riccardo

    2015-02-01

    This in vitro study evaluated the ability of prostate adenocarcinoma (ADC) cells to induce radiation-induced bystander effect (RIBE) exploring the factors that may be responsible and affect its intensity. The idea was to mimic a strong, clinically applicable RIBE that could lead to the development of innovative approaches in modern radiotherapy of prostate cancer, especially for those patients with hormone-refractory ADC in which radiotherapy might have a limited role. Two human prostate cancer cell lines of different differentiation, PC-3 and DU-145, have been irradiated using wide range of doses to obtain radiation-conditioned medium (RCM), which was used to treat the unirradiated cells and to evaluate the cytokines level. Using a trypan blue dye exclusion method, cell growth was assessed. Prostate ADC cells were able to induce RIBE; intensity depended on dose and cell differentiation. RIBE intensity of DU-145 was not correlated with the cytokines level, while for PC-3 Interleukin-6 (IL-6) correlates with strongest RIBE induced by 20 Gy. RIBE can be manipulated by modifying radiation dose and depends on cell differentiation status. IL-6 correlates with RIBE after exposure of PC-3 to a very high dose of radiation, thus indicates its possible involvement in bystander signaling.

  2. Voltage-Gated K+ Channel, Kv3.3 Is Involved in Hemin-Induced K562 Differentiation.

    Science.gov (United States)

    Song, Min Seok; Choi, Seon Young; Ryu, Pan Dong; Lee, So Yeong

    2016-01-01

    Voltage-gated K+ (Kv) channels are well known to be involved in cell proliferation. However, even though cell proliferation is closely related to cell differentiation, the relationship between Kv channels and cell differentiation remains poorly investigated. This study demonstrates that Kv3.3 is involved in K562 cell erythroid differentiation. Down-regulation of Kv3.3 using siRNA-Kv3.3 increased hemin-induced K562 erythroid differentiation through decreased activation of signal molecules such as p38, cAMP response element-binding protein, and c-fos. Down-regulation of Kv3.3 also enhanced cell adhesion by increasing integrin β3 and this effect was amplified when the cells were cultured with fibronectin. The Kv channels, or at least Kv3.3, appear to be associated with cell differentiation; therefore, understanding the mechanisms of Kv channel regulation of cell differentiation would provide important information regarding vital cellular processes.

  3. α2 Integrin, extracellular matrix metalloproteinase inducer, and matrix metalloproteinase-3 act sequentially to induce differentiation of mouse embryonic stem cells into odontoblast-like cells

    Energy Technology Data Exchange (ETDEWEB)

    Ozeki, Nobuaki; Kawai, Rie; Hase, Naoko; Hiyama, Taiki; Yamaguchi, Hideyuki [Department of Endodontics, School of Dentistry, Aichi Gakuin University, Nagoya, Aichi 464-8651 (Japan); Kondo, Ayami [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, Nagoya 464-8650 (Japan); Nakata, Kazuhiko [Department of Endodontics, School of Dentistry, Aichi Gakuin University, Nagoya, Aichi 464-8651 (Japan); Mogi, Makio, E-mail: makio@dpc.agu.ac.jp [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, Nagoya 464-8650 (Japan)

    2015-02-01

    We previously reported that interleukin 1β acts via matrix metalloproteinase (MMP)-3 to regulate cell proliferation and suppress apoptosis in α2 integrin-positive odontoblast-like cells differentiated from mouse embryonic stem (ES) cells. Here we characterize the signal cascade underpinning odontoblastic differentiation in mouse ES cells. The expression of α2 integrin, extracellular matrix metalloproteinase inducer (Emmprin), and MMP-3 mRNA and protein were all potently increased during odontoblastic differentiation. Small interfering RNA (siRNA) disruption of the expression of these effectors potently suppressed the expression of the odontoblastic biomarkers dentin sialophosphoprotein, dentin matrix protein-1 and alkaline phosphatase, and blocked odontoblast calcification. Our siRNA, western blot and blocking antibody analyses revealed a unique sequential cascade involving α2 integrin, Emmprin and MMP-3 that drives ES cell differentiation into odontoblasts. This cascade requires the interaction between α2 integrin and Emmprin and is potentiated by exogenous MMP-3. Finally, although odontoblast-like cells potently express α2, α6, αV, β1, and β3, integrins, we confirmed that β1 integrin acts as the trigger for ES cell differentiation, apparently in complex with α2 integrin. These results demonstrate a unique and unanticipated role for an α2 integrin-, Emmprin-, and MMP-3-mediated signaling cascade in driving mouse ES cell differentiation into odontoblast-like cells. - Highlights: • Odontoblast differentiation requires activation of α2 integrin, Emmprin and MMP-3. • α2 integrin, Emmprin and MMP-3 form a sequential signaling cascade. • β1 integrin acts a specific trigger for odontoblast differentiation. • The role of these effectors is highly novel and unanticipated.

  4. Differential expression of genes during aflatoxin B1-induced hepatocarcinogenesis in tree shrews

    Institute of Scientific and Technical Information of China (English)

    Yuan Li; Dan Luo; Hui-Fen Yue; Li-Sheng Zhang; Jian-Ren Gu; Da-Fang Wan; Jian-Jia Su; Ji Cao; Chao Ou; Xiao-Kun Qiu; Ke-Chen Ban; Chun Yang; Liu-Liang Qin

    2004-01-01

    AIM: Through exploring the regulation of gene expression during hepatocarcinogenesis induced by aflatoxin B1 (AFB1),to find out the responsible genes for hepatocellular carcinoma (HCC) and to further understand the underlying molecular mechanism.METHODS: Tree shrews ( 7upaia belangeri chinensis)were treated with or without AFB1 for about 90 weeks. Liver biopsies were performed regularly during the animal experiment. Eight shares of total RNA were respectively isolated from 2 HCC tissues, 2 HCC-surrounding noncancerous liver tissues, 2 biopsied tissues at the early stage (30th week) of the experiment from the same animals as above, 1 mixed sample of three liver tissues biopsied at the beginning (0th week) of the experiment, and another 1 mixed sample of two liver tissues from the untreated control animals biopsied at the 90th week of the experiment. The samples were then tested with the method of AtlasTM cDNA microarray assay. The levels of gene expression in these tissues taken at different time points during hepatocarcinogenesis were compared.RESULTS: The profiles of differently expressed genes were quite different in different ways of comparison. At the same period of hepatocarcinogenesis, the genes in the same function group usually had the same tendency for up- or down-regulation. Among the checked 588 genes that were known to be related to human cancer, 89 genes (15.1%) were recognized as "important genes" because they showed frequent changes in different ways of comparison. The differentially expressed genes during hepatocarcinogenesis could be classified into four categories: genes up-regulated in HCC tissue, genes with similar expressing levels in both HCC and HCC-surrounding liver tissues which were higher than that in the tissues prior to the development of HCC,genes down-regulated in HCC tissue, and genes up-regulated prior to the development of HCC but down-regulated after the development of HCC.CONCLUSION: A considerable number of genes could change

  5. Differential expression of genes during aflatoxin B1-induced hepatocarcinogenesis in tree shrews

    Science.gov (United States)

    Li, Yuan; Wan, Da-Fang; Su, Jian-Jia; Cao, Ji; Ou, Chao; Qiu, Xiao-Kun; Ban, Ke-Chen; Yang, Chun; Qin, Liu-Liang; Luo, Dan; Yue, Hui-Fen; Zhang, Li-Sheng; Gu, Jian-Ren

    2004-01-01

    AIM: Through exploring the regulation of gene expression during hepatocarcinogenesis induced by aflatoxin B1 (AFB1), to find out the responsible genes for hepatocellular carcinoma (HCC) and to further understand the underlying molecular mechanism. METHODS: Tree shrews (Tupaia belangeri chinensis) were treated with or without AFB1 for about 90 weeks. Liver biopsies were performed regularly during the animal experiment. Eight shares of total RNA were respectively isolated from 2 HCC tissues, 2 HCC-surrounding non-cancerous liver tissues, 2 biopsied tissues at the early stage (30th week) of the experiment from the same animals as above, 1 mixed sample of three liver tissues biopsied at the beginning (0th week) of the experiment, and another 1 mixed sample of two liver tissues from the untreated control animals biopsied at the 90th week of the experiment. The samples were then tested with the method of AtlasTM cDNA microarray assay. The levels of gene expression in these tissues taken at different time points during hepatocarcinogenesis were compared. RESULTS: The profiles of differently expressed genes were quite different in different ways of comparison. At the same period of hepatocarcinogenesis, the genes in the same function group usually had the same tendency for up- or down-regulation. Among the checked 588 genes that were known to be related to human cancer, 89 genes (15.1%) were recognized as “important genes” because they showed frequent changes in different ways of comparison. The differentially expressed genes during hepatocarcinogenesis could be classified into four categories: genes up-regulated in HCC tissue, genes with similar expressing levels in both HCC and HCC-surrounding liver tissues which were higher than that in the tissues prior to the development of HCC, genes down-regulated in HCC tissue, and genes up-regulated prior to the development of HCC but down-regulated after the development of HCC. CONCLUSION: A considerable number of genes could

  6. Differential proteomic analysis of human erythroblasts undergoing apoptosis induced by epo-withdrawal.

    Science.gov (United States)

    Pellegrin, Stéphanie; Heesom, Kate J; Satchwell, Timothy J; Hawley, Bethan R; Daniels, Geoff; van den Akker, Emile; Toye, Ashley M

    2012-01-01

    The availability of Erythropoietin (Epo) is essential for the survival of erythroid progenitors. Here we study the effects of Epo removal on primary human erythroblasts grown from peripheral blood CD34(+) cells. The erythroblasts died rapidly from apoptosis, even in the presence of SCF, and within 24 hours of Epo withdrawal 60% of the cells were Annexin V positive. Other classical hallmarks of apoptosis were also observed, including cytochrome c release into the cytosol, loss of mitochondrial membrane potential, Bax translocation to the mitochondria and caspase activation. We adopted a 2D DIGE approach to compare the proteomes of erythroblasts maintained for 12 hours in the presence or absence of Epo. Proteomic comparisons demonstrated significant and reproducible alterations in the abundance of proteins between the two growth conditions, with 18 and 31 proteins exhibiting altered abundance in presence or absence of Epo, respectively. We observed that Epo withdrawal induced the proteolysis of the multi-functional proteins Hsp90 alpha, Hsp90 beta, SET, 14-3-3 beta, 14-3-3 gamma, 14-3-3 epsilon, and RPSA, thereby targeting multiple signaling pathways and cellular processes simultaneously. We also observed that 14 proteins were differentially phosphorylated and confirmed the phosphorylation of the Hsp90 alpha and Hsp90 beta proteolytic fragments in apoptotic cells using Nano LC mass spectrometry. Our analysis of the global changes occurring in the proteome of primary human erythroblasts in response to Epo removal has increased the repertoire of proteins affected by Epo withdrawal and identified proteins whose aberrant regulation may contribute to ineffective erythropoiesis.

  7. Caffeic acid phenethyl ester inhibits 3-MC-induced CYP1A1 expression through induction of hypoxia-inducible factor-1α

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyung Gyun [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Han, Eun Hee [Division of Life Science, Korea Basic Science Institute, Daejeon (Korea, Republic of); Im, Ji Hye; Lee, Eun Ji; Jin, Sun Woo [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of)

    2015-09-25

    Caffeic acid phenethyl ester (CAPE), a natural component of propolis, is reported to have anticarcinogenic properties, although its precise chemopreventive mechanism remains unclear. In this study, we examined the effects of CAPE on 3-methylcholanthrene (3-MC)-induced CYP1A1 expression and activities. CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. Moreover, CAPE inhibited 3-MC-induced CYP1A1 activity, mRNA expression, protein level, and promoter activity. CAPE treatment also decreased 3-MC-inducible xenobiotic-response element (XRE)-linked luciferase, aryl hydrocarbons receptor (AhR) transactivation and nuclear localization. CAPE induced hypoxia inducible factor-1α (HIF-1α) protein level and HIF-1α responsible element (HRE) transcriptional activity. CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 protein expression. Taken together, CAPE decreases 3-MC-mediated CYP1A1 expression, and this inhibitory response is associated with inhibition of AhR and HIF-1α induction. - Highlights: • CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. • CAPE inhibited 3-MC-induced CYP1A1 expression. • CAPE induced HIF-1α induction. • CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 expression.

  8. Cell-Penetrating Peptide as a Means of Directing the Differentiation of Induced-Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Taku Kaitsuka

    2015-11-01

    Full Text Available Protein transduction using cell-penetrating peptides (CPPs is useful for the delivery of large protein molecules, including some transcription factors. This method is safer than gene transfection methods with a viral vector because there is no risk of genomic integration of the exogenous DNA. Recently, this method was reported as a means for the induction of induced pluripotent stem (iPS cells, directing the differentiation into specific cell types and supporting gene editing/correction. Furthermore, we developed a direct differentiation method to obtain a pancreatic lineage from mouse and human pluripotent stem cells via the protein transduction of three transcription factors, Pdx1, NeuroD, and MafA. Here, we discuss the possibility of using CPPs as a means of directing the differentiation of iPS cells and other stem cell technologies.

  9. Differentiation of Induced Pluripotent Stem Cells to Lentoid Bodies Expressing a Lens Cell-Specific Fluorescent Reporter.

    Directory of Open Access Journals (Sweden)

    Taruna Anand

    Full Text Available Curative approaches for eye cataracts and other eye abnormalities, such as myopia and hyperopia currently suffer from a lack of appropriate models. Here, we present a new approach for in vitro growth of lentoid bodies from induced pluripotent stem (iPS cells as a tool for ophthalmological research. We generated a transgenic mouse line with lens-specific expression of a fluorescent reporter driven by the alphaA crystallin promoter. Fetal fibroblasts were isolated from transgenic fetuses, reprogrammed to iPS cells, and differentiated to lentoid bodies exploiting the specific fluorescence of the lens cell-specific reporter. The employment of cell type-specific reporters for establishing and optimizing differentiation in vitro seems to be an efficient and generally applicable approach for developing differentiation protocols for desired cell populations.

  10. Cell-Penetrating Peptide as a Means of Directing the Differentiation of Induced-Pluripotent Stem Cells.

    Science.gov (United States)

    Kaitsuka, Taku; Tomizawa, Kazuhito

    2015-11-06

    Protein transduction using cell-penetrating peptides (CPPs) is useful for the delivery of large protein molecules, including some transcription factors. This method is safer than gene transfection methods with a viral vector because there is no risk of genomic integration of the exogenous DNA. Recently, this method was reported as a means for the induction of induced pluripotent stem (iPS) cells, directing the differentiation into specific cell types and supporting gene editing/correction. Furthermore, we developed a direct differentiation method to obtain a pancreatic lineage from mouse and human pluripotent stem cells via the protein transduction of three transcription factors, Pdx1, NeuroD, and MafA. Here, we discuss the possibility of using CPPs as a means of directing the differentiation of iPS cells and other stem cell technologies.

  11. PED/PEA-15 induces autophagy and mediates TGF-beta1 effect on muscle cell differentiation.

    Science.gov (United States)

    Iovino, S; Oriente, F; Botta, G; Cabaro, S; Iovane, V; Paciello, O; Viggiano, D; Perruolo, G; Formisano, P; Beguinot, F

    2012-07-01

    TGF-beta1 has been shown to induce autophagy in certain cells but whether and how this action is exerted in muscle and whether this activity relates to TGF-beta1 control of muscle cell differentiation remains unknown. Here, we show that expression of the autophagy-promoting protein phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) progressively declines during L6 and C2C12 skeletal muscle cell differentiation. PED/PEA-15 underwent rapid induction upon TGF-beta1 exposure of L6 and C2C12 myoblasts, accompanied by impaired differentiation into mature myotubes. TGF-beta1 also induced autophagy in the L6 and C2C12 cells through a PP2A/FoxO1-mediated mechanism. Both the TGF-beta1 effect on differentiation and that on autophagy were blocked by specific PED/PEA-15 ShRNAs. Myoblasts stably overexpressing PED/PEA-15 did not differentiate and showed markedly enhanced autophagy. In these same cells, the autophagy inhibitor 3-methyladenine rescued TGF-beta1 effect on both autophagy and myogenesis, indicating that PED/PEA-15 mediates TGF-beta1 effects in muscle. Muscles from transgenic mice overexpressing PED/PEA-15 featured a significant number of atrophic fibers, accompanied by increased light chain 3 (LC3)II to LC3I ratio and reduced PP2A/FoxO1 phosphorylation. Interestingly, these mice showed significantly impaired locomotor activity compared with their non-transgenic littermates. TGF-beta1 causes transcriptional upregulation of the autophagy-promoting gene PED/PEA-15, which in turn is capable to induce atrophic responses in skeletal muscle in vivo.

  12. Growth inhibition and differentiation of promyelocytic cells (HL-60) induced by BC-4, an active principle from Boswellia carterii Birdw.

    Science.gov (United States)

    Jing, Y; Xia, L; Han, R

    1992-03-01

    The induction of cell differentiation and growth inhibition of HL-60 cells by Bc-4, an active principle from Boswellia carterii, was studied in vitro and in vivo. The proliferation of HL-60 cells was found to be inhibited by Bc-4 at a concentration of 5-10 micrograms/ml. Under the action of Bc-4, the acid phosphatase and NBT reduction activities in HL-60 cells were increased, and phagocytosis of cells was also induced. All these activities were concentration dependent. The HL-60 cells were induced by Bc-4 to differentiate into more mature cells morphologically. The in vivo growth of HL-60 cells in mouse subrenal capsules (SRC) and in diffusion chambers inoculated into mice was inhibited by Bc-4 at a dose of 50 mg/kg. The morphology of HL-60 cells treated by Bc-4 in diffusion chambers exhibited characteristics of mature granulocytic cells.

  13. miR-451 Up-regulation, Induce Erythroid Differentiation of CD133+cells Independent of Cytokine Cocktails

    Directory of Open Access Journals (Sweden)

    Fatemeh Kouhkan

    2013-06-01

    Full Text Available   Objective(s: Erythropoiesis is regulated by some extrinsic and intrinsic factors as microRNAs (miRNAs. miRNAs are endogenously small non-coding regulatory RNAs which play vital roles in the variety of cellular fate, critical processes; growth, apoptosis, metabolism, survival of the cells and specially differentiation. Several miRNAs such as miR-16 and miR-451 have been shown to be correlated with erythroid differentiation. Taking into account the importance of miRNAs in cellular differentiation, the goal of the present study was to examine the role of miRNAs in hematopoietic stem cells (HSC differentiation into the erythroid cells in the absence of growth factors and stimulatory cytokines.   Materials and Methods: CD133+ stem cells were infected with lentiviruses containing miR-451/miR-16 precursor sequence, erythroid differentiation was evaluated using RT-PCR for hemoglobin chains and surface antigens, also by banzidine staining. Results: MiR-451up-regulation, but not miR-16, could induce α, β and γ-globin expression in CD133+ cells and have strong correlation with appearance of CD71 and CD235a markers in these cells. Moreover, miR-451 up-regulation increases the banzidine positive cells to ~ %40. Conclusion: Our results provide strong evidence that miR-451 up-regulation strongly induces erythroid differentiation and maturation of CD133+ stem cells. Hence, this method may provide a useful technique for the production of artificial blood RBC and be used as a new strategy for gene therapy of hemoglobinopathies, such as β-thalassemias and sickle cell anemia.

  14. ERK1 and ERK2 are involved in recruitment and maturation of human mesenchymal stem cells induced to adipogenic differentiation

    Institute of Scientific and Technical Information of China (English)

    Elisabetta Donzelli; Caterina Lucchini; Elisa Ballarini; Arianna Scuteri; Fabrizio Carini; Giovanni Tredici; Mariarosaria Miloso

    2011-01-01

    Adipocytes' biology and the mechanisms that control adipogenesis have gained importance because of the need to develop therapeutic strategies to control obesity and the related pathologies. Human mesenchymal stem cells (hMSCs), undifferentiated stem cells present in the bone marrow that are physiological precursors of adipocytes, were induced to adipogenic differentiation. The molecular mechanisms on the basis of the adipogenesis were evaluated, focusing on the MAPKinases ERK1 and ERK2, which are involved in many biological and cellular processes. ERK1 and ERK2 phosphorylation was reduced with different timing and intensity for the two isoforms in treated hMSCs in comparison with control cells until day 10 and then at 14-28 days, it reached the level of untreated cultures. The total amount of ERK1 was also decreased up to day 10 and then was induced to the level of untreated cultures, whereas the expression of ERK2 was not changed following adipogenic induction. Treatment with the specific ERK1/2 inhibitor U0126 during the whole differentiation period hampered hMSCs' adipogenic differentiation, as lipid droplets appeared in very few cells and were reduced in number and size. When U0126 was administered only during the initial phase of differentiation, the number of hMSCs recruited to adipogenesis was reduced while, when it was administered later, hMSCs did not acquire a mature adipocytic phenotype. ERK1 and ERK2 are important for hMSC adipogenic differentiation since any alteration to the correct timing of their phosphorylation affects either the recruitment into the differentiation program and the extent of their maturation.

  15. Osteoblastic differentiation of stem cells from human exfoliated deciduous teeth induced by thermosensitive hydrogels with strontium phosphate

    Energy Technology Data Exchange (ETDEWEB)

    Su, Wen-Ta, E-mail: f10549@ntut.edu.tw [Department of Chemical Engineering and Biotechnology National Taipei University of Technology, Taipei, Taiwan (China); Chou, Wei-Ling [Department of Chemical Engineering and Biotechnology National Taipei University of Technology, Taipei, Taiwan (China); Chou, Chih-Ming [Department of Biochemistry, Taipei Medical University, Taipei, Taiwan (China)

    2015-07-01

    Stem cells from human exfoliated deciduous teeth (SHEDs) are a novel source of multi-potential stem cells for tissue engineering because of their potential to differentiate into multiple cell lineages. Strontium exhibits an important function in bone remodeling because it can simulate bone formation and decrease bone resorption. Hydrogels can mimic the natural cellular environment. The association of hydrogels with cell viability is determined using biological tests, including rheological experiments. In this study, osteogenic differentiation was investigated through SHED encapsulation in hydrogels containing strontium phosphate. Results of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and proliferating cell nuclear antigen (PCNA) immunofluorescence staining indicated that the cells grew well and SHEDs proliferated in the hydrogels. Strontium-loaded chitosan-based hydrogels induced the biomineralization and high expression of alkaline phosphatase. Moreover, the expression levels of bone-related genes, including type-I collagen, Runx2, osteopontin (OP), and osteonectin (ON), were up-regulated during the osteogenic differentiation of SHEDs. This study demonstrated that strontium can be an effective inducer of osteogenesis for SHEDs. Elucidating the function of bioceramics (such as strontium) is useful in designing and developing strategies for bone tissue engineering. - Highlights: • SHEDs have been considered as alternative sources of adult stem cells in tissue engineering. • Strontium phosphate can enhance the osteogenic differentiation of SHEDs. • Hydrogels can mimic the natural cellular environment. • Bioceramics (such as strontium) is useful in designing and developing strategies for bone tissue engineering.

  16. Growth factors and medium hyperglycemia induce Sox9+ ductal cell differentiation into β cells in mice with reversal of diabetes.

    Science.gov (United States)

    Zhang, Mingfeng; Lin, Qing; Qi, Tong; Wang, Tiankun; Chen, Ching-Cheng; Riggs, Arthur D; Zeng, Defu

    2016-01-19

    We previously reported that long-term administration of a low dose of gastrin and epidermal growth factor (GE) augments β-cell neogenesis in late-stage diabetic autoimmune mice after eliminating insulitis by induction of mixed chimerism. However, the source of β-cell neogenesis is still unknown. SRY (sex-determining region Y)-box 9(+) (Sox9(+)) ductal cells in the adult pancreas are clonogenic and can give rise to insulin-producing β cells in an in vitro culture. Whether Sox9(+) ductal cells in the adult pancreas can give rise to β cells in vivo remains controversial. Here, using lineage-tracing with genetic labeling of Insulin- or Sox9-expressing cells, we show that hyperglycemia (>300 mg/dL) is required for inducing Sox9(+) ductal cell differentiation into insulin-producing β cells, and medium hyperglycemia (300-450 mg/dL) in combination with long-term administration of low-dose GE synergistically augments differentiation and is associated with normalization of blood glucose in nonautoimmune diabetic C57BL/6 mice. Short-term administration of high-dose GE cannot augment differentiation, although it can augment preexisting β-cell replication. These results indicate that medium hyperglycemia combined with long-term administration of low-dose GE represents one way to induce Sox9(+) ductal cell differentiation into β cells in adult mice.

  17. Exposure of CD34+ precursors to cytostatic anthraquinone-derivatives induces rapid dendritic cell differentiation: implications for cancer immunotherapy.

    Science.gov (United States)

    van de Ven, Rieneke; Reurs, Anneke W; Wijnands, Pepijn G J T B; van Wetering, Sandra; Kruisbeek, Ada M; Hooijberg, Erik; Scheffer, George L; Scheper, Rik J; de Gruijl, Tanja D

    2012-02-01

    Appropriate activation of dendritic cells (DC) is essential for successful active vaccination and induction of cell-mediated immunity. The scarcity of precursor cells, as well as long culture methods, have hampered wide-scale application of DC vaccines derived from CD34(+) precursors, despite their suggested superior efficacy over the more commonly applied monocyte-derived DC (MoDC). Here, employing the CD34(+)/CD14(+) AML-derived human DC progenitor cell line MUTZ3, we show that cytostatic anthraquinone-derivatives (i.e., the anthracenedione mitoxantrone and the related anthracyclin doxorubicin) induce rapid differentiation of CD34(+) DC precursors into functional antigen-presenting cells (APC) in a three-day protocol. The drugs were found to act specifically on CD34(+), and not on CD14(+) DC precursors. Importantly, these observations were confirmed for primary CD34(+) and CD14(+) DC precursors from peripheral blood. Mitoxantrone-generated DC were fully differentiated within three days and after an additional 24 h of maturation, were as capable as standard 9-day differentiated and matured DC to migrate toward the lymph node-homing chemokines CCL19 and CCL21, to induce primary allogeneic T cell proliferation, and to prime functional MART1-specific CD8(+) T lymphocytes. Our finding that anthraquinone-derivatives like mitoxantrone support rapid high-efficiency differentiation of DC precursors may have consequences for in vitro production of DC vaccines as well as for novel immunochemotherapy strategies.

  18. p-Hydroxylcinnamaldehyde induces the differentiation of oesophageal carcinoma cells via the cAMP-RhoA-MAPK signalling pathway

    Science.gov (United States)

    Ma, Ming; Zhao, Lian-mei; Yang, Xing-xiao; Shan, Ya-nan; Cui, Wen-xuan; Chen, Liang; Shan, Bao-en

    2016-01-01

    p-Hydroxylcinnamaldehyde (CMSP) has been identified as an inhibitor of the growth of various cancer cells. However, its function in oesophageal squamous cell carcinoma (ESCC) and the underlying mechanism remain unclear. The aim of the present study was to characterize the differentiation effects of CMSP, as well as its mechanism in the differentiation of ESCC Kyse30 and TE-13 cells. The function of CMSP in the viability, colony formation, migration and invasion of Kyse30 and TE-13 cells was determined by MTS, colony-formation, wound healing and transwell assays. Western blotting and pull-down assays were used to investigate the effect of CMSP on the expression level of malignant markers of ESCC, as well as the activity of MAPKs, RhoA and GTP-RhoA in Kyse30 and TE-13 cells. We found that CMSP could inhibit proliferation and migration and induce Kyse30 and TE-13 cell differentiation, characterized by dendrite-like outgrowth, decreased expression of tumour-associated antigens, as well as the decreased expression of malignant markers. Furthermore, increased cAMP, p-P38 and decreased activities of ERK, JNK and GTP-RhoA, were detected after treatment with CMSP. These results indicated that CMSP induced the differentiation of Kyse30 and TE-13 cells through mediating the cAMP-RhoA-MAPK axis, which might provide new potential strategies for ESCC treatment. PMID:27501997

  19. PEG-mediated osmotic stress induces premature differentiation of the root apical meristem and outgrowth of lateral roots in wheat.

    Science.gov (United States)

    Ji, Hongtao; Liu, Ling; Li, Kexue; Xie, Qingen; Wang, Zhijuan; Zhao, Xuhua; Li, Xia

    2014-09-01

    Water stress is one of the major environmental stresses causing growth retardation and yield loss of plants. In the past decades, osmotic adjustment, antioxidant protection, and stomatal movement have been extensively studied, but much less attention has been paid to the study of root system reprogramming to maximize water absorption and survival under water stress. Here, it is shown that polyethylene glycol (PEG)-simulated mild and moderate osmotic stress induced premature differentiation of the root apical meristem (RAM). It is demonstrated that RAM premature differentiation is a conserved adaptive mechanism that is widely adopted by various plants to cope with osmotic stress simulated by PEG 8000, and the occurrence of RAM premature differentiation is directly related to stress tolerance of plants. It is shown that the osmotic stress-induced premature differentiation caused growth cessation of primary roots allowing outgrowth of lateral roots. This work has uncovered a key mechanism for controlling the plastic development of the root system by which plants are capable of survival, growth, or reproduction under water stress.

  20. Factors inducing human umbilical cord blood-derived mesenchymal stem cells to differentiate into neuron-like cells

    Institute of Scientific and Technical Information of China (English)

    Nawei Zhang; Fengqing Ji

    2006-01-01

    OBJECTIVE:Human umbilical cord blood-derived mesenchymal stem cells (HUCB-derived MSCs)can differentiate into neuron-like cells,which can be used to treat some central nervous system(CNS)diseases.To investigate the factors,which can induce HUCB-derived MSCs to differentiate into neuron-like cells,so as to find effective methods for future clinical application.DATA SOURCES:Using the key terms"human umbilical cord blood"combined with"mesenchymal stem cells,neuron-like cells,neural cells"respectively,the relevant articles in English published during the period from January 1999 to June 2006 were searched from the Medline database.Meanwhile,relevant Chinese articles published from January 1999 to June 2006 were searched Using the same key terms.STUDY SELECTION: All articles associated with the differentiation from human umbilical cord blood into neuron-like cells were selected firstly.Then the full texts were looked up by searchling Ovid medical Journals full-text database and Elsevier Electrical Journals Full-text Database.Articles with full expeiments,enrolled in inducible factors or involved inducible mechanism were retdeved.DATA EXTRACTION:Among 119 collected correlative articles,29 were involved and 90 were excluded.DATA SYNTHESIS:The inducible factors of HUCB-derived MSCs differentiatling into neuron-like cells included renal endothelial growth factors,fibroblasts,β-mercaptoethanol,dimethyl sulfoxide,butyl hydroxyl anisol,brain-derived neurotrophic factor,Danshen,retinoic acid,sodium ferulate and so on,but its mechanism was unclear.CONCLUSION:Human umbilical cord blood-derived MSCs can differentiate into neuron-like cells,with varied inductors.

  1. A Triterpenoid Inhibited Hormone-Induced Adipocyte Differentiation and Alleviated Dexamethasone-Induced Insulin Resistance in 3T3-L1 adipocytes.

    Science.gov (United States)

    Qin, Ji-Huan; Ma, Jun-Zeng; Yang, Xing-Wei; Hu, Ying-Jie; Zhou, Juan; Fu, Lin-Chun; Tian, Ru-Hua; Liu, Shan; Xu, Gang; Shen, Xiao-Ling

    2015-06-01

    6α-Hydroxylup-20(29)-en-3-on-28-oic acid (1), a natural triterpenoid, was found to possess the ability in a dose-dependent manner inhibiting hormone-induced adipocyte differentiation in 3T3-L1 preadipocytes, and restoring glucose consuming ability in dexamethasone (DXM)-induced insulin resistant 3T3-L1 adipocytes. Compound 1 was also found to ameliorate DXM-induced adipocyte dysfunction in lipolysis and adipokine secretion. Mechanistic studies revealed that 1 inhibited adipocyte differentiation in 3T3-L1 preadipocytes via down-regulating hormone-stimulated gene transcription of peroxisome proliferator-activated receptor γ and CCAAT-enhancer-binding protein alpha which are key factors in lipogenesis, and restored DXM-impaired glucose consuming ability in differentiated 3T3-L1 adipocytes via repairing insulin signaling pathway and activating down-stream signaling transduction by phosphorylation of signaling molecules PI3K/p85, Akt2 and AS160, thus leading to increased translocation of glucose transporter type 4 and transportation of glucose.

  2. Retinoid-induced differentiation of acute promyelocytic leukemia involves PML-RARalpha-mediated increase of type II transglutaminase.

    Science.gov (United States)

    Benedetti, L; Grignani, F; Scicchitano, B M; Jetten, A M; Diverio, D; Lo Coco, F; Avvisati, G; Gambacorti-Passerini, C; Adamo, S; Levin, A A; Pelicci, P G; Nervi, C

    1996-03-01

    All-trans retinoic acid (t-RA) administration leads to complete remission in acute promyelocytic leukemia (APL) patients by inducing growth arrest and differentiation of the leukemic clone. In the present study, we show that t-RA treatment dramatically induced type II transglutaminase (type II TGase) expression in cells carrying the t(15;17) translocation and expressing the PML-RARalpha product such as the APL-derived NB4 cell line and fresh leukemic cells from APL patients. This induction correlated with t-RA-induced growth arrest, granulocytic differentiation, and upregulation of the leukocyte adherence receptor beta subunit (CD18) gene expression. The increase in type II TGase was not abolished by cycloheximide treatment, suggesting that synthesis of a protein intermediate was not required for the induction. t-RA did not significantly alter the rate of growth arrest and did not stimulate differentiation and type II TGase activity in NB4.306 cells, a t-RA-resistant subclone of the NB4 cell line, or in leukemic cells derived from two patients morphologically defined as APL but lacking the t(15;17). However, in NB4.306 cells, t-RA treatment was able to increase CD18 mRNA expression in a manner similar to NB4 cells. The molecular mechanisms involved in the induction of these genes were investigated. In NB4 cells, using novel receptor-selective ligands such as 9-cis-RA, TTNPB, AM580, and SR11217, we found that RAR- and RARalpha-selective retinoids were able to induce growth arrest, granulocytic differentiation, and type II TGase, whereas the RXR-selective retinoid SR11217 was inactive. Moreover, an RAR alpha-antagonist completely inhibited the expression of type II TGase and CD18 induced by these selective retinoids in NB4 cells. In NB4.306 cells, an RARalpha-dependent signaling pathway was found involved in the modulation of CD18 expression. In addition, expression of the PML-RARalpha gene in myeloid U937 precursor cells resulted in the ability of these cells to

  3. Effect of IFN-γ and dexamethasone on TGF-β1-induced human fetal lung fibroblast-myofibroblast differentiation

    Institute of Scientific and Technical Information of China (English)

    Li GU; Yuan-jue ZHU; Zi-jian GUO; Xing-xiang XU; Wen-bing XU

    2004-01-01

    AIM: To study whether Smads signaling pathway was involved in human fetal lung fibroblast-myofibroblast differentiation induced by transforming growth factor (TGF)-β1 and the role of interferon (IFN)-γ, dexamethasone (DEX)in the fibroblast-myofibroblast differentiation. METHODS: α-Smooth muscle actin (α-SMA), Smad2/3, and Smad7protein were assessed by Western blot. Collagen protein was analyzed by measuring hydroxyproline. α-SMA and collagen Ⅲ mRNA were assessed by RT-PCR. Myofibroblasts morphology and Smad2/3 nuclear translocation were assessed by immunohistochemistry. The overexpression of Smad7, a negative mediator of Smads signaling pathway, was acquired by transfection of Smad7 vector. RESULTS: During fibroblast-myofibroblast differentiation induced by TGF-β1, IFN-γ 200 μg/L markedly blocked TGF-β1-induced α-SMA protein expression (P<0.01),collagen protein (P<0.01) and mRNA (P<0.05) expression, and myofibroblasts morphological transformation, but DEX 10 μmol/L augmented TGF-β1-induced α-SMA expression (P<0.01). For myofibroblasts, both IFN-γ 200 μg/L and DEX 10 μmol/L did not inhibit α-SMA expression (P>0.05) and collagen protein (P>0.05) and mRNA expression (P>0.05) and did not change myofibroblasts morphology. Transient transfection of Smad7 vector resulted in significant inhibition of TGF-β1-induced α-SMA expression (P<0.01). IFN-γ 200 μg/L did not block TGF-β1-stimulated Smad2/3 phosphorylation and their nuclear translocation. CONCLUSION: TGF-β1 induced fibroblastmyofibroblast differentiation in a Smad proteins-dependent manner. IFN-γ could block this process but it was not mediated by interrupting smad2/3 phosphorylation and their nuclear translocation and DEX played a synergism with TGF-β1. Differentiated myofibroblasts, however, were resistant to both IFN-γ and DEX.

  4. α-Tocopherol induces hematopoietic stem/progenitor cell expansion and ERK1/2-mediated differentiation.

    Science.gov (United States)

    Nogueira-Pedro, Amanda; Barbosa, Christiano M V; Segreto, Helena Regina Comodo; Lungato, Lisandro; D'Almeida, Vania; Moraes, Andrea Aparecida F S; Miranda, Antonio; Paredes-Gamero, Edgar Julian; Ferreira, Alice Teixeira

    2011-12-01

    Tocopherols promote or inhibit growth in different cell types. In the hematopoietic system, the radioprotective property of tocopherols is thought to act through the expansion of primitive hematopoietic cells. However, the mechanisms activated by tocopherols and which HPs are affected remain poorly understood. To better address these questions, mice were treated with α-tocopherol, and its effects were investigated in the BM microenvironment. α-Tocopherol induced increased proliferation in HSC/HP cells, leading to BM hyperplasia. In addition, differentiation to the granulocytic/monocytic lineage was enhanced by α-tocopherol treatment. α-Tocopherol treatment resulted in decreased basal phosphorylation of ERK1/2, PKC, and STAT-5 in HSC/HP cells. In contrast, α-tocopherol enhanced ERK1/2 activation in response to IL-3 stimulation in HSC/HP cells without altering the expression of IL-3Rs. Moreover, α-tocopherol-induced differentiation and ERK1/2 activation were abolished in mice pretreated with a MEK inhibitor (PD98059); however, pretreatment with PD98059 did not reduce the α-tocopherol-mediated increase in HSC/HP cells but instead, further enhanced their proliferation. Therefore, α-tocopherol induces expansion of HSC/HP cells by a nonidentified intracellular pathway and granulocytic/monocytic differentiation through ERK1/2 activation.

  5. Co-stimulation with bone morphogenetic protein-9 and FK506 induces remarkable osteoblastic differentiation in rat dedifferentiated fat cells.

    Science.gov (United States)

    Nakamura, Toshiaki; Shinohara, Yukiya; Momozaki, Sawako; Yoshimoto, Takehiko; Noguchi, Kazuyuki

    2013-10-18

    Dedifferentiated fat (DFAT) cells, which are isolated from mature adipocytes using the ceiling culture method, exhibit similar characteristics to mesenchymal stem cells, and possess adipogenic, osteogenic, chondrogenic, and myogenic potentials. Bone morphogenetic protein (BMP)-2 and -9, members of the transforming growth factor-β superfamily, exhibit the most potent osteogenic activity of this growth factor family. However, the effects of BMP-2 and BMP-9 on the osteogenic differentiation of DFAT remain unknown. Here, we examined the effects of BMP-2 and BMP-9 on osteoblastic differentiation of rat DFAT (rDFAT) cells in the presence or absence of FK506, an immunosuppressive agent. Co-stimulation with BMP-9 and FK506 induced gene expression of runx2, osterix, and bone sialoprotein, and ALP activity compared with BMP-9 alone, BMP-2 alone and BMP-2+FK506 in rDFAT cells. Furthermore, it caused mineralization of cultures and phosphorylation of smad1/5/8, compared with BMP-9 alone. The ALP activity induced by BMP-9+FK506 was not influenced by addition of noggin, a BMP antagonist. Our data suggest that the combination of BMP-9 and FK506 potently induces osteoblastic differentiation of rDFAT cells.

  6. Magnetorheological elastomer with stiffness-variable characteristics based on induced current applied to differential mount of vehicles

    Science.gov (United States)

    Jeong, Un-Chang; Yoon, Ji-Hyun; Yang, In-Hyung; Jeong, Jae-Eun; Kim, Jin-Su; Chung, Kyung-Ho; Oh, Jae-Eung

    2013-11-01

    A differential mount with elastomers is installed to insulate vibration transmitted from the engine to the body through the propeller shaft. Since existing differential mounts adopt an elastomer of uniform stiffness, it is difficult to meet both the requirements for steering performance and driving comfort at the same time. In order to overcome this limitation, this study suggests a magnetorheological elastomer (MRE)-based stiffness-variable differential mount which allows the mount’s stiffness to vary reversibly or instantly. The stiffness-variable differential mount was designed with a new inner structure where a magnetic field can be induced in the MRE. Further, the geometry of the MRE was optimized by means of the response surface method to achieve a targeted level of stiffness. The variable performance of the stiffness-variable differential mount was evaluated with the dynamic stiffness when a current of 3 A (0.287 T) was applied. As a result, it was found that the average increase in dynamic stiffness was 4.41 kgf mm-1 over an excitation frequency range of 60-100 Hz, a critical point of variation for dynamic stiffness, and 3.60 kgf mm-1 over an excitation frequency range of less than 100 Hz.

  7. Differentiation of induced pluripotent stem cells of swine into rod photoreceptors and their integration into the retina.

    Science.gov (United States)

    Zhou, Liang; Wang, Wei; Liu, Yongqing; Fernandez de Castro, Juan; Ezashi, Toshihiko; Telugu, Bhanu Prakash V L; Roberts, R Michael; Kaplan, Henry J; Dean, Douglas C

    2011-06-01

    Absence of a regenerative pathway for damaged retina following injury or disease has led to experiments using stem cell transplantation for retinal repair, and encouraging results have been obtained in rodents. The swine eye is a closer anatomical and physiological match to the human eye, but embryonic stem cells have not been isolated from pig, and photoreceptor differentiation has not been demonstrated with induced pluripotent stem cells (iPSCs) of swine. Here, we subjected iPSCs of swine to a rod photoreceptor differentiation protocol consisting of floating culture as embryoid bodies followed by differentiation in adherent culture. Real-time PCR and immunostaining of differentiated cells demonstrated loss of expression of the pluripotent genes POU5F1, NANOG, and SOX2 and induction of rod photoreceptor genes RCVRN, NRL, RHO, and ROM1. While these differentiated cells displayed neuronal morphology, culturing on a Matrigel substratum triggered a further morphological change resulting in concentration of rhodopsin (RHO) and rod outer segment-specific membrane protein 1 in outer segment-like projections resembling those on primary cultures of rod photoreceptors. The differentiated cells were transplanted into the subretinal space of pigs treated with iodoacetic acid to eliminate rod photoreceptors. Three weeks after transplantation, engrafted RHO+ cells were evident in the outer nuclear layer where photoreceptors normally reside. A portion of these transplanted cells had generated projections resembling outer segments. These results demonstrate that iPSCs of swine can differentiate into photoreceptors in culture, and these cells can integrate into the damaged swine neural retina, thus, laying a foundation for future studies using the pig as a model for retinal stem cell transplantation.

  8. YAP stabilizes SMAD1 and promotes BMP2-induced neocortical astrocytic differentiation.

    Science.gov (United States)

    Huang, Zhihui; Hu, Jinxia; Pan, Jinxiu; Wang, Ying; Hu, Guoqing; Zhou, Jiliang; Mei, Lin; Xiong, Wen-Cheng

    2016-07-01

    ‪YAP (yes-associated protein), a key transcriptional co-factor that is negatively regulated by the Hippo pathway, is crucial for the development and size control of multiple organs, including the liver. However, its role in the brain remains unclear. Here, we provide evidence for YAP regulation of mouse neocortical astrocytic differentiation and proliferation. YAP was undetectable in neurons, but selectively expressed in neural stem cells (NSCs) and astrocytes. YAP in NSCs was required for neocortical astrocytic differentiation, with no apparent role in self-renewal or neural differentiation. However, YAP in astrocytes was necessary for astrocytic proliferation. Yap (Yap1) knockout, Yap(nestin) conditional knockout and Yap(GFAP) conditional knockout mice displayed fewer neocortical astrocytes and impaired astrocytic proliferation and, consequently, death of neocortical neurons. Mechanistically, YAP was activated by BMP2, and the active/nuclear YAP was crucial for BMP2 induction and stabilization of SMAD1 and astrocytic differentiation. Expression of SMAD1 in YAP-deficient NSCs partially rescued the astrocytic differentiation deficit in response to BMP2. Taken together, these results identify a novel function of YAP in neocortical astrocytic differentiation and proliferation, and reveal a BMP2-YAP-SMAD1 pathway underlying astrocytic differentiation in the developing mouse neocortex.

  9. Selective neuronal differentiation of neural stem cells induced by nanosecond microplasma agitation

    Directory of Open Access Journals (Sweden)

    Z. Xiong

    2014-03-01

    Full Text Available An essential step for therapeutic and research applications of stem cells is their ability to differentiate into specific cell types. Neuronal cells are of great interest for medical treatment of neurodegenerative diseases and traumatic injuries of central nervous system (CNS, but efforts to produce these cells have been met with only modest success. In an attempt of finding new approaches, atmospheric-pressure room-temperature microplasma jets (MPJs are shown to effectively direct in vitro differentiation of neural stem cells (NSCs predominantly into neuronal lineage. Murine neural stem cells (C17.2-NSCs treated with MPJs exhibit rapid proliferation and differentiation with longer neurites and cell bodies eventually forming neuronal networks. MPJs regulate ~75% of NSCs to differentiate into neurons, which is a higher efficiency compared to common protein- and growth factors-based differentiation. NSCs exposure to quantized and transient (~150 ns micro-plasma bullets up-regulates expression of different cell lineage markers as β-Tubulin III (for neurons and O4 (for oligodendrocytes, while the expression of GFAP (for astrocytes remains unchanged, as evidenced by quantitative PCR, immunofluorescence microscopy and Western Blot assay. It is shown that the plasma-increased nitric oxide (NO production is a factor in the fate choice and differentiation of NSCs followed by axonal growth. The differentiated NSC cells matured and produced mostly cholinergic and motor neuronal progeny. It is also demonstrated that exposure of primary rat NSCs to the microplasma leads to quite similar differentiation effects. This suggests that the observed effect may potentially be generic and applicable to other types of neural progenitor cells. The application of this new in vitro strategy to selectively differentiate NSCs into neurons represents a step towards reproducible and efficient production of the desired NSC derivatives.

  10. Interindividual differences in stress sensitivity: basal and stress-induced cortisol levels differentially predict neural vigilance processing under stress.

    Science.gov (United States)

    Henckens, Marloes J A G; Klumpers, Floris; Everaerd, Daphne; Kooijman, Sabine C; van Wingen, Guido A; Fernández, Guillén

    2016-04-01

    Stress exposure is known to precipitate psychological disorders. However, large differences exist in how individuals respond to stressful situations. A major marker for stress sensitivity is hypothalamus-pituitary-adrenal (HPA)-axis function. Here, we studied how interindividual variance in both basal cortisol levels and stress-induced cortisol responses predicts differences in neural vigilance processing during stress exposure. Implementing a randomized, counterbalanced, crossover design, 120 healthy male participants were exposed to a stress-induction and control procedure, followed by an emotional perception task (viewing fearful and happy faces) during fMRI scanning. Stress sensitivity was assessed using physiological (salivary cortisol levels) and psychological measures (trait questionnaires). High stress-induced cortisol responses were associated with increased stress sensitivity as assessed by psychological questionnaires, a stronger stress-induced increase in medial temporal activity and greater differential amygdala responses to fearful as opposed to happy faces under control conditions. In contrast, high basal cortisol levels were related to relative stress resilience as reflected by higher extraversion scores, a lower stress-induced increase in amygdala activity and enhanced differential processing of fearful compared with happy faces under stress. These findings seem to reflect a critical role for HPA-axis signaling in stress coping; higher basal levels indicate stress resilience, whereas higher cortisol responsivity to stress might facilitate recovery in those individuals prone to react sensitively to stress. © The Author (2015). Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

  11. Gravity-induced differentiations and deficiency in flower formation observed on Columbus experiment WAICO1

    Science.gov (United States)

    Scherer, Günther; Pietrzyk, Peter

    formation. When mutants and wt only grown in the 1G centrifuge were compared the mutant leaves and cotyledons were smaller than in wt and hypocotyls were longer, but when the plants in µG for 12d were compared this difference was not found. Hence, gravity had an influence on leaf expansion and hypocotyl length in the mutant. The samples grown for 12d in 1G were kept in µG after 12d on due to a technical failure of the 1G centrifuge. They were retrieved about a year later. They had grown to full senescence and were preserved in a beautiful state as "straw". The observations on the root patterns by the astronaut photos at day 12 could be confirmed but plants had grown on and newer roots made coils just as the plants grown µG. Leaf sizes were different for wt and mutant. The most striking observation was that the mutants had developed small flower stems with a few flower buds but many flowers were incomplete, without the proper sepal or petal number or without gynaecium. The wild type plants had not developed any clear flower stem but only several malformed cell clumps shortly above the rosette. In ground laboratory experiments the mutants flower earlier which might explain why they developed flowers to some extent whereas the wt not at all. Microgravity might be a "stress" for flower formation. Taken together, several gravity-induced (or microgravity-induced) changes in differentiation occurred.

  12. Low electromagnetic field (50 Hz) induces differentiation on primary human oral keratinocytes (HOK).

    Science.gov (United States)

    Manni, Vanessa; Lisi, Antonella; Rieti, Sabrina; Serafino, Annalucia; Ledda, Mario; Giuliani, Livio; Sacco, Donatella; D'Emilia, Enrico; Grimaldi, Settimio

    2004-02-01

    This work concerns the effect of low frequency electromagnetic fields (ELF) on biochemical properties of human oral keratinocytes (HOK). Cells exposed to a 2 mT, 50 Hz, magnetic field, showed by scanning electron microscopy (SEM) modification in shape and morphology; these modifications were also associated with different actin distribution, revealed by phalloidin fluorescence analysis. Moreover, exposed cells had a smaller clonogenic capacity, and decreased cellular growth. Indirect immunofluorescence with fluorescent antibodies against involucrin and beta-catenin, both differentiation and adhesion markers, revealed an increase in involucrin and beta-catenin expression. The advance in differentiation was confirmed by a decrease of expression of epidermal growth factor (EGF) receptor in exposed cells, supporting the idea that exposure to electromagnetic field carries keratinocytes to higher differentiation level. These observations support the hypothesis that 50 Hz electromagnetic fields may modify cell morphology and interfere in differentiation and cellular adhesion of normal keratinocytes.

  13. Donepezil prevents RANK-induced bone loss via inhibition of osteoclast differentiation by downregulating acetylcholinesterase

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Sato

    2015-09-01

    Conclusions: AChE promotes osteoclast differentiation in vitro. Donepezil inhibits osteoclast function in vitro and prevents bone loss by suppressing bone resorption in vivo, suggesting the possibility that donepezil reduces fracture risk in patients with Alzheimer's disease.

  14. Overexpression of insulin-like growth factor-II induces accelerated myoblast differentiation.

    Science.gov (United States)

    Stewart, C E; James, P L; Fant, M E; Rotwein, P

    1996-10-01

    Previous studies have shown that exogenous insulin-like growth factors (IGFs) can stimulate the terminal differentiation of skeletal myoblasts in culture and have established a correlation between the rate and the extent of IGF-II secretion by muscle cell lines and the rate of biochemical and morphological differentiation. To investigate the hypothesis that autocrine secretion of IGF-II plays a critical role in stimulating spontaneous myogenic differentiation in vitro, we have established C2 muscle cell lines that stably express a mouse IGF-II cDNA under control of the strong, constitutively active Moloney sarcoma virus promoter, enabling us to study directly the effects of IGF-II overproduction. Similar to observations with other muscle cell lines, IGF-II overexpressing myoblasts proliferated normally in growth medium containing 20% fetal serum, but they underwent enhanced differentiation compared with controls when incubated in low-serum differentiation medium. Accelerated differentiation of IGF-II overexpressing C2 cells was preceded by the rapid induction of myogenin mRNA and protein expression (within 1 h, compared with 24-48 h in controls) and was accompanied by an enhanced proportion of the retinoblastoma protein in an underphosphrylated and potentially active form, by a marked increase in activity of the muscle-specific enzyme, creatine phosphokinase, by extensive myotube formation by 48 h, and by elevated secretion of IGF binding protein-5 when compared with controls. These results confirm a role for IGF-II as an autocrine/paracrine differentiation factor for skeletal myoblasts, and they define a model cell system that will be useful in determining the biochemical mechanisms of IGF action in cellular differentiation.

  15. Modulation of Mitochondrial DNA Copy Number to Induce Hepatocytic Differentiation of Human Amniotic Epithelial Cells.

    Science.gov (United States)

    Vaghjiani, Vijesh; Cain, Jason E; Lee, William; Vaithilingam, Vijayaganapathy; Tuch, Bernard E; St John, Justin C

    2017-09-05

    Mitochondrial deoxyribonucleic acid (mtDNA) copy number is tightly regulated during pluripotency and differentiation. There is increased demand of cellular adenosine triphosphate (ATP) during differentiation for energy-intensive cell types such as hepatocytes and neurons to meet the cell's functional requirements. During hepatocyte differentiation, mtDNA copy number should be synchronously increased to generate sufficient ATP through oxidative phosphorylation. Unlike bone marrow mesenchymal cells, mtDNA copy number failed to increase by 28 days of differentiation of human amniotic epithelial cells (hAEC) into hepatocyte-like cells (HLC) despite their expression of some end-stage hepatic markers. This was due to higher levels of DNA methylation at exon 2 of POLGA, the mtDNA-specific replication factor. Treatment with a DNA demethylation agent, 5-azacytidine, resulted in increased mtDNA copy number, reduced DNA methylation at exon 2 of POLGA, and reduced hepatic gene expression. Depletion of mtDNA followed by subsequent differentiation did not increase mtDNA copy number, but reduced DNA methylation at exon 2 of POLGA and increased expression of hepatic and pluripotency genes. We encapsulated hAEC in barium alginate microcapsules and subsequently differentiated them into HLC. Encapsulation resulted in no net increase of mtDNA copy number but a significant reduction in DNA methylation of POLGA. RNAseq analysis showed that differentiated HLC express hepatocyte-specific genes but also increased expression of inflammatory interferon genes. Differentiation in encapsulated cells showed suppression of inflammatory genes as well as increased expression of genes associated with hepatocyte function pathways and networks. This study demonstrates that an increase in classical hepatic gene expression can be achieved in HLC through encapsulation, although they fail to effectively regulate mtDNA copy number.

  16. GDNF-induced leukemia inhibitory factor can mediate differentiation via the MEK/ERK pathway in pheochromocytoma cells derived from nf1-heterozygous knockout mice.

    Science.gov (United States)

    Park, Jong-In; Powers, James F; Tischler, Arthur S; Strock, Christopher J; Ball, Douglas W; Nelkin, Barry D

    2005-02-01

    Glial cell line-derived neurotrophic factor (GDNF) can induce neuron-like differentiation of mouse pheochromocytoma (MPC) cell lines derived from mice with a heterozygous knockout mutation of nf1, the murine counterpart of the human gene mutated in neurofibromatosis type 1 (NF1). Here, we show that GDNF-induced differentiation in the MPC 862L cell line is mediated by the MEK/extracellular signal-regulated kinase (ERK) pathway. Neurite outgrowth, increased expression of growth-associated protein 43, and decreased incorporation of bromodeoxyuridine (BrdU) were induced by treatment with GDNF, H-RasV12, or a constitutively active MEK2. GDNF also induces leukemia inhibitory factor (LIF) via the MEK/ERK pathway, and LIF itself can elicit these differentiative changes via a cell-extrinsic autocrine/paracrine pathway. Treatment with anti-LIF neutralizing antibody depleted the differentiative activity of the conditioned medium from cells stimulated for MEK/ERK signaling, while recombinant LIF could induce differentiation in MPC cells, indicating that LIF is the sole factor with differentiative activity. LIF could activate MEK1/2 and STAT3, but LIF-induced differentiation was blocked only by the MEK1/2-specific inhibitor U0126, indicating that the MEK/ERK pathway is necessary for LIF action in MPC cells. Our findings suggest that LIF may be utilized for signaling mediated by GDNF and may be important in the pathobiology of neuroendocrine tumors.

  17. Differentiation-inducing activity of lupeol, a lupane-type triterpene from Chinese dandelion root (Hokouei-kon), on a mouse melanoma cell line.

    Science.gov (United States)

    Hata, K; Ishikawa, K; Hori, K; Konishi, T

    2000-08-01

    We examined the differentiation-inducing effects of extracts of 49 wild plants, 25 types of seaweed and 26 mushrooms in Akita on the human leukemia cell line HL60 and a B16 mouse melanoma-derived sub-clone with high differentiation capability (B16 2F2). Differentiation inducers of HL60 cells such as retinoic acid, showed no effects on the differentiation of B16 2F2 cells. Furthermore, chemical compounds known to be inducers of B16 cells, did not induce differentiation of HL60 cells. Screening tests showed that the differentiation of HL60 cells was induced by extracts of 28 wild plants, 10 types of seaweed and 2 mushrooms, and melanogenesis of B16 2F2 cells was increased by extracts of 21 wild plants, 8 types of seaweed and 7 mushrooms. All of the alcoholic extracts of plants belonging to the subfamily Cichorioideae of the family Compositae caused cell differentiation of the melanoma cell line. The extracts of Chinese dandelion root, also inhibited cell growth and induced melanogenesis of B16 2F2 cells. We isolated the active compound from ethanol extracts of the crude drug. Chemical and physical data for the active compound were identical with those for lupeol, a lupane-type triterpene.

  18. Facilitated Anion Transport Induces Hyperpolarization of the Cell Membrane That Triggers Differentiation and Cell Death in Cancer Stem Cells.

    Science.gov (United States)

    Soto-Cerrato, Vanessa; Manuel-Manresa, Pilar; Hernando, Elsa; Calabuig-Fariñas, Silvia; Martínez-Romero, Alicia; Fernández-Dueñas, Víctor; Sahlholm, Kristoffer; Knöpfel, Thomas; García-Valverde, María; Rodilla, Ananda M; Jantus-Lewintre, Eloisa; Farràs, Rosa; Ciruela, Francisco; Pérez-Tomás, Ricardo; Quesada, Roberto

    2015-12-23

    Facilitated anion transport potentially represents a powerful tool to modulate various cellular functions. However, research into the biological effects of small molecule anionophores is still at an early stage. Here we have used two potent anionophore molecules inspired in the structure of marine metabolites tambjamines to gain insight into the effect induced by these compounds at the cellular level. We show how active anionophores, capable of facilitating the transmembrane transport of chloride and bicarbonate in model phospholipid liposomes, induce acidification of the cytosol and hyperpolarization of plasma cell membranes. We demonstrate how this combined effect can be used against cancer stem cells (CSCs). Hyperpolarization of cell membrane induces cell differentiation and loss of stemness of CSCs leading to effective elimination of this cancer cell subpopulation.

  19. Effects of microsomal enzyme inducers on thyroid follicular cell proliferation and thyroid hormone metabolism.

    Science.gov (United States)

    Klaassen, C D; Hood, A M

    2001-01-01

    The effects of microsomal enzyme inducers on thyroid hormone homeostasis and the thyroid gland are of concern. We have investigated the effects of microsomal enzyme inducers on thyroid follicular cell proliferation and thyroid hormone metabolism in rats. We have shown that small increases in serum TSH can result in large increases in thyroid follicular cell proliferation. Furthermore, only those microsomal enzyme inducers that increase serum TSH--that is, phenobarbital (PB) and pregnenolone-16alpha-carbonitrile (PCN)-increase thyroid follicular cell proliferation, whereas those microsomal enzyme inducers that do not increase serum TSH--that is, 3-methylcholanthrene (3MC) and Aroclor 1254 (PCB)-do not increase thyroid follicular cell proliferation. Deiodination does not appear to be the reason why serum T3 concentrations are maintained in microsomal enzyme inducer-treated rats. We have also shown that those microsomal enzyme inducers that increase serum TSH increase T3 UDP-glucuronosyltransferase (UGT) activity, whereas those microsomal enzyme inducers that do not increase serum TSH do not increase T3 UGT activity. This finding suggests that induction of T3 glucuronidation, rather than T4 glucuronidation, mediates increases in serum TSH of microsomal enzyme inducer treated rats.

  20. Reversible CsA-induced toxic leucencephalopathy - Case report and discussion of differential diognoses; Reversible Cyclosporin-A-induzierte Leukenzephalopathie - Fallbeschreibung und differential-diagnostische Ueberlegungen

    Energy Technology Data Exchange (ETDEWEB)

    Lingesleben, A. [Humboldt-Universitaet, Berlin (Germany). Universitaetsklinikum Charite; Hoffmann, K.T. [Humboldt-Universitaet, Berlin (Germany). Strahlenklinik und Poliklinik; Oppert, M. [Humboldt-Universitaet, Berlin (Germany). Medizinische Klinik m. S. Nephrologie; Irlbacher, K.; Roericht, S.; Meyer, B.U. [Humboldt-Universitaet, Berlin (Germany). Neurologische Klinik

    2000-07-01

    The pathogenesis of CyclosporinA (CsA) induced toxic leucencephalopathy is unclear. CCT and CMRI reveal hypondense respectively hyperintens bilateral and symmetrical changes predominantly in the posterior white matter. We report a patient with a severe CsA induced toxic leucencephalopathy in whom clinical symptoms (complete loss of brain-stem functions, coma) and morphological changes in CCT and CMRI were completely reversible after immunosuppression with CsA has been stopped. We furthermore discuss the differential diagnoses of CCT and CMRI findings. (orig.) [German] Bei der pathogenetisch unklaren Cyclosporin-A-(CsA)-induzierten toxischen Leukenzephalopathie werden in der CCT und CMRT bilateral-symmetrische Manifestationen posterior betonter Marklagerhypodensitaeten bzw. Marklagerhyperintensitaeten beschrieben. Hier wird eine Patientin mit einer klinisch ungewoehnlich schwer verlaufenden durch CsA induzierten Leukenzephalopathie vorgestellt, bei der sowohl die klinische Symptomatik (kompletter Ausfall der Hirnstammfunktionen, Koma), als auch die bildmorphologischen Befunde innerhalb weniger Tage nach Absetzen von CsA komplett reversibel waren. In diesem Fallbericht werden ausserdem differentialdiagnostische Ueberlegungen bei der Beurteilung der CCT und CMRT dargestellt. (orig.)

  1. Three Dimensional Human Neuro-Spheroid Model of Alzheimer’s Disease Based on Differentiated Induced Pluripotent Stem Cells

    Science.gov (United States)

    Lee, Han-Kyu; Velazquez Sanchez, Clara; Chen, Mei; Morin, Peter J.; Wells, John M.; Hanlon, Eugene B.

    2016-01-01

    The testing of candidate drugs to slow progression of Alzheimer’s disease (AD) requires clinical trials that are lengthy and expensive. Efforts to model the biochemical milieu of the AD brain may be greatly facilitated by combining two cutting edge technologies to generate three-dimensional (3D) human neuro-spheroid from induced pluripotent stem cells (iPSC) derived from AD subjects. We created iPSC from blood cells of five AD patients and differentiated them into 3D human neuronal culture. We characterized neuronal markers of our 3D neurons by immunocytochemical staining to validate the differentiation status. To block the generation of pathologic amyloid β peptides (Aβ), the 3D-differentiated AD neurons were treated with inhibitors targeting β-secretase (BACE1) and γ-secretases. As predicted, both BACE1 and γ-secretase inhibitors dramatically decreased Aβ generation in iPSC-derived neural cells derived from all five AD patients, under standard two-dimensional (2D) differentiation conditions. However, BACE1 and γ-secretase inhibitors showed less potency in decreasing Aβ levels in neural cells differentiated under 3D culture conditions. Interestingly, in a single subject AD1, we found that BACE1 inhibitor treatment was not able to significantly reduce Aβ42 levels. To investigate underlying molecular mechanisms, we performed proteomic analysis of 3D AD human neuronal cultures including AD1. Proteomic analysis revealed specific reduction of several proteins that might contribute to a poor inhibition of BACE1 in subject AD1. To our knowledge, this is the first iPSC-differentiated 3D neuro-spheroid model derived from AD patients’ blood. Our results demonstrate that our 3D human neuro-spheroid model can be a physiologically relevant and valid model for testing efficacy of AD drug. PMID:27684569

  2. A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability.

    Directory of Open Access Journals (Sweden)

    Paul W Burridge

    Full Text Available BACKGROUND: The production of cardiomyocytes from human induced pluripotent stem cells (hiPSC holds great promise for patient-specific cardiotoxicity drug testing, disease modeling, and cardiac regeneration. However, existing protocols for the differentiation of hiPSC to the cardiac lineage are inefficient and highly variable. We describe a highly efficient system for differentiation of human embryonic stem cells (hESC and hiPSC to the cardiac lineage. This system eliminated the variability in cardiac differentiation capacity of a variety of human pluripotent stem cells (hPSC, including hiPSC generated from CD34(+ cord blood using non-viral, non-integrating methods. METHODOLOGY/PRINCIPAL FINDINGS: We systematically and rigorously optimized >45 experimental variables to develop a universal cardiac differentiation system that produced contracting human embryoid bodies (hEB with an improved efficiency of 94.7±2.4% in an accelerated nine days from four hESC and seven hiPSC lines tested, including hiPSC derived from neonatal CD34(+ cord blood and adult fibroblasts using non-integrating episomal plasmids. This cost-effective differentiation method employed forced aggregation hEB formation in a chemically defined medium, along with staged exposure to physiological (5% oxygen, and optimized concentrations of mesodermal morphogens BMP4 and FGF2, polyvinyl alcohol, serum, and insulin. The contracting hEB derived using these methods were composed of high percentages (64-89% of cardiac troponin I(+ cells that displayed ultrastructural properties of functional cardiomyocytes and uniform electrophysiological profiles responsive to cardioactive drugs. CONCLUSION/SIGNIFICANCE: This efficient and cost-effective universal system for cardiac differentiation of hiPSC allows a potentially unlimited production of functional cardiomyocytes suitable for application to hPSC-based drug development, cardiac disease modeling, and the future generation of clinically

  3. Progesterone induces cellular differentiation in MDA-MB-231 breast cancer cells transfected with progesterone receptor complementary DNA.

    Science.gov (United States)

    Lin, Valerie Chun-Ling; Jin, Rongxian; Tan, Puay-Hoon; Aw, Swee-Eng; Woon, Chow-Thai; Bay, Boon-Huat

    2003-06-01

    Progesterone is an important regulator of growth and differentiation in breast tissues. In this study, the effect of progesterone on cell differentiation was evaluated in the estrogen receptor-negative and progesterone receptor (PR)-negative MDA-MB-231 cell line which was transfected with PR-complementary DNA. Morphological changes were analyzed at the ultrastructural level by scanning and transmission electron microscopy. Progesterone-treated PR-transfected cells exhibited a more protracted and well spread morphology with an increase in organelles such as mitochondria and rough endoplasmic reticulum as compared to the rounded form of control vehicle (0.1% ethanol)-treated PR-transfected cells. Vehicle and progesterone-treated MDA-MB-231 cells transfected with the pSG5 plasmid (transfection control cells) had similar rounded morphology as control vehicle-treated PR-transfected cells. Immunofluorescence staining revealed that expression of E-cadherin, a differentiation marker, was more prominent in progesterone-treated cells. Expression of keratin and vimentin but not beta-catenin was up-regulated in progesterone treated cells when evaluated by immunoblotting. As signal transducers and activators of transcription (STAT) molecules have been implicated in mammary differentiation, we analyzed the expression of Stat 1, 3, 5a, and 5b proteins and found a significant up-regulation of the Stat 5b protein in progesterone-treated cells. We have provided in vitro evidence of the close association of PR with differentiation in breast cancer. It is likely that the Stat 5b protein may play a major role in progesterone-induced differentiation in breast cancer cells.

  4. 24- and 26-homo-1,25-dihydroxyvitamin D/sub 3/: preferential activity in inducing differentiation of human leukemia cells HL-60 in vitro inducing differentiation of human leukemia cells HL-60 in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Ostrem, V.K.; Tanaka, Y.; Prahl, J.; DeLuca, H.F.; Ikekawa, N.

    1987-05-01

    1,25-Dihydroxyvitamin D/sub 3/, the hormonal form of vitamin D/sub 3/, promotes the differentiation of HL-60 human promyelocytic leukemia cells into monocytes. Differentiation changes include the induction of phagocytosis, the initiation of nitroblue tetrazolium-reducing activity, and the appearance of nonspecific acid esterase. The authors have found that the 24-homo- and 26-homo-1,25-dihydroxyvitamin D/sub 3/ and their ..delta../sup 22/ analogues are 10-fold more potent than 1,25-dihydroxyvitamin D/sub 3/ in inducing differentiation of HL-60 cells in vitro. In vivo, these analogues show activity similar to 1,25-dihydroxy-vitamin D/sub 3/ in stimulating intestinal calcium transport in vitamin D-deficient rats. The 24-homoanalogues are significantly less active, whereas the 26-homo derivatives are more active than the natural hormone in mobilizing calcium from bone. This unusual activity pattern cannot be explained on the basis of the affinity of these analogues for the 1,25-dihydroxy-vitamin D/sub 3/ intracellular receptor: both 24-homo- and 26-homo-1,25-dihydroxyvitamin D/sub 3/ have the same effectiveness as 1,25-dihydroxyvitamin D/sub 3/ in displacing the tritiated hormone from its receptor in rat intestine of HL-60 cells. These analogues of 1,25-dihydroxyvitamin D/sub 3/ may be of some interest as possible therapeutic substances, or as tools in understanding the action of 1,25-dihydroxyvitamin D/sub 3/ in inducing differentiation.

  5. Human induced pluripotent stem cells differentiation into oligodendrocyte progenitors and transplantation in a rat model of optic chiasm demyelination.

    Directory of Open Access Journals (Sweden)

    Alireza Pouya

    Full Text Available BACKGROUND: This study aims to differentiate human induced pluripotent stem cells (hiPSCs into oligodendrocyte precursors and assess their recovery potential in a demyelinated optic chiasm model in rats. METHODOLOGY/PRINCIPAL FINDINGS: We generated a cell population of oligodendrocyte progenitors from hiPSCs by using embryoid body formation in a defined medium supplemented with a combination of factors, positive selection and mechanical enrichment. Real-time polymerase chain reaction and immunofluorescence analyses showed that stage-specific markers, Olig2, Sox10, NG2, PDGFRα, O4, A2B5, GalC, and MBP were expressed following the differentiation procedure, and enrichment of the oligodendrocyte lineage. These results are comparable with the expression of stage-specific markers in human embryonic stem cell-derived oligodendrocyte lineage cells. Transplantation of hiPSC-derived oligodendrocyte progenitors into the lysolecithin-induced demyelinated optic chiasm of the rat model resulted in recovery from symptoms, and integration and differentiation into oligodendrocytes were detected by immunohistofluorescence staining against PLP and MBP, and measurements of the visual evoked potentials. CONCLUSIONS/SIGNIFICANCE: These results showed that oligodendrocyte progenitors generated efficiently from hiPSCs can be used in future biomedical studies once safety issues have been overcome.

  6. Differential effects of multiplicity of infection on Helicobacter pylori-induced signaling pathways and interleukin-8 gene transcription.

    Science.gov (United States)

    Ritter, Birgit; Kilian, Petra; Reboll, Marc Rene; Resch, Klaus; DiStefano, Johanna Kay; Frank, Ronald; Beil, Winfried; Nourbakhsh, Mahtab

    2011-02-01

    Interleukin-8 (IL-8) plays a central role in the pathogenesis of Helicobacter pylori infection. We used four different H. pylori strains isolated from patients with gastritis or duodenal ulcer disease to examine their differential effects on signaling pathways and IL-8 gene response in gastric epithelial cells. IL-8 mRNA level is elevated in response to high (100) multiplicity of infection (MOI) independent of cagA, vacA, and dupA gene characteristics. By lower MOIs (1 or 10), only cagA ( + ) strains significantly induce IL-8 gene expression. This is based on differential regulation of IL-8 promoter activity. Analysis of intracellular signaling pathways indicates that H. pylori clinical isolates induce IL-8 gene transcription through NF-κB p65, but by a MOI-dependent differential activation of MAPK pathways. Thus, the major virulence factors of H. pylori CagA, VacA, and DupA might play a minor role in the level of IL-8 gene response to a high bacterial load.

  7. Rhus javanica Gall Extract Inhibits the Differentiation of Bone Marrow-Derived Osteoclasts and Ovariectomy-Induced Bone Loss

    Directory of Open Access Journals (Sweden)

    Tae-Ho Kim

    2016-01-01

    Full Text Available Inhibition of osteoclast differentiation and bone resorption is a therapeutic strategy for the management of postmenopausal bone loss. This study investigated the effects of Rhus javanica (R. javanica extracts on bone marrow cultures to develop agents from natural sources that may prevent osteoclastogenesis. Extracts of R. javanica (eGr cocoons spun by Rhus javanica (Bell. Baker inhibited the osteoclast differentiation and bone resorption. The effects of aqueous extract (aeGr or 100% ethanolic extract (eeGr on ovariectomy- (OVX- induced bone loss were investigated by various biochemical assays. Furthermore, microcomputed tomography (µCT was performed to study bone remodeling. Oral administration of eGr (30 mg or 100 mg/kg/day for 6 weeks augmented the inhibition of femoral bone mineral density (BMD, bone mineral content (BMC, and other factors involved in bone remodeling when compared to OVX controls. Additionally, eGr slightly decreased bone turnover markers that were increased by OVX. Therefore, it may be suggested that the protective effects of eGr could have originated from the suppression of OVX-induced increase in bone turnover. Collectively, the findings of this study indicate that eGr has potential to activate bone remodeling by inhibiting osteoclast differentiation and bone loss.

  8. Application of Green Tea Catechin for Inducing the Osteogenic Differentiation of Human Dedifferentiated Fat Cells in Vitro

    Directory of Open Access Journals (Sweden)

    Koji Kaida

    2015-11-01

    Full Text Available Despite advances in stem cell biology, there are few effective techniques to promote the osteogenic differentiation of human primary dedifferentiated fat (DFAT cells. We attempted to investigate whether epigallocatechin-3-gallate (EGCG, the main component of green tea catechin, facilitates early osteogenic differentiation and mineralization on DFAT cells in vitro. DFAT cells were treated with EGCG (1.25–10 μM in osteogenic medium (OM with or without 100 nM dexamethasone (Dex for 12 days (hereafter two osteogenic media were designated as OM(Dex and OM. Supplementation of 1.25 μM EGCG to both the media effectively increased the mRNA expression of collagen 1 (COL1A1 and runt-related transcription factor 2 (RUNX2 and also increased proliferation and mineralization. Compared to OM(Dex with EGCG, OM with EGCG induced earlier expression for COL1A1 and RUNX2 at day 1 and higher mineralization level at day 12. OM(Dex with 10 μM EGCG remarkably hampered the proliferation of the DFAT cells. These results suggest that OM(without Dex with EGCG might be a preferable medium to promote proliferation and to induce osteoblast differentiation of DFAT cells. Our findings provide an insight for the combinatory use of EGCG and DFAT cells for bone regeneration and stem cell-based therapy.

  9. Application of Green Tea Catechin for Inducing the Osteogenic Differentiation of Human Dedifferentiated Fat Cells in Vitro.

    Science.gov (United States)

    Kaida, Koji; Honda, Yoshitomo; Hashimoto, Yoshiya; Tanaka, Masahiro; Baba, Shunsuke

    2015-11-25

    Despite advances in stem cell biology, there are few effective techniques to promote the osteogenic differentiation of human primary dedifferentiated fat (DFAT) cells. We attempted to investigate whether epigallocatechin-3-gallate (EGCG), the main component of green tea catechin, facilitates early osteogenic differentiation and mineralization on DFAT cells in vitro. DFAT cells were treated with EGCG (1.25-10 μM) in osteogenic medium (OM) with or without 100 nM dexamethasone (Dex) for 12 days (hereafter two osteogenic media were designated as OM(Dex) and OM). Supplementation of 1.25 μM EGCG to both the media effectively increased the mRNA expression of collagen 1 (COL1A1) and runt-related transcription factor 2 (RUNX2) and also increased proliferation and mineralization. Compared to OM(Dex) with EGCG, OM with EGCG induced earlier expression for COL1A1 and RUNX2 at day 1 and higher mineralization level at day 12. OM(Dex) with 10 μM EGCG remarkably hampered the proliferation of the DFAT cells. These results suggest that OM(without Dex) with EGCG might be a preferable medium to promote proliferation and to induce osteoblast differentiation of DFAT cells. Our findings provide an insight for the combinatory use of EGCG and DFAT cells for bone regeneration and stem cell-based therapy.

  10. Inhibition of ref-1 stimulates the production of reactive oxygen species and induces differentiation in adult cardiac stem cells.

    Science.gov (United States)

    Gurusamy, Narasimman; Mukherjee, Subhendu; Lekli, Istvan; Bearzi, Claudia; Bardelli, Silvana; Das, Dipak K

    2009-03-01

    Redox effector protein-1 (Ref-1) plays an essential role in DNA repair and redox regulation of several transcription factors. In the present study, we examined the role of Ref-1 in maintaining the redox status and survivability of adult cardiac stem cells challenged with a subtoxic level of H2O2 under inhibition of Ref-1 by RNA interference. Treatment of cardiac stem cells with a low concentration of H2O2 induced Ref-1-mediated survival signaling through phosphorylation of Akt. However, Ref-1 inhibition followed by H2O2 treatment extensively induced the level of intracellular reactive oxygen species (ROS) through activation of the components of NADPH oxidase, like p22( phox ), p47( phox ), and Nox4. Cardiac differentiation markers (Nkx2.5, MEF2C, and GATA4), and cell death by apoptosis were significantly elevated in Ref-1 siRNA followed by H2O2-treated stem cells. Further, inhibition of Ref-1 increased the level of p53 but decreased the phosphorylation of Akt, a molecule involved in survival signaling. Treatment with ROS scavenger N-acetyl-L-cysteine attenuated Ref-1 siRNA-mediated activation of NADPH oxidase and cardiac differentiation. Taken together, these results indicate that Ref-1 plays an important role in maintaining the redox status of cardiac stem cells and protects them from oxidative injury-mediated cell death and differentiation.

  11. Potent inhibitory effect of Foeniculum vulgare Miller extract on osteoclast differentiation and ovariectomy-induced bone loss.

    Science.gov (United States)

    Kim, Tae-Ho; Kim, Hyun-Ju; Lee, Sang-Han; Kim, Shin-Yoon

    2012-06-01

    Inhibition of osteoclast differentiation and bone resorption is considered an effective therapeutic approach to the treatment of postmenopausal bone loss. To find natural compounds that may inhibit osteoclastogenesis, we screened herbal extracts on bone marrow cultures. In this study, we found that an aqueous extract of Foeniculum vulgare Miller seed (FvMs) at low concentration, which has traditionally been used as a treatment for a variety of ailments, inhibits the osteoclast differentiation and bone resorptive activity of mature osteoclasts. We further investigated the effects of FvMs on ovariectomy (OVX)-induced bone loss using microcomputed tomography, biomechanical tests and serum marker assays for bone remodeling. Oral administration of FvMs (30 mg or 100 mg/kg/day) for 6 weeks had an intermediary effect on the prevention of femoral bone mineral density (BMD), bone mineral content (BMC), and other parameters compared to OVX controls. In addition, FvMs slightly decreased bone turnover markers that were accelerated by OVX. The bone-protective effects of FvMs may be due to suppression of an OVX-induced increase in bone turnover. Collectively, our findings indicate that FvMs have potential in preventing bone loss in postmenopausal osteoporosis by reducing both osteoclast differentiation and function.

  12. Insights into the osteoblast precursor differentiation towards mature osteoblasts induced by continuous BMP-2 signaling

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    Omar F. Zouani

    2013-07-01

    Mature osteoblasts are the cells responsible for bone formation and are derived from precursor osteoblasts. However, the mechanisms that control this differentiation are poorly understood. In fact, unlike the majority of organs in the body, which are composed of “soft” tissue from which cells can easily be isolated and studied, the “hard” mineralized tissue of bone has made it difficult to study the function of bone cells. Here, we established an in vitro model that mimics this differentiation under physiological conditions. We obtained mature osteoblasts and characterized them on the basis of the following parameters: the strong expression of osteoblastic markers, such as Runx2 and Col-I; the achievement of specific dimensions (the cell volume increases 26-fold compared to the osteoblast precursors; and the production of an abundant extracellular matrix also called osteoid. We demonstrated that the differentiation of osteoblast precursors into mature osteoblasts requires the continuous activation of Bone Morphogenetic Protein (BMP receptors, which we established with the immobilization of a BMP-2mimetic peptide on a synthetic matrix mimicking in vivo microenvironment. Importantly, we demonstrated that the organization of the F-actin network and acetylated microtubules of the cells were modified during the differentiation process. We showed that the perturbation of the F-actin cytoskeleton organization abolished the differentiation process. In addition, we demonstrated that expression of the Runx2 gene is required for this differentiation. These findings demonstrate the retro-regulation of cytoplasmic and genic components due to the continuous induction of BMP-2 and also provide more detailed insights into the correct signaling of BMPs for cell differentiation in bone tissue.

  13. Propofol inhibits T-helper cell type-2 differentiation by inducing apoptosis via activating gamma-aminobutyric acid receptor.

    Science.gov (United States)

    Meng, Jingxia; Xin, Xin; Liu, Zhen; Li, Hao; Huang, Bo; Huang, Yuguang; Zhao, Jing

    2016-12-01

    Propofol has been shown to attenuate airway hyperresponsiveness in asthma patients. Our previous study showed that it may alleviate lung inflammation in a mouse model of asthma. Given the critical role of T-helper cell type-2 (Th2) differentiation in asthma pathology and the immunomodulatory role of the gamma-aminobutyric acid type A (GABAA) receptor, we hypothesized that propofol could alleviate asthma inflammation by inhibiting Th2 cell differentiation via the GABA receptor. For in vivo testing, chicken ovalbumin-sensitized and challenged asthmatic mice were used to determine the effect of propofol on Th2-type asthma inflammation. For in vitro testing, Th2-type cytokines as well as the cell proliferation and apoptosis were measured to assess the effects of propofol on Th2 cell differentiation and determine the underlying mechanisms. We found that propofol significantly decreased inflammatory cell counts and interleukin-4 and inflammation score in vivo. Propofol, but not intralipid, significantly reduced the Th2-type cytokine interleukin-5 secretion and caused Th2 cell apoptosis without obvious inhibition of proliferation in vitro. A GABA receptor agonist simulated the effect of propofol, whereas pretreatment with an antagonist reversed this effect. This study demonstrates that the antiinflammatory effects of propofol on Th2-type asthma inflammation in mice are mediated by inducing apoptosis without compromising proliferation during Th2 cell differentiation via activation of the GABA receptor. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Leucine-rich repeat kinase 2 modulates retinoic acid-induced neuronal differentiation of murine embryonic stem cells.

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    Cathrin Schulz

    Full Text Available BACKGROUND: Dominant mutations in the leucine-rich repeat kinase 2 (LRRK2 gene are the most prevalent cause of Parkinson's disease, however, little is known about the biological function of LRRK2 protein. LRRK2 is expressed in neural precursor cells suggesting a role in neurodevelopment. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, differential gene expression profiling revealed a faster silencing of pluripotency-associated genes, like Nanog, Oct4, and Lin28, during retinoic acid-induced neuronal differentiation of LRRK2-deficient mouse embryonic stem cells compared to wildtype cultures. By contrast, expression of neurotransmitter receptors and neurotransmitter release was increased in LRRK2+/- cultures indicating that LRRK2 promotes neuronal differentiation. Consistently, the number of neural progenitor cells was higher in the hippocampal dentate gyrus of adult LRRK2-deficient mice. Alterations in phosphorylation of the putative LRRK2 substrates, translation initiation factor 4E binding protein 1 and moesin, do not appear to be involved in altered differentiation, rather there is indirect evidence that a regulatory signaling network comprising retinoic acid receptors, let-7 miRNA and downstream target genes/mRNAs may be affected in LRRK2-deficient stem cells in culture. CONCLUSION/SIGNIFICANCE: Parkinson's disease-linked LRRK2 mutations that associated with enhanced kinase activity may affect retinoic acid receptor signaling during neurodevelopment and/or neuronal maintenance as has been shown in other mouse models of chronic neurodegenerative diseases.

  15. Arsenic Trioxide Induced Differentiation and Apoptosis in Human Nasopharyngeal Carcinoma Xenografts in BALB/C Nude Mice

    Institute of Scientific and Technical Information of China (English)

    ZHENGYuwu; DUCaiwen; LIDerui; LINYingcheng; WUMingyao

    2004-01-01

    To study the effect of arsenic trioxide (As2O3) on human poorly differentiated nasopharyngeal cancer cell line, CSNE-1, in vivo and its possible mechanism of action. Methods: CSNE-1 cells were established as xenografts in BALB/C nude mice. The tumor-bearing mice were treated with As2O3 at the dose of 5 mg/kg every day. The tumor growth was observed by tumor-growth curve. Morphologic changes were studied under light microscopy and electron microscopy. TUNEL was used to detect apoptosis. The expression of PCNA, p53, Bcl-2 and Bax were determined by immunohistochemistry. Results: The cell growth and proliferate activity were significantly inhibited by As203 at the dose of 5 mg/kg every day. Morphologic changes such as the formation of keratinization of tumor cells, decreased ratio of nuclear/cytoplasm, increased organelle and plasmic fibril in cytoplasm were identified. Cytodesma, desmosomes and micro-process were seen under light microscopy and transmission electron microscopy, which revealed that the cancer cells underwent differentiation. In addition, remarkable cell apoptosis were observed by TUNEL assay. Over expression of p53 and Bax was detected in the As203 treatment group when compared with control group. Conclusion: As203 inhibited proliferation of human poorly differentiated nasopharyngeal cancer cell CSNE-1 by inducing differentiation and apoptosis, which may be related to the up-regulation of p53 and Bax expression.

  16. Shape-induced terminal differentiation of human epidermal stem cells requires p38 and is regulated by histone acetylation.

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    John T Connelly

    Full Text Available Engineered model substrates are powerful tools for examining interactions between stem cells and their microenvironment. Using this approach, we have previously shown that restricted cell adhesion promotes terminal differentiation of human epidermal stem cells via activation of serum response factor (SRF and transcription of AP-1 genes. Here we investigate the roles of p38 MAPK and histone acetylation. Inhibition of p38 activity impaired SRF transcriptional activity and shape-induced terminal differentiation of human keratinocytes. In addition, inhibiting p38 reduced histone H3 acetylation at the promoters of SRF target genes, FOS and JUNB. Although histone acetylation correlated with SRF transcriptional activity and target gene expression, treatment with the histone de-acetylase inhibitor, trichostatin A (TSA blocked terminal differentiation on micro-patterned substrates and in suspension. TSA treatment simultaneously maintained expression of LRIG1, TP63, and ITGB1. Therefore, global histone de-acetylation represses stem cell maintenance genes independent of SRF. Our studies establish a novel role for extrinsic physical cues in the regulation of chromatin remodeling, transcription, and differentiation of human epidermal stem cells.

  17. Enhanced growth and osteogenic differentiation of Induced Pluripotent Stem cells by Extremely Low-Frequency Electromagnetic Field.

    Science.gov (United States)

    Ardeshirylajimi, A; Soleimani, M

    2015-03-09

    It is accepted that induced pluripotent stem cells (iPSCs) have a great osteogenic potential differentiation, in the present study, we tried to improve this potentials using mechanical and biological stimulation. To achieve this goal, the influence of prolonged pulsed extremely low frequency electromagnetic field (ELF—EMF) (50 Hz and 1.5 mT) was investigated on cultured iPSCs. After evaluation of iPSCs biological behavior under radiation using MTT assay, osteogenic differentiation of stem cells was investigated via common important osteogenic markers such as alkaline phosphatase (ALP) activity, calcium mineral deposition and important bone—related genes. MTT result showed that proliferation rate of iPSCs significantly increased followed by stimulate with ELF—EMF. Osteogenic differentiation characterization demonstrated that potential of stem cells also was significantly increased while these cells cultured under both ELF—EMF and osteogenic medium (OM) in comparison to cultured under ELF—EMF or OM alone. According to the results, concluded that combination of OM and ELF—EMF can be a great supplement for bone differentiation of stem cells and appropriate candidate for use in the treatment of bone defects and osteoporosis patients by accelerating healing process.

  18. Photo-induced in situ crosslinking of polymer brushes with dimethyl maleimide moieties for dynamically stimulating stem cell differentiation.

    Science.gov (United States)

    Arisaka, Yoshinori; Nishijima, Yuka; Yusa, Shin-Ichi; Takeda, Naoya

    2016-09-01

    We designed photo-crosslinkable polymer brushes with dimethylmaleimide moieties, in order to demonstrate dynamic stimulation of cell differentiation in mesenchymal stem cells (MSCs). The polymer brushes were synthesized by surface-initiated reversible addition fragmentation chain transfer polymerization using dimethylmaleimide ethyl methacrylate and methyl methacrylate on a chain transfer agent-immobilized glass surface. The polymer brushes were crosslinked by photodimerization of the dimethylmaleimide moieties within polymer chains with stem cells present on the surface. In order to evaluate the effects of in situ photo-induced crosslinking of the polymer brushes on gene expression of stem cells, human bone marrow MSCs were cultured under static and dynamic culture conditions for 7 days. Expression of the osteocalcin (Ocn) gene in MSCs was used as an indicator of osteoblast differentiation under dynamic culture conditions. Structural conversion from non-crosslinked polymer brushes to crosslinked polymer brushes increased the expression of Ocn by 1.4-fold in the presence of adhered cells, compared with non-crosslinked polymer brushes under static culture conditions. These results suggest that MSCs recognized surface conversion from non-crosslinked to crosslinked structures, which resulted in altered differentiation lineages. Therefore, photo-crosslinkable surfaces with dimethyl maleimide moieties are potential novel materials for dynamically stimulating MSC differentiation.

  19. Maternal Obesity Induces Epigenetic Modifications to Facilitate Zfp423 Expression and Enhance Adipogenic Differentiation in Fetal Mice

    Science.gov (United States)

    Yang, Qi-Yuan; Liang, Jun-Fang; Rogers, Carl J.; Zhao, Jun-Xing; Zhu, Mei-Jun; Du, Min

    2013-01-01

    Maternal obesity (MO) predisposes offspring to obesity and type 2 diabetes despite poorly defined mechanisms. Zfp423 is the key transcription factor committing cells to the adipogenic lineage, with exceptionally dense CpG sites in its promoter. We hypothesized that MO enhances adipogenic differentiation during fetal development through inducing epigenetic changes in the Zfp423 promoter and elevating its expression. Female mice were subjected to a control (Con) or obesogenic (OB) diet for 2 months, mated, and maintained on their diets during pregnancy. Fetal tissue was harvested at embryonic day 14.5 (E14.5), when the early adipogenic commitment is initiated. The Zfp423 expression was 3.6-fold higher and DNA methylation in the Zfp423 promoter was lower in OB compared with Con. Correspondingly, repressive histone methylation (H3K27me3) was lower in the Zfp423 promoter of OB fetal tissue, accompanied by reduced binding of enhancer of zeste 2 (EZH2). Gain- and loss-of-function analysis showed that Zfp423 regulates early adipogenic differentiation in fetal progenitor cells. In summary, MO enhanced Zfp423 expression and adipogenic differentiation during fetal development, at least partially through reducing DNA methylation in the Zfp423 promoter, which is expected to durably elevate adipogenic differentiation of progenitor cells in adult tissue, programming adiposity and metabolic dysfunction later in life. PMID:23884886

  20. Eosinophils from Murine Lamina Propria Induce Differentiation of Naive T Cells into Regulatory T Cells via TGF-β1 and Retinoic Acid.

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    Hong-Hu Chen

    Full Text Available Treg cells play a crucial role in immune tolerance, but mechanisms that induce Treg cells are poorly understood. We here have described eosinophils in lamina propria (LP that displayed high aldehyde dehydrogenase (ALDH activity, a rate-limiting step during all-trans retinoic acid (ATRA synthesis, and expressed TGF-β1 mRNA and high levels of ATRA. Co-incubation assay confirmed that LP eosinophils induced the differentiation of naïve T cells into Treg cells. Differentiation promoted by LP eosinophils were inhibited by blocked either TGF-β1 or ATRA. Peripheral blood (PB eosinophils did not produce ATRA and could not induce Treg differentiation. These data identifies LP eosinophils as effective inducers of Treg cell differentiation through a mechanism dependent on TGF-β1 and ATRA.

  1. Physalis angulata induces in vitro differentiation of murine bone marrow cells into macrophages.

    Science.gov (United States)

    da Silva, Bruno José Martins; Rodrigues, Ana Paula D; Farias, Luis Henrique S; Hage, Amanda Anastácia P; Do Nascimento, Jose Luiz M; Silva, Edilene O

    2014-10-03

    The bone marrow is a hematopoietic tissue that, in the presence of cytokines and growth factors, generates all of the circulating blood cells. These cells are important for protecting the organism against pathogens and for establishing an effective immune response. Previous studies have shown immunomodulatory effects of different products isolated from plant extracts. This study aimed to evaluate the immunomodulatory properties of aqueous Physalis angulata (AEPa) extract on the differentiation of bone marrow cells. Increased cellular area, higher spreading ability and several cytoplasmatic projections were observed in the treated cells, using optical microscopy, suggesting cell differentiation. Furthermore, AEPa did not promote the proliferation of lymphocytes and polymorphonuclear leukocytes, however promotes increased the number of macrophages in the culture. The ultrastructural analysis by Transmission Electron Microscopy of treated cells showed spreading ability, high number of cytoplasmatic projections and increase of autophagic vacuoles. Moreover, a high level of LC3b expression by treated cells was detected by flow cytometry, suggesting an autophagic process. Cell surface expression of F4/80 and CD11b also indicated that AEPa may stimulate differentiation of bone marrow cells mainly into macrophages. In addition, AEPa did not differentiate cells into dendritic cells, as assessed by CD11c analysis. Furthermore, no cytotoxic effects were observed in the cells treated with AEPa. Results demonstrate that AEPa promotes the differentiation of bone marrow cells, particularly into macrophages and may hold promise as an immunomodulating agent.

  2. Induced Pluripotent Stem (iPS) Cell Culture Methods and Induction of Differentiation into Endothelial Cells

    Science.gov (United States)

    Chatterjee, Ishita; Li, Fei; Kohler, Erin E.; Rehman, Jalees; Malik, Asrar B.; Wary, Kishore K.

    2015-01-01

    Summary The studies of stem cell behavior and differentiation in a developmental context is complex, time-consuming and expensive, and for this reason, cell culture remains a method of choice for developmental and regenerative biology and mechanistic studies. Similar to ES cells, iPS cells have the ability to differentiate into endothelial cells (ECs), and the route for differentiation appears to mimic the developmental process that occurs during the formation of an embryo. Traditional EC induction methods from embryonic stem (ES) cells rely mostly on the formation the embryoid body (EB), which employs feeder or feeder-free conditions in the presence or absence of supporting cells. Similar to ES cells, iPS cells can be cultured in feeder-layer or feeder-free conditions. Here, we describe the iPS cell culture methods and induction differentiation of these cells into ECs. We use anti-mouse Flk1 and anti-mouse VE-cadherin to isolate and characterize mouse ECs, because these antibodies are commercially available and their use has been described in the literature, including by our group. The ECs produced by this method have been used by our laboratory, and we have demonstrated their in vivo potential. We also discuss how iPS cells differ in their ability to differentiate into endothelial cells in culture. PMID:25687301

  3. Downregulation of Col1a1 induces differentiation in mouse spermatogonia

    Institute of Scientific and Technical Information of China (English)

    Sun-Hong Chen; Ding Li; Chen Xu

    2012-01-01

    Col1a1 (one of the subunit of collagen type Ⅰ) is a collagen,which belongs to a family of extracellular matrix (ECM) proteins that play an important role in cellular proliferation and differentiation.However,the role of Col1a1 in spermatogenesis,especially in the control of proliferation and differentiation of spermatogonial stem cells (SSCs),remains unknown.In this study,we explored effects of downregulation of Col1a1 on differentiation and proliferation of mouse spermatogonia.Loss-of-function study revealed that Oct4 and Plzf,markers of SSC self-renewal,were significantly decreased,whereas the expression of c-kit and haprin,hallmarks of SSC differentiation,was enhanced after Col1a1 knockdown.Cell cycle analyses indicated that two-thirds of spermatogonia were arrested in S phase after Col1a1 knockdown.In vivo experiments,DNA injection and electroporation of the testes showed that spermatogonia self-renewal ability was impaired remarkably with the loss-of-function of Col1a1.Our data suggest that silencing of Col1a1 can suppress spermatogonia self-renewal and promote spermatogonia differentiation.

  4. Induced Pluripotent Stem (iPS) Cell Culture Methods and Induction of Differentiation into Endothelial Cells.

    Science.gov (United States)

    Chatterjee, Ishita; Li, Fei; Kohler, Erin E; Rehman, Jalees; Malik, Asrar B; Wary, Kishore K

    2016-01-01

    The study of stem cell behavior and differentiation in a developmental context is complex, time-consuming, and expensive, and for this reason, cell culture remains a method of choice for developmental and regenerative biology and mechanistic studies. Similar to ES cells, iPS cells have the ability to differentiate into endothelial cells (ECs), and the route for differentiation appears to mimic the developmental process that occurs during the formation of an embryo. Traditional EC induction methods from embryonic stem (ES) cells rely mostly on the formation of embryoid body (EB), which employs feeder or feeder-free conditions in the presence or absence of supporting cells. Similar to ES cells, iPS cells can be cultured in feeder layer or feeder-free conditions. Here, we describe the iPS cell culture methods and induction differentiation of these cells into ECs. We use anti-mouse Flk1 and anti-mouse VE-cadherin to isolate and characterize mouse ECs, because these antibodies are commercially available and their use has been described in the literature, including by our group. The ECs produced by this method have been used by our laboratory, and we have demonstrated their in vivo potential. We also discuss how iPS cells differ in their ability to differentiate into endothelial cells in culture.

  5. Differential modulation of bradykinin-induced relaxation of endothelin-1 and phenylephrine contractions of rat aorta by antioxidants

    Institute of Scientific and Technical Information of China (English)

    Ogechukwu ANOZIE; Richonda ROSS; Adebayo O OYEKAN; Momoh A YAKUBU

    2007-01-01

    Aim: We tested the hypothesis that bradykinin (BK)-induced relaxation of phenylephrine (PE) and endothelin-1 (ET-1) contractions can be differentially modulated by reactive oxygen species (ROS). Methods: Aortic rings isolated from Sprague-Dawley rats were used for the study. The contribution of ROS to PE(1×10-9-1×10-5 mol/L)- and ET-1 (1×10-10-1×10-8 mol/L)-induced contractions and the influence of ROS in B K (1×10-9-1×10mol/L) relaxation of PE (1×10-7 mol/L) or ET-1 (1×10-9 mol/L)-induced tension was evaluated in the aorta in the presence or absence of the following antioxidants: catalase (CAT, 300 U/mL), superoxide dismutase (SOD, 300 U/mL), and vitamin C (1×10-4 mol/L). Results: Tension generated by ET-1 (1 × 10-9 mol/L) or PE (1 × 10-7 mol/L) was differentially relaxed by BK(1 × 10-5 mol/L), producing a maximal relaxation of 75 %±5 % and 35±4%, respectively.The BK (1×10-5 mol/L)-induced relaxation of PE (1×10-7 mol/L) tension was signifi-cantly enhanced from 35%±4% (control) to 56%±9%, 60%±5%, and 49%±6% by SOD, CAT, and vitamin C, respectively (P<0.05, n=8). However, the relaxation of ET-1 (1×10-9 mol/L) tension was significantly attenuated from 75%±5% (control)to 37%±9%, 63%±4%, and 39%±7% by SOD, CAT, and vitamin C, respectively(P<0.05, n=8). On the other hand, CAT had no effect on PE-induced tension, while SOD enhanced PE-induced tension (36%,P<0.05, n=10) and vitamin C attenuated(66%, P<0.05, n=8) the tension induced by PE. By contrast, SOD or vitamin C had no effect, but CAT attenuated (44%, P<0.05, n=9) the tension induced by ET-1.Conclusion: We have demonstrated that O2- and H2O2 differentially modulate BK relaxation in an agonist-specific manner. O2- attenuates BK-induced relaxation of PE contraction, but contributes to the relaxation of ET-1 contraction. O2- seems to inhibit PE contraction, while H2O2 contributes to ET-1-induced contraction. Thus,ROS differentially modulate vascular tone depending on the vasoactive agent that

  6. E2F1 impairs all-trans retinoic acid-induced osteogenic differentiation of osteosarcoma via promoting ubiquitination-mediated degradation of RARα

    Science.gov (United States)

    Zhang, Lei; Zhou, Qian; Zhang, Ning; Li, Weixu; Ying, Meidan; Ding, Wanjing; Yang, Bo; He, Qiaojun

    2014-01-01

    All-trans retinoic acid (ATRA) is a widely used differentiation drug that can effectively induce osteogenic differentiation of osteosarcoma cells, but the underlying mechanism remains elusive, which limits the clinical application for ATRA in osteosarcoma patients. In this study, we identified E2F1 as a novel regulator involved in ATRA-induced osteogenic differentiation of osteosarcoma cells. We observed that osteosarcoma cells are coupled with individual differences in the expression levels of E2F1 in patients, and E2F1 impairs ATRA-induced differentiation of osteosarcoma cells. Moreover, remarkable anti-proliferative and differentiation-inducing effects of ATRA treatment are only observed in E2F1 low to negative expressed primary osteosarcoma cultures. These results strongly suggested that E2F1 may serve as a potent indicator for the effectiveness of ATRA treatment in osteosarcoma. Interestingly, E2F1 is found to downregulate retinoic acid receptor α (RARα), a key factor determines the effectiveness of ATRA. E2F1 specifically binds to RARα and promotes its ubiquitination-mediated degradation; as a consequence, RARα-mediated differentiation is inhibited in osteosarcoma. Therefore, our studies present E2F1 as a potent biomarker, as well as a therapeutic target for ATRA-based differentiation therapeutics, and raise the hope of using differentiation-based approaches for osteosarcoma patients. PMID:24608861

  7. The urokinase plasminogen activator receptor (UPAR) is preferentially induced by nerve growth factor in PC12 pheochromocytoma cells and is required for NGF-driven differentiation.

    Science.gov (United States)

    Farias-Eisner, R; Vician, L; Silver, A; Reddy, S; Rabbani, S A; Herschman, H R

    2000-01-01

    Nerve growth factor (NGF)-driven differentiation of PC12 pheochromocytoma cells is a well studied model used both to identify molecular, biochemical, and physiological correlates of neurotrophin-driven neuronal differentiation and to determine the causal nature of specific events in this differentiation process. Although epidermal growth factor (EGF) elicits many of the same early biochemical and molecular changes in PC12 cells observed in response to NGF, EGF does not induce molecular or morphological differentiation of PC12 cells. The identification of genes whose expression is differentially regulated by NGF versus EGF in PC12 cells has, therefore, been considered a source of potential insight into the molecular specificity of neurotrophin-driven neuronal differentiation. A "second generation" representational difference analysis procedure now identifies the urokinase plasminogen activator receptor (UPAR) as a gene that is much more extensively induced by NGF than by EGF in PC12 cells. Both an antisense oligonucleotide for the UPAR mRNA and an antibody directed against UPAR protein block NGF-induced morphological and biochemical differentiation of PC12 cells; NGF-induced UPAR expression is required for subsequent NGF-driven differentiation.

  8. Potential therapeutic applications of differentiated induced pluripotent stem cells (iPSCs) in the treatment of neurodegenerative diseases.

    Science.gov (United States)

    Gao, Aijing; Peng, Yuhua; Deng, Yulin; Qing, Hong

    2013-01-01

    Difficulties in realizing persistent neurogenesis, inabilities in modeling pathogenesis of most cases, and a shortage of disease material for screening therapeutic agents restrict our progress to overcome challenges presented by neurodegenerative diseases. We propose that reprogramming primary somatic cells of patients into induced pluripotent stem cells (iPSCs) provides a new avenue to overcome these impediments. Their abilities in self-renewal and differentiation into various cell types will enable disease investigation and drug development. In this review, we introduce efficient approaches to generate iPSCs and distinct iPSCs differentiation stages, and critically discuss paradigms of iPSCs technology application to investigate neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). Although iPSCs technology is in its infancy and faces many obstacles, it has great potential in helping to identify therapeutic targets for treating neurodegenerative diseases.

  9. Dopaminergic neurons differentiating from LRRK2 G2019S induced pluripotent stem cells show early neuritic branching defects.

    Science.gov (United States)

    Borgs, Laurence; Peyre, Elise; Alix, Philippe; Hanon, Kevin; Grobarczyk, Benjamin; Godin, Juliette D; Purnelle, Audrey; Krusy, Nathalie; Maquet, Pierre; Lefebvre, Philippe; Seutin, Vincent; Malgrange, Brigitte; Nguyen, Laurent

    2016-09-19

    Some mutations of the LRRK2 gene underlie autosomal dominant form of Parkinson's disease (PD). The G2019S is a common mutation that accounts for about 2% of PD cases. To understand the pathophysiology of this mutation and its possible developmental implications, we developed an in vitro assay to model PD with human induced pluripotent stem cells (hiPSCs) reprogrammed from skin fibroblasts of PD patients suffering from the LRKK2 G2019S mutation. We differentiated the hiPSCs into neural stem cells (NSCs) and further into dopaminergic neurons. Here we show that NSCs bearing the mutation tend to differentiate less efficiently into dopaminergic neurons and that the latter exhibit significant branching defects as compared to their controls.

  10. Electrospun fibrous scaffolds combined with nanoscale hydroxyapatite induce osteogenic differentiation of human periodontal ligament cells

    Directory of Open Access Journals (Sweden)

    Wu XN

    2014-08-01

    extended gradually with stretched filopodia, indicating an ability to fill the fiber pores. A Cell Counting Kit-8 assay showed that both scaffolds supported cell proliferation. However, real-time quantitative polymerase chain reaction analysis showed that expression of the bone-related markers, alkaline phosphatase and osteocalcin, was upregulated only on the COL/PCL/nHA-SBF scaffold, indicating that this scaffold had the ability to induce osteogenic differentiation of periodontal ligament cells. In this study, COL/PCL/nHA-SBF produced by electrospinning followed by biomimetic mineralization had combined electrospun fibers with nHA in it. This scaffold has good biocompatibility and osteoinductive ability as a result of the characteristics of nHA, so could be innovatively applied to periodontal tissue engineering as a potential scaffold. Keywords: nanoscale hydroxyapatite, electrospinning, periodontal ligament cells 

  11. Amyloid-β-induced reactive oxygen species production and priming are differentially regulated by ion channels in microglia.

    Science.gov (United States)

    Schilling, Tom; Eder, Claudia

    2011-12-01

    Production of reactive oxygen species (ROS) by microglial cells and subsequent oxidative stress are strongly implicated in the pathogenesis of Alzheimer's disease. Although it is recognized that amyloid-β (Aβ) plays a major role in inducing and regulating microglial ROS production in Alzheimer's disease, to date little is known about cellular mechanisms underlying Aβ-stimulated ROS production. Here, we identified ion channels involved in Aβ-induced microglial ROS production and in Aβ-induced microglial priming. Acute stimulation of microglial cells with either fibrillar Aβ(1-42) (fAβ(1-42) ) or soluble Aβ(1-42) (sAβ(1-42) ) caused significant increases in microglial ROS production, which were abolished by inhibition of TRPV1 cation channels with 5-iodo-resiniferatoxin (I-RTX), but were unaffected by inhibition of K(+) channels with charybdotoxin (CTX). Furthermore, pretreatment with either fAβ(1-42) or sAβ(1-42) induced microglial priming, that is, increased ROS production upon secondary stimulation with the phorbol ester PMA. Microglial priming induced by fAβ(1-42) or sAβ(1-42) remained unaffected by TRPV1 channel inhibition with I-RTX. However, sAβ(1-42) -induced priming was inhibited by CTX and margatoxin, but not by TRAM-34 or paxilline, indicating a role of Kv1.3 voltage-gated K(+) channels, but not of Ca(2+) -activated K(+) channels, in the priming process. In summary, our data suggest that in microglia Aβ-induced ROS production and priming are differentially regulated by ion channels, and that TRPV1 cation channels and Kv1.3 K(+) channels may provide potential therapeutic targets to reduce microglia-induced oxidative stress in Alzheimer's disease.

  12. Hydrogen sulfide suppresses transforming growth factor-β1-induced differentiation of human cardiac fibroblasts into myofibroblasts.

    Science.gov (United States)

    Zhang, YouEn; Wang, JiaNing; Li, Hua; Yuan, LiangJun; Wang, Lei; Wu, Bing; Ge, JunBo

    2015-11-01

    In heart disease, transforming growth factor-β1 (TGF-β1) converts fibroblasts into myofibroblasts, which synthesize and secrete fibrillar type I and III collagens. The purpose of the present study was to investigate how hydrogen sulfide (H2S) suppresses TGF-β1-induced differentiation of human cardiac fibroblasts to myofibroblasts. Human cardiac fibroblasts were serum-starved in fibroblast medium for 16 h before exposure to TGF-β1 (10 ng mL(-1)) for 24 h with or without sodium hydrosulfide (NaHS, 100 µmol L(-1), 30 min pretreatment) treatment. NaHS, an exogenous H2S donor, potently inhibited the proliferation and migration of TGF-β1-induced human cardiac fibroblasts and regulated their cell cycle progression. Furthermore, NaHS treatment led to suppression of fibroblast differentiation into myofibroblasts, and reduced the levels of collagen, TGF-β1, and activated Smad3 in TGF-β1-induced human cardiac fibroblasts in vitro. We therefore conclude that H2S suppresses TGF-β1-stimulated conversion of fibroblasts to myofibroblasts by inhibiting the TGF-β1/Smad3 signaling pathway, as well as by inhibiting the proliferation, migration, and cell cycle progression of human cardiac myofibroblasts. These effects of H2S may play significant roles in cardiac remodeling associated with heart failure.

  13. Connective tissue growth factor stimulates the proliferation, migration and differentiation of lung fibroblasts during paraquat-induced pulmonary fibrosis.

    Science.gov (United States)

    Yang, Zhizhou; Sun, Zhaorui; Liu, Hongmei; Ren, Yi; Shao, Danbing; Zhang, Wei; Lin, Jinfeng; Wolfram, Joy; Wang, Feng; Nie, Shinan

    2015-07-01

    It is well established that paraquat (PQ) poisoning can cause severe lung injury during the early stages of exposure, finally leading to irreversible pulmonary fibrosis. Connective tissue growth factor (CTGF) is an essential growth factor that is involved in tissue repair and pulmonary fibrogenesis. In the present study, the role of CTGF was examined in a rat model of pulmonary fibrosis induced by PQ poisoning. Histological examination revealed interstitial edema and extensive cellular thickening of interalveolar septa at the early stages of poisoning. At 2 weeks after PQ administration, lung tissue sections exhibited a marked thickening of the alveolar walls with an accumulation of interstitial cells with a fibroblastic appearance. Masson's trichrome staining revealed a patchy distribution of collagen deposition, indicating pulmonary fibrogenesis. Western blot analysis and immunohistochemical staining of tissue samples demonstrated that CTGF expression was significantly upregulated in the PQ-treated group. Similarly, PQ treatment of MRC-5 human lung fibroblast cells caused an increase in CTGF in a dose-dependent manner. Furthermore, the addition of CTGF to MRC-5 cells triggered cellular proliferation and migration. In addition, CTGF induced the differentiation of fibroblasts to myofibroblasts, as was evident from increased expression of α-smooth muscle actin (α-SMA) and collagen. These findings demonstrate that PQ causes increased CTGF expression, which triggers proliferation, migration and differentiation of lung fibroblasts. Therefore, CTGF may be important in PQ-induced pulmonary fibrogenesis, rendering this growth factor a potential pharmacological target for reducing lung injury.

  14. Fibroblast growth factor-2 induces osteogenic differentiation through a Runx2 activation in vascular smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Nakahara, Takehiro; Sato, Hiroko; Shimizu, Takehisa; Tanaka, Toru; Matsui, Hiroki; Kawai-Kowase, Keiko; Sato, Mahito; Iso, Tatsuya; Arai, Masashi [Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511 (Japan); Kurabayashi, Masahiko, E-mail: mkuraba@med.gunma-u.ac.jp [Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511 (Japan)

    2010-04-02

    Expression of bone-associated proteins and osteoblastic transcription factor Runx2 in arterial cells has been implicated in the development of vascular calcification. However, the signaling