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Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature,differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF,NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases. View Publication

The hepatocyte growth factor (HGF)/MNNG HOS transforming gene (MET) pathway regulates cell growth,survival,and migration. MET is mutated or amplified in several malignancies. In myeloma,MET is not mutated,but patients have high plasma concentrations of HGF,high levels of MET expression,and gene copy number,which are associated with poor prognosis and advanced disease. Our previous studies demonstrated that MET is critical for myeloma cell survival and its knockdown induces apoptosis. In our current study,we tested tivantinib (ARQ 197),a small-molecule pharmacological MET inhibitor. At clinically achievable concentrations,tivantinib induced apoptosis by textgreater50% in all 12 human myeloma cell lines tested. This biologic response was associated with down-regulation of MET signaling and inhibition of the mitogen-activated protein kinase and phosphoinositide 3-kinase pathways,which are downstream of the HGF/MET axis. Tivantinib was equally effective in inducing apoptosis in myeloma cell lines resistant to standard chemotherapy (melphalan,dexamethasone,bortezomib,and lenalidomide) as well as in cells that were co-cultured with a protective bone marrow microenvironment or with exogenous cytokines. Tivantinib induced apoptosis in CD138+ plasma cells from patients and demonstrated efficacy in a myeloma xenograft mouse model. On the basis of these data,we initiated a clinical trial for relapsed/refractory multiple myeloma (MM). In conclusion,MET inhibitors may be an attractive target-based strategy for the treatment of MM. View Publication

Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations are responsive to EGFR-tyrosine kinase inhibitor (EGFR-TKI). However,NSCLC patients with secondary somatic EGFR mutations are resistant to EGFR-TKI treatment. In this study,we investigated the effect of TG101348 (a JAK2 inhibitor) on the tumor growth of erlotinib-resistant NSCLC cells. Cell proliferation,apoptosis,gene expression and tumor growth were evaluated by diphenyltetrazolium bromide (MTT) assay,flow cytometry,terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining,Western Blot and a xenograft mouse model,respectively. Results showed that erlotinib had a stronger impact on the induction of apoptosis in erlotinib-sensitive PC-9 cells but had a weaker effect on erlotinib-resistant H1975 and H1650 cells than TG101348. TG101348 significantly enhanced the cytotoxicity of erlotinib to erlotinib-resistant NSCLC cells,stimulated erlotinib-induced apoptosis and downregulated the expressions of EGFR,p-EGFR,p-STAT3,Bcl-xL and survivin in erlotinib-resistant NSCLC cells. Moreover,the combined treatment of TG101348 and erlotinib induced apoptosis,inhibited the activation of p-EGFR and p-STAT3,and inhibited tumor growth of erlotinib-resistant NSCLC cells in vivo. Our results indicate that TG101348 is a potential adjuvant for NSCLC patients during erlotinib treatment. View Publication

Manipulation of genes in human embryonic stem cells (hESCs) is imperative for their highly potential applications; however,the transduction efficiency remains very low. Although existing evidence revealed the type,size,and zeta potential of vector affect gene transfection efficiency in cells,the systematic study in hESCs is scarce. In this study,using poly(amidoamine) (PAMAM) dendrimers ended with amine,hydroxyl,or carboxyl as model,we tested the influences of size and surface group as well as cytotoxicity and endocytosis on hESC gene transfection. We found that in culture medium of mTeSR the particle sizes of G5,G7,G4.5COOH,and G5OH were around 5 nm and G1 had a smaller size of 3.14 nm. G5 and G7 had a slight and significant positive zeta potential,respectively,whereas G1 was slightly negative,and G4.5COOH and G5OH were significantly negative. We demonstrated that only amine-terminated dendrimers accomplished gene transfection in hESCs,which is greater than that from Lipofectamine 2000 transfection. Ten micromolar G5 had the greatest efficiency and was better than 1000 μM G1. Only a low concentration (0.5 and 1 μM) of G7 realized gene delivery. Amine-ended dendrimers,especially with higher generations,were detrimental to the growth and pluripotent maintenance of hESCs. In contrast,similarly sized hydroxyl- and carboxyl-terminated dendrimers exerted much lower cytotoxicity,in which carboxyl-terminated dendrimer maintained pluripotency of hESCs. We also confirmed the endocytosis into and significant exocytosis from hESCs using FITC-labeled G5 dendrimer. These results suggested that careful considerations of size,concentration,and zeta potential,particularly the identity and position of groups,as well as minimized exocytosis in the design of a vector for hESC gene delivery are necessary,which helps to better design an effective vector in hESC gene transduction. View Publication

The activation of the complement system is a key initiating step in the protective innate immune-inflammatory response against injury,although it may also cause harm if left unchecked. The structurally related soluble complement inhibitors C4b-binding protein (C4BP) and factor H (FH) exert a tight regulation of the classical/lectin and alternative pathways of complement activation,respectively,attenuating the activity of the C3/C5 convertases and,consequently,avoiding serious damage to host tissues. We recently reported that the acute-phase C4BP isoform C4BP lacking the β-chain plays a pivotal role in the modulation of the adaptive immune responses. In this study,we demonstrate that FH acts in the early stages of monocyte to dendritic cell (DC) differentiation and is able to promote a distinctive tolerogenic and anti-inflammatory profile on monocyte-derived DCs (MoDCs) challenged by a proinflammatory stimulus. Accordingly,FH-treated and LPS-matured MoDCs are characterized by altered cytoarchitecture,resembling immature MoDCs,lower expression of the maturation marker CD83 and the costimulatory molecules CD40,CD80,and CD86,decreased production of key proinflammatory Th1-cytokines (IL-12,TNF-α,IFN-γ,IL-6,and IL-8),and preferential production of immunomodulatory mediators (IL-10 and TGF-β). Moreover,FH-treated MoDCs show low Ag uptake and,when challenged with LPS,display reduced CCR7 expression and chemotactic migration,impaired CD4(+) T cell alloproliferation,inhibition of IFN-γ secretion by the allostimulated T cells,and,conversely,induction of CD4(+)CD127(low/negative)CD25(high)Foxp3(+) regulatory T cells. Thus,this novel noncanonical role of FH as an immunological brake able to directly affect the function of MoDCs in an inflammatory environment may exhibit therapeutic potential in hypersensitivity,transplantation,and autoimmunity. View Publication

Functional hepatocytes derived from human pluripotent stem cells (hPSCs) have potential as tools for predicting drug-induced hepatotoxicity in the early phases of drug development. However,the propensity of hPSC lines to differentiate into specific lineages is reported to differ. The ability to predict low propensity of hPSCs to differentiate into hepatocytes would facilitate the selection of useful hPSC clones and substantially accelerate development of hPSC-derived hepatocytes for pharmaceutical research. In this study,we compared the expression of genes associated with hepatic differentiation in five hPSC lines including human ES cell line,H9,which is known to differentiate into hepatocytes,and an hPSC line reported with a poor propensity for hepatic differentiation. Genes distinguishing between undifferentiated hPSCs,hPSC-derived hepatoblast-like differentiated cells,and primary human hepatocytes were drawn by two-way cluster analysis. The order of expression levels of genes in undifferentiated hPSCs was compared with that in hPSC-derived hepatoblast-like cells. Three genes were selected as predictors of low propensity for hepatic differentiation. Expression of these genes was investigated in 23 hPSC clones. Review of representative cells by induction of hepatic differentiation suggested that low prediction scores were linked with low hepatic differentiation. Thus,our model using gene expression ranking and bioinformatic analysis could reasonably predict poor differentiation propensity of hPSC lines. View Publication

To develop a purification strategy for isolating the most primitive hematopoietic stem cells present in normal human marrow we have combined cell separation techniques with an assay for cells that initiate sustained hematopoiesis in vitro in the presence of irradiated human marrow adherent cells. These feeders" were established by subculturing 2- to 6-week-old primary long-term marrow culture adherent layers at a density of 3 x 10(4) irradiated cells per square centimeter. Test "long-term culture (LTC)-initiating cells" were plated on top of the feeders and the cocultures then maintained as standard long-term marrow cultures with half-media changes and removal of half of the nonadherent cells each week. The total number of myeloid View Publication

Insulin-like growth factor (IGF) is involved in regulating many processes during neural development,and IGF binding protein-4 (IGFBP4) functions as a modulator of IGF actions or in an IGF-independent manner (e.g.,via inhibiting Wnt/β-catenin signaling). In the present study,neural progenitor cells (NPCs) were isolated from the forebrain of newborn mice to investigate effects of IGFBP4 on the proliferation and differentiation of NPCs. The proliferation of NPCs was evaluated using Cell Counting Kit-8 (CCK-8) after treatment with or without IGFBP4 as well as blockers of IGF-IR and β-catenin. Phosphorylation levels of Akt,Erk1,2 and p38 were analyzed by Western blotting. The differentiation of NPCs was evaluated using immunofluorescence and Western blotting. It was shown that exogenous IGFBP4 significantly inhibited the proliferation of NPCs and it did not induce a more pronounced inhibition of cell proliferation after blockade of IGF-IR but it did after antagonism of β-catenin. Akt phosphorylation was significantly decreased and phosphorylation levels of Erk1,2 and p38 were not significantly changed in IGFBP4-treated NPCs. Excessive IGFBP4 significantly promoted NPCs to differentiate into astrocytes and neurons. These data suggested that exogenous IGFBP4 inhibits proliferation and promotes differentiation of neural progenitor cells mainly through IGF-IR signaling pathway. View Publication

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Pluripotent embryonic stem cells (ESC,ES cells) of line D3 were differentiated in vitro and via embryo-like aggregates (embryoid bodies) of defined cell number into spontaneously beating cardiomyocytes. By using RT-PCR technique,alpha- and beta-cardiac myosin heavy chain (MHC) genes were found to be expressed in embryoid bodies of early to terminal differentiation stages. The exclusive expression of the beta-cardiac MHC gene detected in very early differentiated embryoid bodies proved to be dependent on the number of ES cells developing in the embryoid body. Cardiomyocytes enzymatically isolated from embryoid body outgrowths at different stages of development were further characterized by immunocytological and electrophysiological techniques. All cardiomyocytes appeared to be positive in immunofluorescence assays with monoclonal antibodies against cardiac-specific alpha-cardiac MHC,as well as muscle-specific sarcomeric myosin heavy chain and desmin. The patch-clamp technique allowed a more detailed characterization of the in vitro differentiated cardiomyocytes which were found to represent phenotypes corresponding to sinusnode,atrium or ventricle of the heart. The cardiac cells of early differentiated stage expressed pacemaker-like action potentials similar to those described for embryonic cardiomyocytes. The action potentials of terminally differentiated cells revealed shapes,pharmacological characteristics and hormonal regulation inherent to adult sinusnodal,atrial or ventricular cells. In cardiomyocytes of intermediate differentiation state,action potentials of very long duration (0.3-1 s) were found,which may represent developmentally controlled transitions between different types of action potentials. Therefore,the presented ES cell differentiation system permits the investigation of commitment and differentiation of embryonic cells into the cardiomyogenic lineage in vitro. View Publication

Neurogenesis in the adult brain is important for memory and learning,and the alterations in neural stem cells (NSCs) may be an important part of Alzheimer's disease pathogenesis. The phosphatidylinositol 3-kinase (PI3K) pathway has been suggested to play an important role in neuronal cell survival and is highly involved in adult neurogenesis. Recently,coenzyme Q10 (CoQ10) was found to affect the PI3K pathway. We investigated whether CoQ10 could restore amyloid β (Aβ)25-35 oligomer-inhibited proliferation of NSCs by focusing on the PI3K pathway. To evaluate the effects of CoQ10 on Aβ25-35 oligomer-inhibited proliferation of NSCs,NSCs were treated with several concentrations of CoQ10 and/or Aβ25-35 oligomers. BrdU labeling,Colony Formation Assays,and immunoreactivity of Ki-67,a marker of proliferative activity,showed that NSC proliferation decreased with Aβ25-35 oligomer treatment,but combined treatment with CoQ10 restored it. Western blotting showed that CoQ10 treatment increased the expression levels of p85α PI3K,phosphorylated Akt (Ser473),phosphorylated glycogen synthase kinase-3β (Ser9),and heat shock transcription factor,which are proteins related to the PI3K pathway in Aβ25-35 oligomers-treated NSCs. To confirm a direct role for the PI3K pathway in CoQ10-induced restoration of proliferation of NSCs inhibited by Aβ25-35 oligomers,NSCs were pretreated with a PI3K inhibitor,LY294002; the effects of CoQ10 on the proliferation of NSCs inhibited by Aβ25-35 oligomers were almost completely blocked. Together,these results suggest that CoQ10 restores Aβ25-35 oligomer-inhibited proliferation of NSCs by activating the PI3K pathway. View Publication

Cancer stem cells (CSC) are resistant to radiation and chemotherapy and play a significant role in cancer recurrence and metastatic disease. It is therefore important to identify alternative strategies,such as immunotherapies that can be used to control this refractory population. A CD44(+)CD24(-/low) subpopulation of cells within the B6 PyMT-MMTV transgenic mouse-derived AT-3 mammary carcinoma cell line was identified,which had CSC-like characteristics,including pluripotency and a resistance to chemo- and radiotherapy. Therefore,unlike xenograph models that require immunocompromised settings,this novel system may provide a means to study immune-mediated responses against CSC-like cells. The immunobiology of the AT-3 CSC-like cell population was studied by their surface molecule expression profile and their sensitivity to specified cell death pathways. Comparable levels of Rae-1,CD155,CD54 and higher levels of Fas and DR5 were expressed on the AT-3 CSC-like cells compared to non-CSC-like tumor cells. Expression correlated with an in vitro sensitivity to cell death by NK cells or through the ligation of the death receptors (Fas or DR5),by their ligands or anti-Fas and anti-DR5 mAbs. Indeed,compared to the rest of the AT-3 tumor cells,the CD44(+)CD24(-/low) subpopulation of cells were more sensitive to both Fas- and TRAIL-mediated cell death pathways. Therefore,despite the refractory nature of CSC to other conventional therapies,these CSC-like cells were not inherently resistant to specified forms of immune-mediated cell death. These results encourage the continued investigation into immunotherapeutic strategies as a means of controlling breast CSC,particularly through their cell death pathways. View Publication