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

The tumor suppressor gene phosphatase and tensin homolog (PTEN) is inactivated in many human cancers. However,it is unknown whether PTEN functions as a tumor suppressor in human Philadelphia chromosome-positive leukemia that includes chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL) and is induced by the BCR-ABL oncogene. By using our mouse model of BCR-ABL-induced leukemias,we show that Pten is down-regulated by BCR-ABL in leukemia stem cells in CML and that PTEN deletion causes acceleration of CML development. In addition,overexpression of PTEN delays the development of CML and B-ALL and prolongs survival of leukemia mice. PTEN suppresses leukemia stem cells and induces cell-cycle arrest of leukemia cells. Moreover,PTEN suppresses B-ALL development through regulating its downstream gene Akt1. These results demonstrate a critical role of PTEN in BCR-ABL-induced leukemias and suggest a potential strategy for the treatment of Philadelphia chromosome-positive leukemia. View Publication

In vitro differentiation of embryonic stem cells is tightly regulated by the same key signaling pathways that control pattern formation during embryogenesis. Small molecules that selectively target these developmental pathways,including Wnt,and BMP signaling may be valuable for directing differentiation of pluripotent stem cells toward many desired tissue types,but to date only few such compounds have been shown to promote cardiac differentiation. Here,we show that XAV939,a recently discovered small molecule inhibitor of Wnt/β-catenin signaling,can robustly induce cardiomyogenesis in mouse ES cells. Our results suggest that a timely administration of XAV939 immediately following the formation of mesoderm progenitor cells promotes cardiomyogenic development at the expense of other mesoderm derived lineages,including the endothelial,smooth muscle,and hematopoietic lineages. Given the critical role that Wnt/β-catenin signaling plays in many aspects of embryogenesis and tissue regeneration,XAV939 is a valuable chemical probe to dissect in vitro differentiation of stem cells and to explore their regenerative potential in a variety of contexts. View Publication

Mast cells (MCs) play an important role in the regulation of protective adaptive immune responses against pathogens. However,it is still unclear whether MCs promote such host defense responses via direct effects on T cells or rather by modifying the functions of antigen-presenting cells. To identify the underlying mechanisms of the immunoregulatory capacity of MCs,we investigated the impact of MCs on dendritic cell (DC) maturation and function. We found that murine peritoneal MCs underwent direct crosstalk with immature DCs that induced DC maturation as evidenced by enhanced expression of costimulatory molecules. Furthermore,the MC/DC interaction resulted in the release of the T-cell modulating cytokines IFN-γ,IL-2,IL-6 and TGF-β into coculture supernatants and increased the IL-12p70,IFN-γ,IL-6 and TGF-β secretion of LPS-matured DCs. Such MC-primed" DCs subsequently induced efficient CD4+ T-cell proliferation. Surprisingly� View Publication

Canonical Wnt/β-catenin signaling has been suggested to promote self-renewal of pluripotent mouse and human embryonic stem cells. Here,we show that SB-216763,a glycogen synthase kinase-3 (GSK3) inhibitor,can maintain mouse embryonic stem cells (mESCs) in a pluripotent state in the absence of exogenous leukemia inhibitory factor (LIF) when cultured on mouse embryonic fibroblasts (MEFs). MESCs maintained with SB-216763 for one month were morphologically indistinguishable from LIF-treated mESCs and expressed pluripotent-specific genes Oct4,Sox2,and Nanog. Furthermore,Nanog immunostaining was more homogenous in SB-216763-treated colonies compared to LIF. Embryoid bodies (EBs) prepared from these mESCs expressed early-stage markers for all three germ layers,and could efficiently differentiate into cardiac-like cells and MAP2-immunoreactive neurons. To our knowledge,SB-216763 is the first GSK3 inhibitor that can promote self-renewal of mESC co-cultured with MEFs for more than two months. View Publication

We examined the signaling pathways and cell type-specific responses of IFN regulatory factor (IRF) 5,an immune-regulatory transcription factor. We show that the protein kinases IKK$\alpha$,IKK$\beta$,IKK$\epsilon$,and TANK-binding kinase 1 each confer IRF5 phosphorylation/dimerization,thus extending the family of IRF5 activator kinases. Among primary human immune cell subsets,we found that IRF5 is most abundant in plasmacytoid dendritic cells (pDCs). Flow cytometric cell imaging revealed that IRF5 is specifically activated by endosomal TLR signaling. Comparative analyses revealed that IRF3 is activated in pDCs uniquely through RIG-I-like receptor (RLR) signaling. Transcriptomic analyses of pDCs show that the partitioning of TLR7/IRF5 and RLR/IRF3 pathways confers differential gene expression and immune cytokine production in pDCs,linking IRF5 with immune regulatory and proinflammatory gene expression. Thus,TLR7/IRF5 and RLR-IRF3 partitioning serves to polarize pDC response outcome. Strategies to differentially engage IRF signaling pathways should be considered in the design of immunotherapeutic approaches to modulate or polarize the immune response for specific outcome. View Publication

Due to viral envelope glycoprotein D binding to cellular membrane HVEM receptor,HSV-1 can infect certain dendritic cells,which becomes an event in the viral strategy to interfere with the host's immune system. We previously generated the HSV-1 mutant strain M6,which produced an attenuated phenotype in mice and rhesus monkeys. The attenuated M6 strain was used to investigate how HSV-1 infection of dendritic cells interferes with both innate and adaptive immunity. Our study showed that dendritic cells membrane HVEM receptors could mediate infection of the wild-type strain and attenuated M6 strain and that dendritic cells infected by both viruses in local tissues of animals exhibited changes in transcriptional profiles associated with innate immune and inflammatory responses. The infection of pDCs and cDCs by the two strains promoted cell differentiation to the CD103+ phenotype,but varied transcriptional profiles were observed,implying a strategy that the HSV-1 wild-type strain interferes with antiviral immunity,probably due to viral modification of the immunological phenotype of dendritic cells during processing and presentation of antigen to T cells,leading to a series of deviations in immune responses,ultimately generating the deficient immune phenotype observed in infected individuals in the clinical. View Publication

BACKGROUND Two opposing B cell subsets have been defined based on their cytokine profile: IL-6 producing effector B cells (B-effs) versus IL-10 producing regulatory B cells (B-regs) that respectively positively or negatively regulate immune responses. B-regs are decreased and/or impaired in many autoimmune diseases and inflammatory conditions. Since there is increasing evidence that links B cells and B cell-rich lymphoid follicles to the pathogenesis of COPD,the aim of this study was to investigate the presence and function of B-regs in COPD. METHODS First,presence of IL-10 producing regulatory B cells in human lung tissue was determined by immunohistochemistry. Secondly,quantification of IL-10??+??B-regs and IL-6??+??B-effs in peripheral blood mononuclear cells (PBMCs) from healthy controls,smokers without airflow limitation,and COPD patients (GOLD stage I-IV) was performed by flow cytometry. Thirdly,we exposed blood-derived B cells from COPD patients in vitro to cigarette smoke extract (CSE) and quantified IL-10??+??B-regs and IL-6??+??B-effs. Furthermore,we aimed at restoring the perturbed IL10 production by blocking BAFF. Fourthly,we determined mRNA expression of transcription factors involved in IL-10 production in FACS sorted memory- and naive B cells upon exposure to medium or CSE. RESULTS The presence of IL-10 producing regulatory B cells in parenchyma and lymphoid follicles in lungs was confirmed by immunohistochemistry. The percentage of IL-10??+??B-regs was significantly decreased in blood-derived memory B cell subsets from smokers without airflow limitation and patients with COPD,compared to never smokers. Furthermore,the capacity of B cells to produce IL-10 was reduced upon in vitro exposure to CSE and this could not be restored by BAFF-blockade. Finally,upon CSE exposure,mRNA levels of the transcription factors IRF4 and HIF-1$\alpha$,were decreased in memory B cells. CONCLUSION Decreased numbers and impaired function of B-regs in smokers and patients with COPD might contribute to the initiation and progression of the disease. View Publication

BACKGROUND Human pancreata contain many types of cells,such as endocrine islets,acinar,ductal,fat,and mesenchymal stromal cells (MSCs). MSCs are important and shown to have a promising therapeutic potential to treat various disease conditions. METHODS We investigated intra-pancreatic tissue-derived (IPTD) MSCs isolated from tissue fractions that are routinely discarded during pancreatic islet isolation of human cadaveric donors. Furthermore,whether pro-angiogenic and anti-inflammatory properties of these cells could be enhanced was investigated. RESULTS IPTD-MSCs were expanded in GMP-compatible CMRL-1066 medium supplemented with 5{\%} human platelet lysate (hPL). IPTD-MSCs were found to be highly pure,with {\textgreater} 95{\%} positive for CD90,CD105,and CD73,and negative for CD45,CD34,CD14,and HLA-DR. Immunofluorescence staining of pancreas tissue demonstrated the presence of CD105+ cells in the vicinity of islets. IPTD-MSCs were capable of differentiation into adipocytes,chondrocytes,and osteoblasts in vitro,underscoring their multipotent features. When these cells were cultured in the presence of a low dose of TNF-$\alpha$,gene expression of tumor necrosis factor alpha-stimulated gene-6 (TSG-6) was significantly increased,compared to control. In contrast,treating cells with dimethyloxallyl glycine (DMOG) (a prolyl 4-hydroxylase inhibitor) enhanced mRNA levels of nuclear factor erythroid 2-related factor 2 (NRF2) and vascular endothelial growth factor (VEGF). Interestingly,a combination of TNF-$\alpha$ and DMOG stimulated the optimal expression of all three genes in IPTD-MSCs. Conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-$\alpha$ contained higher levels of pro-angiogenic (VEGF,IL-6,and IL-8) compared to controls,promoting angiogenesis of human endothelial cells in vitro. In contrast,levels of MCP-1,a pro-inflammatory cytokine,were reduced in the conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-$\alpha$. CONCLUSIONS The results demonstrate that IPTD-MSCs reside within the pancreas and can be separated as part of a standard islet-isolation protocol. These IPTD-MSCs can be expanded and potentiated ex vivo to enhance their anti-inflammatory and pro-angiogenic profiles. The fact that IPTD-MSCs are generated in a GMP-compatible procedure implicates a direct clinical application. View Publication

Cancer stem cells (CSCs) residing in colorectal cancer tissues have tumorigenic capacity and contribute to chemotherapeutic resistance and disease relapse. It is well known that the survival of colorectal CSCs after 5-fluorouracil (5-FU)-based therapy leads to cancer recurrence. Thus CSCs represent a promising drug target. Here,we designed and synthesized novel hybrid molecules linking 5-FU with the plant-derived compound thymoquinone (TQ) and tested the potential of individual compounds and their combination to eliminate colorectal CSCs. Both,Combi and SARB hybrid showed augmented cytotoxicity against colorectal cancer cells,but were non-toxic to organoids prepared from healthy murine small intestine. NanoString analysis revealed a unique signature of deregulated gene expression in response to the combination of TQ and 5-FU (Combi) and SARB treatment. Importantly,two principle stem cell regulatory pathways WNT/{\ss}-Catenin and PI3K/AKT were found to be downregulated after Combi and hybrid treatment. Furthermore,both treatments strikingly eliminated CD133+ CSC population,accompanying the depleted self-renewal capacity by eradicating long-term propagated 3D tumor cell spheres at sub-toxic doses. In vivo xenografts on chicken eggs of SARB-treated HCT116 cells showed a prominent nuclear {\ss}-Catenin and E-cadherin staining. This was in line with the reduced transcriptional activity of {\ss}-Catenin and diminished cell adhesion under SARB exposure. In contrast to 5-FU,both,Combi and SARB treatment effectively reduced the angiogenic capacity of the remaining resistant tumor cells. Taken together,combination or hybridization of single compounds target simultaneously a broader spectrum of oncogenic pathways leading to an effective eradication of colorectal cancer cells. View Publication

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by anti-nuclear autoantibodies whose production is promoted by autoreactive T follicular helper (TFH) cells. During SLE pathogenesis,basophils accumulate in secondary lymphoid organs (SLO),amplify autoantibody production and disease progression through mechanisms that remain to be defined. Here,we provide evidence for a direct functional relationship between TFH cells and basophils during lupus pathogenesis,both in humans and mice. PD-L1 upregulation on basophils and IL-4 production are associated with TFH and TFH2 cell expansions and with disease activity. Pathogenic TFH cell accumulation,maintenance,and function in SLO were dependent on PD-L1 and IL-4 in basophils,which induced a transcriptional program allowing TFH2 cell differentiation and function. Our study establishes a direct mechanistic link between basophils and TFH cells in SLE that promotes autoantibody production and lupus nephritis. Basophils have been implicated in systemic lupus erythematosus (SLE),as evidenced by the fact that basophil-deficient mice do not develop the disease. Here,the authors demonstrate that PD-L1 and IL-4 expression in basophils promotes the pathogenic accumulation of follicular helper T cells in patients with SLE and murine models. View Publication

Toll-like receptor 9 (TLR9) recognizes bacterial,viral and self DNA and play an important role in immunity and inflammation. However,the role of TLR9 in obesity is less well-studied. Here,we generate B-cell-specific Tlr9-deficient (Tlr9fl/fl/Cd19Cre+/-,KO) B6 mice and model obesity using a high-fat diet. Compared with control mice,B-cell-specific-Tlr9-deficient mice exhibited increased fat tissue inflammation,weight gain,and impaired glucose and insulin tolerance. Furthermore,the frequencies of IL-10-producing-B cells and marginal zone B cells were reduced,and those of follicular and germinal center B cells were increased. This was associated with increased frequencies of IFNγ-producing-T cells and increased follicular helper cells. In addition,gut microbiota from the KO mice induced a pro-inflammatory state leading to immunological and metabolic dysregulation when transferred to germ-free mice. Using 16 S rRNA gene sequencing,we identify altered gut microbial communities including reduced Lachnospiraceae,which may play a role in altered metabolism in KO mice. We identify an important network involving Tlr9,Irf4 and Il-10 interconnecting metabolic homeostasis,with the function of B and T cells,and gut microbiota in obesity. Although the function of Toll-like receptor 9 (TLR9) in immunity and inflammation is well-established,its role in obesity is less well-studied. In this study,the authors demonstrate that TLR9 deficiency in B cells is associated with obesity in mice and results in altered frequencies of T and B lymphocyte subsets and gut microbiome dysbiosis. View Publication

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition that affects communication,social interaction,and behavior. Calcium (Ca2+) signaling dysregulation has been frequently highlighted in genetic studies as a contributing factor to aberrant developmental processes in ASD. Herein,we used ASD and control induced pluripotent stem cells (iPSCs) to investigate transcriptomic and functional Ca2+ dynamics at various stages of differentiation to cortical neurons. Idiopathic ASD and control iPSC lines underwent the dual SMAD inhibition differentiation protocol to direct their fate toward cortical neurons. Samples from multiple time points along the course of differentiation were processed for bulk RNA sequencing,spanning the following sequential stages: the iPSC stage,neural induction (NI) stage,neurosphere (NSP) stage,and differentiated cortical neuron (Diff) stage. Our transcriptomic analyses suggested that the numbers of Ca2+ signaling-relevant differentially expressed genes between ASD and control samples were higher in the iPSC and Diff stages. Accordingly,samples from the iPSC and Diff stages were processed for Ca2+ imaging studies. Results revealed that iPSC-stage ASD samples displayed elevated maximum Ca2+ levels in response to ATP compared to controls. By contrast,in the Diff stage,ASD neurons showed reduced maximum Ca2+ levels in response to ATP but increased maximum Ca2+ levels in response to KCl and DHPG relative to controls. Considering the distinct functional signaling contexts of these stimuli,this differential profile of receptor- and ionophore-mediated Ca2+ response suggests that aberrant calcium homeostasis underlies the pathophysiology of ASD neurons. Our data provides functional evidence for Ca2+ signaling dysregulation during neurogenesis in idiopathic ASD. View Publication