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

BACKGROUND B1a and B1b lymphocytes produce IgM that inactivates oxidation-specific epitopes (IgMOSE) on LDL (low-density lipoprotein) and protects against atherosclerosis. Loss of ID3 (inhibitor of differentiation 3) in B cells selectively promotes B1b but not B1a cell numbers,leading to higher IgMOSE production and reduction in atherosclerotic plaque formation. Yet,the mechanism underlying this regulation remains unexplored. METHODS Bulk RNA sequencing was utilized to identify differentially expressed genes in B1a and B1b cells from Id3KO and Id3WT mice. CRISPR/Cas9 and lentiviral genome editing coupled with adoptive transfer were used to identify key Id3-dependent signaling pathways regulating B1b cell proliferation and the impact on atherosclerosis. Biospecimens from humans with advanced coronary artery disease imaging were analyzed to translate murine findings to human subjects with coronary artery disease. RESULTS Through RNA sequencing,P62 was found to be enriched in Id3KO B1b cells. Further in vitro characterization reveals a novel role for P62 in mediating BAFF (B-cell activating factor)-induced B1b cell proliferation through interacting with TRAF6 (tumor necrosis factor receptor 6) and activating NF-$\kappa$B (nuclear factor kappa B),leading to subsequent C-MYC (C-myelocytomatosis) upregulation. Promoter-reporter assays reveal that Id3 inhibits the E2A protein from activating the P62 promoter. Mice adoptively transferred with B1 cells overexpressing P62 exhibited an increase in B1b cell number and IgMOSE levels and were protected against atherosclerosis. Consistent with murine mechanistic findings,P62 expression in human B1 cells was significantly higher in subjects harboring a function-impairing single nucleotide polymorphism (SNP) at rs11574 position in the ID3 gene and directly correlated with plasma IgMOSE levels. CONCLUSIONS This study unveils a novel role for P62 in driving BAFF-induced B1b cell proliferation and IgMOSE production to attenuate diet-induced atherosclerosis. Results identify a direct role for Id3 in antagonizing E2A from activating the p62 promoter. Moreover,analysis of putative human B1 cells also implicates these pathways in coronary artery disease subjects,suggesting P62 as a new immunomodulatory target for treating atherosclerosis. View Publication

Despite the clinical success of cancer immunotherapies,the majority of patients fail to respond or develop resistance through disruption of pathways that promote neo-antigen presentation on MHC I molecules. Here,we conducted a series of unbiased,genome-wide CRISPR/Cas9 screens to identify genes that limit natural killer (NK) cell anti-tumor activity. We identified that genes associated with antigen presentation and/or interferon-$\gamma$ (IFN-$\gamma$) signaling protect tumor cells from NK cell killing. Indeed,Jak1-deficient melanoma cells were sensitized to NK cell killing through attenuated NK cell-derived IFN-$\gamma$-driven transcriptional events that regulate MHC I expression. Importantly,tumor cells that became resistant to T cell killing through enrichment of MHC I-deficient clones were highly sensitive to NK cell killing. Taken together,we reveal the genes targeted by tumor cells to drive checkpoint blockade resistance but simultaneously increase their vulnerability to NK cells,unveiling NK cell-based immunotherapies as a strategy to antagonize tumor immune escape. View Publication

Tolerogenic dendritic cells (tolDCs) that dampen T cell responses can be induced from blood monocytes in vitro using factors such as Vitamin D3 (VitD),dexamethasone,IL‐10,or rapamycin. However,challenges remain in obtaining robust and efficient generation of cell therapy‐based tolDCs without compromising their viability. We recently reported that CCR2‐dependent recruitment of monocytic cells,with the capacity to dampen T‐helper responses,occurs in mice treated with a single‐stranded oligonucleotide (ssON). Here,we investigated the effects of this immunomodulatory noncoding ssON on differentiating human monocytes towards DC in the presence of IL‐4 and GM‐CSF (moDC). The moDC differentiated in the presence of ssON upregulated CD1a but also increased their expression of PD‐L1. The differentiation of monocytes to moDC in the presence of ssON introduced transcriptomic changes,many of which overlapped with VitD‐moDC and resulted in moDCs with altered lipopolysaccharide (LPS)‐responsiveness. Moreover,ssON‐moDC exhibited a low capacity to stimulate alloreactive T cells in vitro and instead promoted the induction of CD4+FoxP3+CD25+ T cells. Experiments using chemical reagents support a role for PPAR‐γ in the generation of ssON‐moDC. Collectively,our data show that monocytes differentiated with IL‐4,GM‐CSF,and ssON generate cells with phenotypic and functional characteristics of tolDCs. In this article,the authors elucidated the immunoregulatory role of an oligonucleotide (ssON) that favors the induction of human tolerogenic dendritic cells (DC). The tolerogenic profile was evidenced by reduced responsiveness to lipopolysaccharides (LPS) (A). Importantly,the tolerogenic DCs had upregulated PD‐L1 molecules and functionally inhibited the proliferation of alloreactive T cells and induced FoxP3+ Tregs (B). This study envisions the development of ssON as therapeutic for rebalancing overactive T‐helper cell responses. View Publication

Cancer stem cells were prominent responsible for cancer initiation,metastasis,and invasion as well as therapeutic resistance in colorectal cancer (CRC). The extracellular axon guidance factor netrin-1 has been found to be overexpressed in several malignant cancers such as glioma,lung cancers,and colorectal cancer. However,the role of netrin-1 on cancer stemness in CRC remains unveiled. Our study revealed high expression of netrin-1 in colorectal cancer tissues and its ability to promote cancer stemness by interacting with receptors UNC5B and neogenin on murine colorectal cancer cell. Mechanistically,the netrin-1-UNC5B/neogenin axis activates the downstream NF-κB and ERK1/2 signaling pathways,reinforcing the stemness properties of tumor cells,and further exacerbating tumor progression. Clinically,netrin-1 expression associated with poor survival and high CD133 expression in patients with CRC. Taken together,these results suggest that netrin-1 blockade could be a compelling therapeutic strategy to improve the poor outcomes and trigger cancer stemness inhibition in CRC treatment. View Publication

The lectin galectin-9 may help establish and maintain chronic hepatitis C virus infection. Galectin-9 is elevated in the liver and sera of hepatitis C virus patients,induces apoptosis of hepatitis C virus-specific T cells,and increases inhibitory regulatory T cells. Kupffer cells stain strongly for galectin-9 protein in hepatitis C virus patients. In the current study,we determined stimuli that induce galectin-9 production by monocytes and macrophages in hepatitis C virus infection. With the use of real-time PCR and flow cytometry,we analyzed galectin-9 mRNA and protein from human monocytes cocultured with hepatitis C virus-infected cells or noninfectious hepatitis C virus subgenomic replicon cells. We focused on finding the stimuli for galectin-9 production. Additionally,we measured galectin-9 during monocyte-to-macrophage maturation. Finally,we examined galectin-9 in peripheral monocytes from hepatitis C virus patients using flow cytometry. Galectin-9 mRNA increased 8-fold when primary monocytes were exposed to hepatitis C virus--infected cells. Maximum induction required proximity or contact and did not require IFN-γ or hepatitis C virus virions. Coculture of monocytes with subgenomic replicon cells increased galectin-9 5-fold,and purified exosomes from infected cells stimulated galectin-9 production. Stimulation of monocyte TLR3,-7,and -8 increased galectin-9 production. Differentiation of monocytes to macrophages increased galectin-9,and nonclassic monocytes from hepatitis C virus patients had the highest levels of galectin-9. Hepatitis C virus-infected cells stimulated monocytes to produce galectin-9 in close proximity,possibly,in part,as a result of exosomes and endosomal TLRs. Differentiation of monocytes to macrophages increased galectin-9. Nonclassic monocytes from hepatitis C virus patients express the highest galectin-9 levels,suggesting they may contribute to elevated galectin-9 and adaptive immune inhibition in hepatitis C virus infection. View Publication

Inductive signals and transcription factors involved in motor neuron generation have been identified,raising the question of whether these developmental insights can be used to direct stem cells to a motor neuron fate. We show that developmentally relevant signaling factors can induce mouse embryonic stem (ES) cells to differentiate into spinal progenitor cells,and subsequently into motor neurons,through a pathway recapitulating that used in vivo. ES cell-derived motor neurons can populate the embryonic spinal cord,extend axons,and form synapses with target muscles. Thus,inductive signals involved in normal pathways of neurogenesis can direct ES cells to form specific classes of CNS neurons. View Publication

A phenotypic cell-based screen of a large combinatorial chemical library led to the identification of a class of diaminopyrimidine compounds (cardiogenol A-D) which can selectively and efficiently induce mouse embryonic stem cells (ESCs) to differentiate into cardiomyocytes. ESC-derived cardiomyocytes were shown to express multiple cardiac muscle markers,including myosin heavy chain,GATA-4,MEF2,and Nkx2.5,and spontaneously form beating regions. Such small molecules will serve as useful chemical probes to study cardiac muscle differentiation and may ultimately facilitate the therapeutic application of ESCs for cardiac repair. View Publication

Chronic treatment with antidepressants increases neurogenesis in the adult hippocampus. This increase in the production of new neurons may be required for the behavioral effects of antidepressants. However,it is not known which class of cells within the neuronal differentiation cascade is targeted by the drugs. We have generated a reporter mouse line,which allows identification and classification of early neuronal progenitors. It also allows accurate quantitation of changes induced by neurogenic agents in these distinct subclasses of neuronal precursors. We use this line to demonstrate that the selective serotonin reuptake inhibitor antidepressant fluoxetine does not affect division of stem-like cells in the dentate gyrus but increases symmetric divisions of an early progenitor cell class. We further demonstrate that these cells are the sole class of neuronal progenitors targeted by fluoxetine in the adult brain and suggest that the fluoxetine-induced increase in new neurons arises as a result of the expansion of this cell class. This finding defines a cellular target for antidepressant drug therapies. View Publication

OBJECTIVE: Studies in pulmonary arteries (PA) of patients with chronic obstructive pulmonary disease (COPD) suggest that bone marrow-derived endothelial progenitor cells (CD133(+)) may infiltrate the intima and differentiate into smooth muscle cells (SMC). This study aimed to evaluate the plasticity of CD133(+) cells to differentiate into SMC and endothelial cells (EC) in both cell culture and human isolated PA. METHODS: Plasticity of granulocyte-colony stimulator factor (G-CSF)-mobilized peripheral blood CD133(+) cells was assessed in co-cultures with primary lines of human PA endothelial cells (PAEC) or SMC (PASMC) and in isolated human PA. We also evaluated if the phenotype of differentiated progenitor cells was acquired by fusion or differentiation. RESULTS: The in vitro studies demonstrated CD133(+) cells may acquire the morphology and phenotype of the cells they were co-cultured with. CD133(+) cells co-incubated with human isolated PA were able to migrate into the intima and differentiate into SMC. Progenitor cell differentiation was produced without fusion with mature cells. CONCLUSIONS: We provide evidence of plasticity of CD133(+) cells to differentiate into both endothelial cells and SMC,reinforcing the idea of their potential role in the remodeling process of PA in COPD. This process was conducted by transdifferentiation and not by cell fusion. View Publication

Rats have important advantages over mice as an experimental system for physiological and pharmacological investigations. The lack of rat embryonic stem (ES) cells has restricted the availability of transgenic technologies to create genetic models in this species. Here,we show that rat ES cells can be efficiently derived,propagated,and genetically manipulated in the presence of small molecules that specifically inhibit GSK3,MEK,and FGF receptor tyrosine kinases. These rat ES cells express pluripotency markers and retain the capacity to differentiate into derivatives of all three germ layers. Most importantly,they can produce high rates of chimerism when reintroduced into early stage embryos and can transmit through the germline. Establishment of authentic rat ES cells will make possible sophisticated genetic manipulation to create models for the study of human diseases. View Publication

Aldehyde dehydrogenase-1A1 (ALDH1A1) expression characterizes a subpopulation of cells with tumor-initiating or cancer stem cell properties in several malignancies. Our goal was to characterize the phenotype of ALDH1A1-positive ovarian cancer cells and examine the biological effects of ALDH1A1 gene silencing. In our analysis of multiple ovarian cancer cell lines,we found that ALDH1A1 expression and activity was significantly higher in taxane- and platinum-resistant cell lines. In patient samples,72.9% of ovarian cancers had ALDH1A1 expression in which the percentage of ALDH1A1-positive cells correlated negatively with progression-free survival (6.05 vs. 13.81 months; P textless 0.035). Subpopulations of A2780cp20 cells with ALDH1A1 activity were isolated for orthotopic tumor-initiating studies,where tumorigenicity was approximately 50-fold higher with ALDH1A1-positive cells. Interestingly,tumors derived from ALDH1A1-positive cells gave rise to both ALDH1A1-positive and ALDH1A1-negative populations,but ALDH1A1-negative cells could not generate ALDH1A1-positive cells. In an in vivo orthotopic mouse model of ovarian cancer,ALDH1A1 silencing using nanoliposomal siRNA sensitized both taxane- and platinum-resistant cell lines to chemotherapy,significantly reducing tumor growth in mice compared with chemotherapy alone (a 74%-90% reduction; P textless 0.015). These data show that the ALDH1A1 subpopulation is associated with chemoresistance and outcome in ovarian cancer patients,and targeting ALDH1A1 sensitizes resistant cells to chemotherapy. ALDH1A1-positive cells have enhanced,but not absolute,tumorigenicity but do have differentiation capacity lacking in ALDH1A1-negative cells. This enzyme may be important for identification and targeting of chemoresistant cell populations in ovarian cancer. View Publication