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

Many pathogens evade cytotoxic T lymphocytes (CTLs) by downregulating HLA molecules on infected cells,but the loss of HLA can trigger NK cell-mediated lysis. HIV-1 is thought to subvert CTLs while preserving NK cell inhibition by Nef-mediated downregulation of HLA-A and -B but not HLA-C molecules. We find that HLA-C is downregulated by most primary HIV-1 clones,including transmitted founder viruses,in contrast to the laboratory-adapted NL4-3 virus. HLA-C reduction is mediated by viral Vpu and reduces the ability of HLA-C restricted CTLs to suppress viral replication in CD4+ cells in vitro. HLA-A/B are unaffected by Vpu,and primary HIV-1 clones vary in their ability to downregulate HLA-C,possibly in response to whether CTLs or NK cells dominate immune pressure through HLA-C. HIV-2 also suppresses HLA-C expression through distinct mechanisms,underscoring the immune pressure HLA-C exerts on HIV. This viral immune evasion casts new light on the roles of CTLs and NK cells in immune responses against HIV. View Publication

PI3K activity determines positive and negative selection of B cells,a key process for immune tolerance and B cell maturation. Activation of PI3K is balanced by phosphatase and tensin homolog (Pten),the PI3K's main antagonistic phosphatase. Yet,the extent of feedback regulation between PI3K activity and Pten expression during B cell development is unclear. Here,we show that PI3K control of this process is achieved post-transcriptionally by an axis composed of a transcription factor (c-Myc),a microRNA (miR17-92),and Pten. Enhancing activation of this axis through overexpression of miR17-92 reconstitutes the impaired PI3K activity for positive selection in CD19-deficient B cells and restores most of the B cell developmental impairments that are evident in CD19-deficient mice. Using a genetic approach of deletion and complementation,we show that the c-Myc/miR17-92/Pten axis critically controls PI3K activity and the sensitivity of immature B cells to negative selection imposed by activation-induced cell death. View Publication

The epigenetic background of pluripotent stem cells can influence transcriptional and functional behavior. Most of these data have been obtained in standard monolayer cell culture systems. In this study,we used exome sequencing,array comparative genomic hybridization (CGH),miRNA array,DNA methylation array,three-dimensional (3D) tissue engineering,and immunostaining to conduct a comparative analysis of two induced pluripotent stem cell (iPSC) lines used in engineering of 3D human epidermal equivalent (HEE),which more closely approximates epidermis. Exome sequencing and array CGH suggested that their genome was stable following 3 months of feeder-free culture. While the miRNAome was also not affected,≈7% of CpG sites were differently methylated between the two lines. Analysis of the epidermal differentiation complex,a region on chromosome 1 that contains multiple genes involved in skin barrier maturation (including trichohyalin,TCHH),found that in one of the iPSC clones (iKCL004),TCHH retained a DNA methylation signature characteristic of the original somatic cells,whereas in other iPSC line (iKCL011),the TCHH methylation signature matched that of the human embryonic stem cell line KCL034. The difference between the two iPSC clones in TCHH methylation did not have an obvious effect on its expression in 3D HEE,suggesting that differentiation and tissue formation may mitigate variations in the iPSC methylome. View Publication

Inflammation is characterized by the recruitment of leukocytes from the bloodstream. The rapid arrival of neutrophils is followed by a wave of inflammatory lymphocyte antigen 6 complex (Ly6C)-positive monocytes. In contrast Ly6C(low) monocytes survey the endothelium in the steady state,but their role in inflammation is still unclear. Here,using confocal intravital microscopy,we show that upon Toll-like receptor 7/8 (TLR7/8)-mediated inflammation of mesenteric veins,platelet activation drives the rapid mobilization of Ly6C(low) monocytes to the luminal side of the endothelium. After repeatedly interacting with platelets,Ly6C(low) monocytes commit to a meticulous patrolling of the endothelial wall and orchestrate the subsequent arrival and extravasation of neutrophils through the production of proinflammatory cytokines and chemokines. At a molecular level,we show that cysteine-rich protein 61 (CYR61)/CYR61 connective tissue growth factor nephroblastoma overexpressed 1 (CCN1) protein is released by activated platelets and enables the recruitment of Ly6C(low) monocytes upon vascular inflammation. In addition endothelium-bound CCN1 sustains the adequate patrolling of Ly6C(low) monocytes both in the steady state and under inflammatory conditions. Blocking CCN1 or platelets with specific antibodies impaired the early arrival of Ly6C(low) monocytes and abolished the recruitment of neutrophils. These results refine the leukocyte recruitment cascade model by introducing endothelium-bound CCN1 as an inflammation mediator and by demonstrating a role for platelets and patrolling Ly6C(low) monocytes in acute vascular inflammation. View Publication

Structural cardiac remodeling,accompanying cytoskeletal reorganization of cardiac cells,is a major clinical outcome of diastolic heart failure. A highly local Ca(2+) influx across the plasma membrane has been suggested to code signals to induce Rho GTPase-mediated fibrosis,but it is obscure how the heart specifically decodes the local Ca(2+) influx as a cytoskeletal reorganizing signal under the conditions of the rhythmic Ca(2+) handling required for pump function. We found that an inhibition of transient receptor potential canonical 3 (TRPC3) channel activity exhibited resistance to Rho-mediated maladaptive fibrosis in pressure-overloaded mouse hearts. Proteomic analysis revealed that microtubule-associated Rho guanine nucleotide exchange factor,GEF-H1,participates in TRPC3-mediated RhoA activation induced by mechanical stress in cardiomyocytes and transforming growth factor (TGF) β stimulation in cardiac fibroblasts. We previously revealed that TRPC3 functionally interacts with microtubule-associated NADPH oxidase (Nox) 2,and inhibition of Nox2 attenuated mechanical stretch-induced GEF-H1 activation in cardiomyocytes. Finally,pharmacological TRPC3 inhibition significantly suppressed fibrotic responses in human cardiomyocytes and cardiac fibroblasts. These results strongly suggest that microtubule-localized TRPC3-GEF-H1 axis mediates fibrotic responses commonly in cardiac myocytes and fibroblasts induced by physico-chemical stimulation. View Publication

A murine leukocyte surface glycoprotein (Mr = 95 000) has been defined by means of xenogeneic monoclonal antibodies. In normal hematopoietic tissues,the glycoprotein is found in highest amounts in the bone marrow. Flow cytometric analysis shows that essentially all bone-marrow cells express the glycoprotein and that it is a major component of a subpopulation of cells containing predominantly granulocytic precursors. In contrast,only about 5 percent of thymocytes express sufficient glycoprotein to be detected by flow cytometric analysis,although under stringent conditions up to 20 percent of thymocytes are susceptible to complement-mediated cytotoxicity using a monoclonal antibody against the glycoprotein. Functional assays showed that both prothymocytes and colony forming unit-spleen express the glycoprotein which is broadly distributed on murine hematopoietic tumor cell lines. However,although some Thy-1+ (T) cell lymphomas express large amounts of the glycoprotein,others do not express detectable quantities of the molecule. The glycoprotein is not restricted to hematopoietic cells and can be detected on lung,kidney,brain,and liver as well as cultured fibroblasts. Monoclonal antibodies against the glycoprotein cross-react with an antigen present on human cells. As described in the accompanying paper,the glycoprotein exists in two antithetical allelic forms and we show that it is identical to a polymorphic surface molecule independently characterized by Colombatti and co-workers. View Publication

Stroma-dependent long-term bone marrow cultures (LTBMC) assay the ability of primitive haematopoietic stem cells (HSC) for long-term production of clonable progenitors. We have developed a limiting dilution type LTBMC assay allowing frequency analysis of transiently repopulating HSC and long-term culture initiating cells (LTC-IC) without the necessity to replate large numbers of wells. Normal or 5-FU-treated Ficoll bone marrow cells (BMC),or BMCs sorted on CD34 or HLA-DR expression,or Rh123 retention,(input range 40-70,000 CFU-GM/BFU-E/10(5) cells) were plated at limiting dilution on unirradiated adherent layers formed by a novel murine preadipose cell line (FBMD-1). The percentage of wells with at least one phase-dark haematopoietic clone (cobblestone area,CA) beneath the stromal layer was weekly determined for at least 8 weeks,and CA-forming cell (CAFC) frequencies were calculated using Poisson statistics. Parallel LTBMCs of the same samples were weekly assessed for supernate CFU-GM/BFU-E production. Weekly addition of rhIL-3 with rhG-CSF supported a high average clonogenic output per CA and dramatically increased CA size,but did not significantly alter the apparent CAFC frequency. The generation of CFU-GM per CA was constant over a period of 6 weeks with weekly means of eight normal BM samples,ranging between 5-16. At week 6 the mean CAFC frequency was 29 (1 SEM,8.8)/10(5). Early appearing CAFC were highly sensitive to 5-FU,and were contained over the full Rh123 and HLA-DR fluorescence profile of CD34pos cells,whereas CAFC week 5-8 were predominantly contained in the CD34pos Rh123dull HLA-DRlow fraction in agreement with previously reported LTC-IC characteristics. In conclusion,the CAFC assay enumerates LTC-IC using a direct visual endpoint and allows study of LTC-IC heterogeneity with respect to progenitor cell generation per stem cell clone in various haematologic diseases. View Publication

We have systematically optimized the concentrations of 20 components of a previously published serum-free medium (Brewer and Cotman,Brain Res 494: 65-74,1989) for survival of rat embryonic hippocampal neurons after 4 days in culture. This serum-free medium supplement,B27,produced neuron survival above 60%,independent of plating density above 160 plated cells/mm2. For isolated cells (textless 100 cells/mm2),survival at 4 days was still above 45%,but could be rescued to the 60% level at 40 cells/mm2 by simply applying a coverslip on top of the cells. This suggests a need for additional trophic factors. High survival was achieved with osmolarity lower than found in Dulbecco's Modified Eagle's Medium (DMEM),and by reducing cysteine and glutamine concentrations and by the elimination of toxic ferrous sulphate found in DME/F12. Neurobasal is a new medium that incorporates these modifications to DMEM. In B27/Neurobasal,glial growth is reduced to less than 0.5% of the nearly pure neuronal population,as judged by immunocytochemistry for glial fibrillary acidic protein and neuron-specific enolase. Excellent long-term viability is achieved after 4 weeks in culture with greater than 90% viability for cells plated at 640/mm2 and greater than 50% viability for cells plated at 160/mm2. Since the medium also supports the growth of neurons from embryonic rat striatum,substantia nigra,septum,and cortex,and neonatal dentate gyrus and cerebellum (Brewer,in preparation),support for other neuron types is likely. B27/Neurobasal should be useful for in vitro studies of neuronal toxicology,pharmacology,electrophysiology,gene expression,development,and effects of growth factors and hormones. View Publication

Increasing evidence supports the importance of cell extrinsic regulation in stem cell fate control. Hematopoietic stem cells (HSC) are responsive to local signals from their niche and to systemic feedback from progenitors and mature cells. The Notch ligand Delta-1 (DL1),a key component of the stem cell niche,regulates human hematopoietic lineage development in a dose-dependent manner and has been used clinically for primitive progenitor expansion. How DL1 acts to regulate HSC fate and whether these actions are related to its lineage skewing effects are poorly understood. Here we demonstrate that,although DL1 activates signal transducer and activator of transcription 3 signaling similarly to the gp130-activating cytokine interleukin-6 (IL-6),it has opposite effects on myeloid cell production. Mechanistically,these different outcomes are attributable to a DL1-mediated reduction in membrane (m)-bound IL-6 receptor (R) expression,converting progenitor cells from being directly IL-6 responsive to requiring both IL-6 and soluble (s) IL-6R for activation. Concomitant reduction of both mIL-6R (by DL1 supplementation) and sIL-6R (using dynamically fed cultures) reduced myeloid cell production and led to enhanced outputs of human HSCs. This work describes a new mode of cytokine action in which DL1 changes cytokine receptor distributions on hematopoietic cells,altering feedback networks and their impact on stem cell fate. View Publication

The first step in developing regenerative medicine approaches to treat renal diseases using pluripotent stem cells must be the generation of intermediate mesoderm (IM),an embryonic germ layer that gives rise to kidneys. In order to achieve this goal,establishing an efficient,stable and low-cost method for differentiating IM cells using small molecules is required. In this study,we identified two retinoids,AM580 and TTNPB,as potent IM inducers by high-throughput chemical screening,and established rapid (five days) and efficient (80% induction rate) IM differentiation from human iPSCs using only two small molecules: a Wnt pathway activator,CHIR99021,combined with either AM580 or TTNPB. The resulting human IM cells showed the ability to differentiate into multiple cell types that constitute adult kidneys,and to form renal tubule-like structures. These small molecule differentiation methods can bypass the mesendoderm step,directly inducing IM cells by activating Wnt,retinoic acid (RA),and bone morphogenetic protein (BMP) pathways. Such methods are powerful tools for studying kidney development and may potentially provide cell sources to generate renal lineage cells for regenerative therapy. View Publication

The mammalian immune system constitutively senses vast quantities of commensal bacteria and their products through pattern recognition receptors,yet excessive immune reactivity is prevented under homeostasis. The intestinal microbiome can influence host susceptibility to extra-intestinal autoimmune disorders. Here we report that polysaccharide A (PSA),a symbiosis factor for the human intestinal commensal Bacteroides fragilis,protects against central nervous system demyelination and inflammation during experimental autoimmune encephalomyelitis (EAE),an animal model for multiple sclerosis,through Toll-like receptor 2 (TLR2). TLR2 mediates tissue-specific expansion of a critical regulatory CD39(+) CD4 T-cell subset by PSA. Ablation of CD39 signalling abrogates PSA control of EAE manifestations and inflammatory cytokine responses. Further,CD39 confers immune-regulatory phenotypes to total CD4 T cells and Foxp3(+) CD4 Tregs. Importantly,CD39-deficient CD4 T cells show an enhanced capability to drive EAE progression. Our results demonstrate the therapeutic potential and underlying mechanism by which an intestinal symbiont product modulates CNS-targeted demyelination. View Publication

Tivantinib (ARQ197) was first reported as a highly selective inhibitor of c-MET and is currently being investigated in a phase III clinical trial. However,as recently reported by us and another group,tivantinib showed cytotoxic activity independent of cellular c-MET status and also disrupted microtubule dynamics. To investigate if tivantinib exerts its cytotoxic activity by disrupting microtubules,we quantified polymerized tubulin in cells and xenograft tumors after tivantinib treatment. Consistent with our previous report,tivantinib reduced tubulin polymerization in cells and in mouse xenograft tumors in vivo. To determine if tivantinib directly binds to tubulin,we performed an in vitro competition assay. Tivantinib competitively inhibited colchicine but not vincristine or vinblastine binding to purified tubulin. These results imply that tivantinib directly binds to the colchicine binding site of tubulin. To predict the binding mode of tivantinib with tubulin,we performed computer simulation of the docking pose of tivantinib with tubulin using GOLD docking program. Computer simulation predicts tivantinib fitted into the colchicine binding pocket of tubulin without steric hindrance. Furthermore,tivantinib showed similar IC50 values against parental and multidrug-resistant cells. In contrast,other microtubule-targeting drugs,such as vincristine,paclitaxel,and colchicine,could not suppress the growth of cells overexpressing ABC transporters. Moreover,the expression level of ABC transporters did not correlate with the apoptosis-inducing ability of tivantinib different from other microtubule inhibitor. These results suggest that tivantinib can overcome ABC transporter-mediated multidrug-resistant tumor cells and is potentially useful against various tumors. View Publication