搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

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. View Publication

Obesity increases the risk of developing multiple myeloma (MM). Adiponectin is a cytokine produced by adipocytes,but paradoxically decreased in obesity,that has been implicated in MM progression. Herein,we evaluated how prolonged exposure to adiponectin affected the survival of MM cells as well as putative signaling mechanisms. Adiponectin activates protein kinase A (PKA),which leads to decreased AKT activity and increased AMP-activated protein kinase (AMPK) activation. AMPK,in turn,induces cell cycle arrest and apoptosis. Adiponectin-induced apoptosis may be mediated,at least in part,by the PKA/AMPK-dependent decline in the expression of the enzyme acetyl-CoA-carboxylase (ACC),which is essential to lipogenesis. Supplementation with palmitic acid,the preliminary end product of fatty acid synthesis,rescues MM cells from adiponectin-induced apoptosis. Furthermore,5-(tetradecyloxy)-2-furancarboxylic acid (TOFA),an ACC inhibitor,exhibited potent antiproliferative effects on MM cells that could also be inhibited by fatty acid supplementation. Thus,adiponectin's ability to reduce survival of MM cells appears to be mediated through its ability to suppress lipogenesis. Our findings suggest that PKA/AMPK pathway activators,or inhibitors of ACC,may be useful adjuvants to treat MM. Moreover,the antimyeloma effect of adiponectin supports the concept that hypoadiponectinemia,as occurs in obesity,promotes MM tumor progression. View Publication

BACKGROUND: Airway remodeling is a proposed mechanism that underlies the persistent loss of lung function associated with childhood asthma. Previous studies have demonstrated that human lung fibroblasts (HLFs) co-cultured with primary human bronchial epithelial cells (BECs) from asthmatic children exhibit greater expression of extracellular matrix (ECM) components compared to co-culture with BECs derived from healthy children. Myofibroblasts represent a population of differentiated fibroblasts that have greater synthetic activity. We hypothesized co-culture with asthmatic BECs would lead to greater fibroblast to myofibroblast transition (FMT) compared to co-culture with healthy BECs. METHODS: BECs were obtained from well-characterized asthmatic and healthy children and were proliferated and differentiated at an air-liquid interface (ALI). BEC-ALI cultures were co-cultured with HLFs for 96 hours. RT-PCR was performed in HLFs for alpha smooth muscle actin ($$-SMA) and flow cytometry was used to assay for $$-SMA antibody labeling of HLFs. RT-PCR was also preformed for the expression of tropomyosin-I as an additional marker of myofibroblast phenotype. In separate experiments,we investigated the role of TGF$$2 in BEC-HLF co-cultures using monoclonal antibody inhibition. RESULTS: Expression of $$-SMA by HLFs alone was greater than by HLFs co-cultured with healthy BECs,but not different than $$-SMA expression by HLFs co-cultured with asthmatic BECs. Flow cytometry also revealed significantly less $$-SMA expression by healthy co-co-cultures compared to asthmatic co-cultures or HLF alone. Monoclonal antibody inhibition of TGF$$2 led to similar expression of $$-SMA between healthy and asthmatic BEC-HLF co-cultures. Expression of topomyosin-I was also significantly increased in HLF co-cultured with asthmatic BECs compared to healthy BEC-HLF co-cultures or HLF cultured alone. CONCLUSION: These findings suggest dysregulation of FMT in HLF co-cultured with asthmatic as compared to healthy BECs. Our results suggest TGF$$2 may be involved in the differential regulation of FMT by asthmatic BECs. These findings further illustrate the importance of BEC-HLF cross-talk in asthmatic airway remodeling. View Publication

CD8(+) T cells have a central role in antitumour immunity,but their activity is suppressed in the tumour microenvironment. Reactivating the cytotoxicity of CD8(+) T cells is of great clinical interest in cancer immunotherapy. Here we report a new mechanism by which the antitumour response of mouse CD8(+) T cells can be potentiated by modulating cholesterol metabolism. Inhibiting cholesterol esterification in T cells by genetic ablation or pharmacological inhibition of ACAT1,a key cholesterol esterification enzyme,led to potentiated effector function and enhanced proliferation of CD8(+) but not CD4(+) T cells. This is due to the increase in the plasma membrane cholesterol level of CD8(+) T cells,which causes enhanced T-cell receptor clustering and signalling as well as more efficient formation of the immunological synapse. ACAT1-deficient CD8(+) T cells were better than wild-type CD8(+) T cells at controlling melanoma growth and metastasis in mice. We used the ACAT inhibitor avasimibe,which was previously tested in clinical trials for treating atherosclerosis and showed a good human safety profile,to treat melanoma in mice and observed a good antitumour effect. A combined therapy of avasimibe plus an anti-PD-1 antibody showed better efficacy than monotherapies in controlling tumour progression. ACAT1,an established target for atherosclerosis,is therefore also a potential target for cancer immunotherapy. View Publication

Bone marrow-derived mesenchymal stem cells (BM-MSCs) can be induced to differentiate into myogenic cells. Despite their potential,previous studies have not been successful in producing a high percentage of cardiac-like cells with a muscle phenotype. We hypothesized that cardiac lineage development in BM-MSC is related to cell passage,culture milieu,and enrichment for specific cell subtypes before and during differentiation. Our study demonstrated that Lin(-) BM-MSC at an intermediate passage (IP; P8-P12) expressed cardiac troponin T (cTnT) after 21 days in culture. Cardiac TnT expression was similar whether IP cells were differentiated in media containing 5-azacytidine+2% FBS (AZA; 14%) or 2% FBS alone (LS; 12%) and both were significantly higher than AZA+5% FBS. This expression was potentiated by first enriching for CD117/Sca-1 cells followed by differentiation (AZA,39% and LS,28%). A second sequential enrichment for the dihydropyridine receptor subunit alpha2delta1 (DHPR-alpha2) resulted in cardiac TnT expressed in 54% of cultured cells compared to 28% of cells after CD117/Sca-1(+) enrichment. Cells enriched for CD117/Sca-1 and subjected to differentiation displayed spontaneous intracellular Ca(2+) transients with an increase in transient frequency and a 60% decrease in the transient duration amplitude between days 14 and 29. In conclusion,IP CD117/Sca-1(+) murine BM-MSCs display robust cardiac muscle lineage development that can be induced independent of AZA but is diminished under higher serum concentrations. Furthermore,temporal changes in calcium kinetics commensurate with increased cTnT expression suggest progressive maturation of a cardiac muscle lineage. Enrichment with CD117/Sca-1 to establish lineage commitment followed by DHPR-alpha2 in lineage developing cells may enhance the therapeutic potential of these cells for transplantation. View Publication

Protein kinase CK2 (formerly casein kinase II) is a serine/threonine kinase overexpressed in many human tumors,transformed cell lines,and rapidly proliferating tissues. Recent data have shown that many cancers involve inappropriate reactivation of Wnt signaling through ectopic expression of Wnts themselves,as has been seen in a number of human breast cancers,or through mutation of intermediates in the Wnt pathway,such as adenomatous polyposis coli or beta-catenin,as described in colon and other cancers. Wnts are secreted factors that are important in embryonic development,but overexpression of certain Wnts,such as Wnt-1,leads to proliferation and transformation of cells. We report that upon stable transfection of Wnt-1 into the mouse mammary epithelial cell line C57MG,morphological changes and increased proliferation are accompanied by increased levels of CK2,as well as of beta-catenin. CK2 and beta-catenin co-precipitate with the Dvl proteins,which are Wnt signaling intermediates. A major phosphoprotein of the size of beta-catenin appears in in vitro kinase reactions performed on the Dvl immunoprecipitates. In vitro translated beta-catenin,Dvl-2,and Dvl-3 are phosphorylated by CK2. The selective CK2 inhibitor apigenin blocks proliferation of Wnt-1-transfected cells,abrogates phosphorylation of beta-catenin,and reduces beta-catenin and Dvl protein levels. These results demonstrate that endogenous CK2 is a positive regulator of Wnt signaling and growth of mammary epithelial cells. View Publication

Embryonic stem (ES) cells are clonal cell lines derived from the inner cell mass of the developing blastocyst that can proliferate extensively in vitro and are capable of adopting all the cell fates in a developing embryo. Clinical interest in the use of ES cells has been stimulated by studies showing that isolated human cells with ES properties from the inner cell mass or developing germ cells can provide a source of somatic precursors. Previous studies have defined in vitro conditions for promoting the development of specific somatic fates,specifically,hematopoietic,mesodermal,and neurectodermal. In this study,we present a method for obtaining dopaminergic (DA) and serotonergic neurons in high yield from mouse ES cells in vitro. Furthermore,we demonstrate that the ES cells can be obtained in unlimited numbers and that these neuron types are generated efficiently. We generated CNS progenitor populations from ES cells,expanded these cells and promoted their differentiation into dopaminergic and serotonergic neurons in the presence of mitogen and specific signaling molecules. The differentiation and maturation of neuronal cells was completed after mitogen withdrawal from the growth medium. This experimental system provides a powerful tool for analyzing the molecular mechanisms controlling the functions of these neurons in vitro and in vivo,and potentially for understanding and treating neurodegenerative and psychiatric diseases. View Publication

Premature proliferative arrest in benign or early-stage tumors induced by oncoproteins,chromosomal instability,or DNA damage is associated with p53/p21 activation,culminating in either senescence or apoptosis,depending on cell context. Growth hormone (GH) elicits direct peripheral metabolic actions as well as growth effects mediated by insulin-like growth factor 1 (IGF1). Locally produced peripheral tissue GH,in contrast to circulating pituitary-derived endocrine GH,has been proposed to be both proapoptotic and prooncogenic. Pituitary adenomas expressing and secreting GH are invariably benign and exhibit DNA damage and a senescent phenotype. We therefore tested effects of nutlin-induced p53-mediated senescence in rat and human pituitary cells. We show that DNA damage senescence induced by nutlin triggers the p53/p21 senescent pathway,with subsequent marked induction of intracellular pituitary GH in vitro. In contrast,GH is not induced in cells devoid of p53. Furthermore we show that p53 binds specific GH promoter motifs and enhances GH transcription and secretion in senescent pituitary adenoma cells and also in nonpituitary (human breast and colon) cells. In vivo,treatment with nutlin results in up-regulation of both p53 and GH in the pituitary gland,as well as increased GH expression in nonpituitary tissues (lung and liver). Intracrine GH acts in pituitary cells as an apoptosis switch for p53-mediated senescence,likely protecting the pituitary adenoma from progression to malignancy. Unlike in the pituitary,in nonpituitary cells GH exerts antiapoptotic properties. Thus,the results show that GH is a direct p53 transcriptional target and fulfills criteria as a p53 target gene. Induced GH is a readily measurable cell marker for p53-mediated cellular senescence. View Publication

Neurogenesis decreases during aging causing a progressive cognitive decline but it is still controversial whether proliferation defects in neurogenic niches result from a loss of neural stem cells or from an impairment of their progression through the cell cycle. Using an accurate fluorescence-activated cell sorting technique,we show that the pool of neural stem cells is maintained in the subventricular zone of middle-aged mice while they have a reduced proliferative potential eventually leading to the subsequent decrease of their progeny. In addition,we demonstrate that the G1 phase is lengthened during aging specifically in activated stem cells,but not in transit-amplifying cells,and directly impacts on neurogenesis. Finally,we report that inhibition of TGFβ signaling restores cell cycle progression defects in stem cells. Our data highlight the significance of cell cycle dysregulation in stem cells in the aged brain and provide an attractive foundation for the development of anti-TGFβ regenerative therapies based on stimulating endogenous neural stem cells. View Publication

Regeneration of the adult intestinal epithelium is mediated by a pool of cycling stem cells,which are located at the base of the crypt,that express leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5). The Frizzled (FZD) 7 receptor (FZD7) is enriched in LGR5+ intestinal stem cells and plays a critical role in their self-renewal. Yet,drug discovery approaches and structural bases for targeting specific FZD isoforms remain poorly defined. FZD proteins interact with Wnt signaling proteins via,in part,a lipid-binding groove on the extracellular cysteine-rich domain (CRD) of the FZD receptor. Here we report the identification of a potent peptide that selectively binds to the FZD7 CRD at a previously uncharacterized site and alters the conformation of the CRD and the architecture of its lipid-binding groove. Treatment with the FZD7-binding peptide impaired Wnt signaling in cultured cells and stem cell function in intestinal organoids. Together,our data illustrate that targeting the lipid-binding groove holds promise as an approach for achieving isoform-selective FZD receptor inhibition. View Publication