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

Removal of sialic acid from glycoconjugates on the surface of monocytes enhances their response to bacterial LPS. We tested the hypothesis that endogenous sialidase activity creates a permissive state for LPS-induced cytokine production in human monocyte-derived DCs. Of the four genetically distinct sialidases (Neu1-4),Neu1,Neu3,and Neu4 are expressed in human monocytes,but only Neu1 and Neu3 are up-regulated as cells differentiate into DCs. Neu1 and Neu3 are present on the surface of monocytes and DCs and are also present intracellularly. DCs contain a greater amount of sialic acid than monocytes,but the amount of sialic acid/mg total protein declines during differentiation to DCs. This relative hyposialylation of cells does not occur in mature DCs grown in the presence of zanamivir,a pharmacologic inhibitor of Neu3 but not Neu1,or DANA,an inhibitor of Neu1 and Neu3. Inhibition of sialidase activity during differentiation to DCs causes no detectable change in cell viability or expression of DC surface markers. Differentiation of monocytes into DCs in the presence of zanamivir results in reduced LPS- induced expression of IL-6,IL-12p40,and TNF-α by mature DCs,demonstrating a role for Neu3 in cytokine production. A role for Neu3 is supported by inhibition of cytokine production by DANA in DCs from Neu1�?�/�?� and WT mice. We conclude that sialidase-mediated change in sialic acid content of specific cell surface glycoconjugates in DCs regulates LPS-induced cytokine production,thereby contributing to development of adaptive immune responses. View Publication

Resident tissue macrophages (Mφs) continually survey the microenvironment,ingesting Ags and presenting them on their surface for recognition by T cells. Because these Ags can be either host cell- or pathogen-derived,Mφs must be able to distinguish whether a particular Ag should provoke an immune response or be tolerated. However,the mechanisms that determine whether Mφs promote or inhibit T cell activation are not well understood. To investigate this,we first determined the mechanism by which murine resident peritoneal Mφs suppress in vitro T cell proliferation in the absence of pathogens and then explored the effects of different pathogen-derived molecules on Mφ immunosuppression. Our results suggest that,in response to IFN-γ,which is secreted by TCR-activated T cells,resident peritoneal Mφs acquire immunosuppressive properties that are mediated by NO. However,pretreatment of Mφs with LPS or dsRNA,but not CpG or peptidoglycan,eliminates their suppressive properties,in part via the induction of autocrine-acting IFN-β. These results suggest TLR agonists that activate TRIF,and consequently induce IFN-β,but not those that exclusively signal through MyD88,abrogate the immunosuppressive properties of Mφs,and thus promote T cell expansion and elimination of invading microorganisms. View Publication

The conversion of lineage-committed cells to induced pluripotent stem cells (iPSCs) by reprogramming is accompanied by a global remodeling of the epigenome,resulting in altered patterns of gene expression. Here we characterize the transcriptional reorganization of large intergenic non-coding RNAs (lincRNAs) that occurs upon derivation of human iPSCs and identify numerous lincRNAs whose expression is linked to pluripotency. Among these,we defined ten lincRNAs whose expression was elevated in iPSCs compared with embryonic stem cells,suggesting that their activation may promote the emergence of iPSCs. Supporting this,our results indicate that these lincRNAs are direct targets of key pluripotency transcription factors. Using loss-of-function and gain-of-function approaches,we found that one such lincRNA (lincRNA-RoR) modulates reprogramming,thus providing a first demonstration for critical functions of lincRNAs in the derivation of pluripotent stem cells. View Publication

Chromatin regulation is critical for differentiation and disease. However,features linking the chromatin environment of stem cells with disease remain largely unknown. We explored chromatin accessibility in embryonic and multipotent stem cells and unexpectedly identified widespread chromatin accessibility at repetitive elements. Integrating genomic and biochemical approaches,we demonstrate that these sites of increased accessibility are associated with well-positioned nucleosomes marked by distinct histone modifications. Differentiation is accompanied by chromatin remodeling at repetitive elements associated with altered expression of genes in relevant developmental pathways. Remarkably,we found that the chromatin environment of Ewing sarcoma,a mesenchymally derived tumor,is shared with primary mesenchymal stem cells (MSCs). Accessibility at repetitive elements in MSCs offers a permissive environment that is exploited by the critical oncogene responsible for this cancer. Our data demonstrate that stem cells harbor a unique chromatin landscape characterized by accessibility at repetitive elements,a feature associated with differentiation and oncogenesis. View Publication

The regulatory properties of B cells have been studied in autoimmune diseases; however,their role in allergic diseases is poorly understood. We demonstrate that Semaphorin 4C (Sema4C),an axonal guidance molecule,plays a crucial role in B cell regulatory function. Mice deficient in Sema4C exhibited increased airway inflammation after allergen exposure,with massive eosinophilic lung infiltrates and increased Th2 cytokines. This phenotype was reproduced by mixed bone marrow chimeric mice with Sema4C deficient only in B cells,indicating that B lymphocytes were the key cells affected by the absence of Sema4C expression in allergic inflammation. We determined that Sema4C-deficient CD19(+)CD138(+) cells exhibited decreased IL-10 and increased IL-4 expression in vivo and in vitro. Adoptive transfer of Sema4c(-/-) CD19(+)CD138(+) cells induced marked pulmonary inflammation,eosinophilia,and increased bronchoalveolar lavage fluid IL-4 and IL-5,whereas adoptive transfer of wild-type CD19(+)CD138(+)IL-10(+) cells dramatically decreased allergic airway inflammation in wild-type and Sema4c(-/-) mice. This study identifies a novel pathway by which Th2-mediated immune responses are regulated. It highlights the importance of plasma cells as regulatory cells in allergic inflammation and suggests that CD138(+) B cells contribute to cytokine balance and are important for maintenance of immune homeostasis in allergic airways disease. Furthermore,we demonstrate that Sema4C is critical for optimal regulatory cytokine production in CD138(+) B cells. View Publication

The aldehyde dehydrogenase (ALDH) superfamily is composed of nicotinamide adenine dinucleotide (phosphate) (NAD(P)(+))-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. To date,24 ALDH gene families have been identified in the eukaryotic genome. In addition to aldehyde metabolizing capacity,ALDHs have additional catalytic (e.g. esterase and reductase) and non-catalytic activities. The latter include functioning as structural elements in the eye (crystallins) and as binding molecules to endobiotics and xenobiotics. Mutations in human ALDH genes and subsequent inborn errors in aldehyde metabolism are the molecular basis of several diseases. Most recently ALDH polymorphisms have been associated with gout and osteoporosis. Aldehyde dehydrogenase enzymes also play important roles in embryogenesis and development,neurotransmission,oxidative stress and cancer. This article serves as a comprehensive review of the current state of knowledge regarding the ALDH superfamily and the contribution of ALDHs to various physiological and pathophysiological processes. View Publication

Many long noncoding RNA (lncRNA) species have been identified in mammalian cells,but the genomic origin and regulation of these molecules in individual cell types is poorly understood. We have generated catalogs of lncRNA species expressed in human and murine embryonic stem cells and mapped their genomic origin. A surprisingly large fraction of these transcripts (textgreater60%) originate from divergent transcription at promoters of active protein-coding genes. The divergently transcribed lncRNA/mRNA gene pairs exhibit coordinated changes in transcription when embryonic stem cells are differentiated into endoderm. Our results reveal that transcription of most lncRNA genes is coordinated with transcription of protein-coding genes. View Publication

Induced pluripotent stem cell (iPS cell) holds great potential for applications in regenerative medicine,drug discovery,and disease modeling. We describe here a practical method to generate human iPS cells from urine-derived cells (UCs) under feeder-free,virus-free,serum-free condition and without oncogene c-MYC. We showed that this approach could be applied in a large population with different genetic backgrounds. UCs are easily accessible and exhibit high reprogramming efficiency,offering advantages over other cell types used for the purpose of iPS generation. Using the approach described in this study,we have generated 93 iPS cell lines from 20 donors with diverse genetic backgrounds. The non-viral iPS cell bank with these cell lines provides a valuable resource for iPS cells research,facilitating future applications of human iPS cells. View Publication

Experiments were conducted to isolate and characterize the gene and gene product of a human hematopoietic colony-stimulating factor with pluripotent biological activities. This factor has the ability to induce differentiation of a murine myelomonocytic leukemia cell line WEHI-3B(D+) and cells from patients with newly diagnosed acute nonlymphocytic leukemia (ANLL). A complementary DNA copy of the gene encoding a pluripotent human granulocyte colony-stimulating factor (hG-CSF) was cloned and expressed in Escherichia coli. The recombinant form of hG-CSF is capable of supporting neutrophil proliferation in a CFU-GM assay. In addition,recombinant hG-CSF can support early erythroid colonies and mixed colony formation. Competitive binding studies done with 125I-labeled hG-CSF and cell samples from two patients with newly diagnosed human leukemias as well as WEHI-3B(D+) cells showed that one of the human leukemias (ANLL,classified as M4) and the WEHI-3B(D+) cells have receptors for hG-CSF. Furthermore,the murine WEHI-3B(D+) cells and human leukemic cells classified as M2,M3,and M4 were induced by recombinant hG-CSF to undergo terminal differentiation to macrophages and granulocytes. The secreted form of the protein produced by the bladder carcinoma cell line 5637 was found to be O-glycosylated and to have a molecular weight of 19,600. View Publication

A 4-kilobase and a 2-kilobase cDNA clone encoding a murine macrophage colony-stimulating factor have been isolated. Except for 2 amino acid residue differences,these two clones encode the same 520 amino acid residue protein,which is preceded by a 32-amino acid residue signal peptide. The two clones,whose molecular masses correspond to the two transcripts observed in murine L929 fibroblasts,contain 3' untranslated regions that are markedly different in sequence and length. Both clones can be expressed in COS cells and the recombinant protein is active in a mouse bone marrow colony assay. View Publication

BACKGROUND: Airway remodeling might explain lung function decline among asthmatic children. Extracellular matrix (ECM) deposition by human lung fibroblasts (HLFs) is implicated in airway remodeling. Airway epithelial cell (AEC) signaling might regulate HLF ECM expression. OBJECTIVES: We sought to determine whether AECs from asthmatic children differentially regulate HLF expression of ECM constituents. METHODS: Primary AECs were obtained from well-characterized atopic asthmatic (n = 10) and healthy (n = 10) children intubated during anesthesia for an elective surgical procedure. AECs were differentiated at an air-liquid interface for 3 weeks and then cocultured with HLFs from a healthy child for 96 hours. Collagen I (COL1A1),collagen III (COL3A1),hyaluronan synthase (HAS) 2,and fibronectin expression by HLFs and prostaglandin E2 synthase (PGE2S) expression by AECs were assessed by using RT-PCR. TGF-$$1 and TGF-$$2 concentrations in media were measured by using ELISA. RESULTS: COL1A1 and COL3A1 expression by HLFs cocultured with AECs from asthmatic patients was greater than that by HLFs cocultured with AECs from healthy subjects (2.2-fold,P textless .02; 10.8-fold,P textless .02). HAS2 expression by HLFs cocultured with AECs from asthmatic patients was 2.5-fold higher than that by HLFs cocultured with AECs from healthy subjects (P textless .002). Fibronectin expression by HLFs cocultured with AECs from asthmatic patients was significantly greater than that by HLFs alone. TGF-$$2 activity was increased in cocultures of HLFs with AECs from asthmatic patients (P textless .05),whereas PGES2 was downregulated in AEC-HLF cocultures (2.2-fold,P textless .006). CONCLUSIONS: HLFs cocultured with AECs from asthmatic patients showed differential expression of the ECM constituents COL1A1 and COL3A1 and HAS2 compared with HLFs cocultured with AECs from healthy subjects. These findings support a role for altered ECM production in asthmatic airway remodeling,possibly regulated by unbalanced AEC signaling. View Publication

It remains a challenge to differentiate human induced pluripotent stem cells (iPSCs) or embryonic stem (ES) cells to Purkinje cells. In this study,we derived iPSCs from human fibroblasts and directed the specification of iPSCs first to Purkinje progenitors,by adding Fgf2 and insulin to the embryoid bodies (EBs) in a time-sensitive manner,which activates the endogenous production of Wnt1 and Fgf8 from EBs that further patterned the cells towards a midbrain-hindbrain-boundary tissue identity. Neph3-positive human Purkinje progenitors were sorted out by using flow cytometry and cultured either alone or with granule cell precursors,in a 2-dimensional or 3-dimensional environment. However,Purkinje progenitors failed to mature further under above conditions. By co-culturing human Purkinje progenitors with rat cerebellar slices,we observed mature Purkinje-like cells with right morphology and marker expression patterns,which yet showed no appropriate membrane properties. Co-culture with human fetal cerebellar slices drove the progenitors to not only morphologically correct but also electrophysiologically functional Purkinje neurons. Neph3-posotive human cells could also survive transplantation into the cerebellum of newborn immunodeficient mice and differentiate to L7- and Calbindin-positive neurons. Obtaining mature human Purkinje cells in vitro has significant implications in studying the mechanisms of spinocerebellar ataxias and other cerebellar diseases. View Publication