搜索结果: 'gmp t cell activator'

The development of embryonic stem cell (ESC) therapies requires the establishment of efficient methods to differentiate ESCs into specific cell lineages. Here,we report the in vitro differentiation of common marmoset (CM) (Callithrix jacchus) ESCs into hematopoietic cells after exogenous gene transfer using vesicular stomatitis virus-glycoprotein-pseudotyped lentiviral vectors. We transduced hematopoietic genes,including tal1/scl,gata1,gata2,hoxB4,and lhx2,into CM ESCs. By immunochemical and morphological analyses,we demonstrated that overexpression of tal1/scl,but not the remaining genes,dramatically increased hematopoiesis of CM ESCs,resulting in multiple blood-cell lineages. Furthermore,flow cytometric analysis demonstrated that CD34,a hematopoietic stem/progenitor cell marker,was highly expressed in tal1/scl-overexpressing embryoid body cells. Similar results were obtained from three independent CM ESC lines. These results suggest that transduction of exogenous tal1/scl cDNA into ESCs is a promising method to induce the efficient differentiation of CM ESCs into hematopoietic stem/progenitor cells. View Publication

We recently reported that chronic lymphocytic leukemia (CLL) subgroups with distinct clonotypic BCRs present discrete patterns of TLR expression,function,and/or tolerance. In this study,to explore whether specific types of BCR/TLR collaboration exist in CLL,we studied the effect of single versus concomitant BCR and/or TLR stimulation on CLL cells from mutated (M-CLL) and unmutated CLL (U-CLL) cases. We stimulated negatively isolated CLL cells by using anti-IgM,imiquimod,and CpG oligodeoxynucleotide for BCR,TLR7,and TLR9,respectively,alone or in combination for different time points. After in vitro culture in the absence of stimulation,differences in p-ERK were identified at any time point,with higher p-ERK levels in U-CLL versus M-CLL. Pronounced p-ERK induction was seen by single stimulation in U-CLL,whereas BCR/TLR synergism was required in View Publication

textlessptextgreaterStem cell phenotypes are reflected by post-translational histone modifications,and this chromatin-related memory must be mitotically inherited to maintain cell identity through proliferative expansion. In human embryonic stem cells (hESCs),bivalent genes with both activating (H3K4me3) and repressive (H3K27me3) histone modifications are essential to sustain pluripotency. Yet the molecular mechanisms by which this epigenetic landscape is transferred to progeny cells remains to be established. By mapping genomic enrichment of H3K4me3/H3K27me3 in pure populations of hESCs in G2,mitotic,and G1 phases of the cell cycle,we found striking variations in the levels of H3K4me3 through the G2-M-G1 transition. Analysis of a representative set of bivalent genes revealed that chromatin modifiers involved in H3K4 methylation/demethylation are recruited to bivalent gene promoters in a cell cycle–dependent fashion. Interestingly,bivalent genes enriched with H3K4me3 exclusively during mitosis undergo the strongest upregulation after induction of differentiation. Furthermore,the histone-modification signature of genes that remain bivalent in differentiated cells resolves into a cell cycle–independent pattern after lineage commitment. These results establish a new dimension of chromatin regulation important in maintenance of pluripotency.textless/ptextgreater View Publication

Studies have suggested involvement of interleukin 17 (IL-17) in autoimmune diseases,although its effect on B cell biology has not been clearly established. Here we demonstrate that IL-17 alone or in combination with B cell-activating factor controlled the survival and proliferation of human B cells and their differentiation into immunoglobulin-secreting cells. This effect was mediated mainly through the nuclear factor-kappaB-regulated transcription factor Twist-1. In support of the relevance of our observations and the potential involvement of IL-17 in B cell biology,we found that the serum of patients with systemic lupus erythematosus had higher concentrations of IL-17 than did the serum of healthy people and that IL-17 abundance correlated with the disease severity of systemic lupus erythematosus. View Publication

BACKGROUND Autologous bone marrow mononuclear cell (ABMMNC) therapy has shown promise in patients with heart failure (HF). Cell function analysis may be important in interpreting trial results. METHODS In this prospective study,we evaluated the safety and efficacy of the transendocardial delivery of ABMMNCs in no-option patients with chronic HF. Efficacy was assessed by maximal myocardial oxygen consumption,single photon emission computed tomography,2-dimensional echocardiography,and quality-of-life assessment (Minnesota Living with Heart Failure and Short Form 36). We also characterized patients' bone marrow cells by flow cytometry,colony-forming unit,and proliferative assays. RESULTS Cell-treated (n = 20) and control patients (n = 10) were similar at baseline. The procedure was safe; adverse events were similar in both groups. Canadian Cardiovascular Society angina score improved significantly (P = .001) in cell-treated patients,but function was not affected. Quality-of-life scores improved significantly at 6 months (P = .009 Minnesota Living with Heart Failure and P = .002 physical component of Short Form 36) over baseline in cell-treated but not control patients. Single photon emission computed tomography data suggested a trend toward improved perfusion in cell-treated patients. The proportion of fixed defects significantly increased in control (P = .02) but not in treated patients (P = .16). Function of patients' bone marrow mononuclear cells was severely impaired. Stratifying cell results by age showed that younger patients (%60 years) had significantly more mesenchymal progenitor cells (colony-forming unit fibroblasts) than patients<60 years (20.16 ± 14.6 vs 10.92 ± 7.8,P = .04). Furthermore,cell-treated younger patients had significantly improved maximal myocardial oxygen consumption (15 ± 5.8,18.6 ± 2.7,and 17 ± 3.7 mL/kg per minute at baseline,3 months,and 6 months,respectively) compared with similarly aged control patients (14.3 ± 2.5,13.7 ± 3.7,and 14.6 ± 4.7 mL/kg per minute,P = .04). CONCLUSIONS ABMMNC therapy is safe and improves symptoms,quality of life,and possibly perfusion in patients with chronic HF. View Publication

The murine heat-stable antigen (HSA) is a glycosyl-phosphatidylinositol-linked cell surface protein which has been implicated in cellular adhesion processes,the co-stimulation of CD4+ T cells,and B cell memory. We have recently demonstrated a significant reduction in pro-B and pre-B lymphocytes in transgenic mice that overexpress HSA. We now report that cross-linking HSA with the M1/69 monoclonal antibody induces the apoptosis of cultured B cell precursors in a stomal cell and cytokine-independent manner and that sensitivity to HSA-mediated cell death increases with developmental maturity. The cross-linking of HSA does not induce apoptosis in mature splenic B cells,but instead inhibits their ability to proliferate in response to anti-CD40 + IL-4. Taken together,these data implicate HSA as a potent negative regulator of B cell development and activation. View Publication

The concept of exploiting tumor intrinsic deficiencies in DNA damage repair mechanisms by inhibiting compensatory DNA repair pathways is well established. For example,ATM-deficient cells show increased sensitivity to the ATR inhibitor ceralasertib. DNA damage response (DDR)-deficient cells are also more sensitive to DNA damaging agents like the DNA crosslinker pyrrolobenzodiazepine (PBD) SG-3199. However,additional antitumor benefits from targeting the DDR pathways,which could operate through the activation of the innate immune system are less well studied. DNA accumulation in the cytosol acts as an immunogenic danger signal,inducing the expression of type-I interferon (IFN) stimulated genes (ISGs) by the activation of the cGAS-STING pathway. Here,we demonstrate that ATM -/- FaDu tumor cells have higher basal expression of ISGs when compared to WT cells and respond to ceralasertib and PBD SG-3199 by inducing higher levels of ISGs in a cGAS-STING-dependent manner. We show that sensitive tumor cells treated with ceralasertib and PBD SG-3199 activate dendritic cells (DCs) via a type-I IFN-dependent mechanism. However,STING deficiency in tumor cells does not prevent DC activation,suggesting that transactivation of the STING pathway occurs within DCs. Furthermore,depletion of the cytosolic DNA exonuclease TREX1 in tumor cells increases DC activation in response to PBD SG-3199-treated tumor cells,indicating that an increase in tumor-derived cytosolic DNA may further enhance DC activation. In summary,in this study,we show that ceralasertib and PBD SG-3199 treatment not only intrinsically target tumor cells but also extrinsically increase tumor cell immunogenicity by inducing DC activation,which is enhanced in ATM-deficient cells. View Publication

Numerous publications have described the importance of bone morphogenetic protein (BMP) signaling in the specification of hematopoietic tissue in developing embryos. Here we investigate the full role of canonical BMP signaling in both adult and fetal liver hematopoiesis using conditional knockout strategies because conventional disruption of components of the BMP signaling pathway result in early death of the embryo. By targeting both Smad1 and Smad5,we have generated a double-knockout mouse with complete disruption of canonical BMP signaling. Interestingly,concurrent deletion of Smad1 and Smad5 results in death because of extrahematopoietic pathologic changes in the colon. However,Smad1/Smad5-deficient bone marrow cells can compete normally with wild-type cells and display unaffected self-renewal and differentiation capacity when transplanted into lethally irradiated recipients. Moreover,although BMP receptor expression is increased in fetal liver,fetal liver cells deficient in both Smad1 and Smad5 remain competent to long-term reconstitute lethally irradiated recipients in a multilineage manner. In conclusion,canonical BMP signaling is not required to maintain either adult or fetal liver hematopoiesis,despite its crucial role in the initial patterning of hematopoiesis in early embryonic development. View Publication

In recent years,mesenchymal stem cells (MSCs) have been shown to inhibit T-lymphocyte proliferation induced by alloantigens or mitogens. However,no substantial information is available regarding their effect on natural killer (NK) cells. Here we show that MSCs sharply inhibit IL-2-induced proliferation of resting NK cells,whereas they only partially affect the proliferation of activated NK cells. In addition,we show that IL-2-activated NK cells (but not freshly isolated NK cells) efficiently lyse autologous and allogeneic MSCs. The activating NK receptors NKp30,NKG2D,and DNAM-1 represented the major receptors responsible for the induction of NK-mediated cytotoxicity against MSCs. Accordingly,MSCs expressed the known ligands for these activating NK receptors-ULBPs,PVR,and Nectin-2. Moreover,NK-mediated lysis was inhibited when IFN-gamma-exposed MSCs were used as target cells as a consequence of the up-regulation of HLA class I molecules at the MSC surface. The interaction between NK cells and MSCs resulted not only in the lysis of MSCs but also in cytokine production by NK cells. These results should be taken into account when evaluating the possible use of MSCs in novel therapeutic strategies designed to improve engraftment or to suppress graft-versus-host disease (GVHD) in bone marrow transplantation. View Publication

Metabolic labeling with stable isotopes is a prominent technique for comparative quantitative proteomics,and stable isotope labeling with amino acids in cell culture (SILAC) is the most commonly used approach. SILAC is,however,traditionally limited to simple tissue culture regimens and only rarely employed in the context of complex culturing conditions as those required for human embryonic stem cells (hESCs). Classic hESC culture is based on the use of mouse embryonic fibroblasts (MEFs) as a feeder layer,and as a result,possible xenogeneic contamination,contribution of unlabeled amino acids by the feeders,interlaboratory variability of MEF preparation,and the overall complexity of the culture system are all of concern in conjunction with SILAC. We demonstrate a feeder-free SILAC culture system based on a customized version of a commonly used,chemically defined hESC medium developed by Ludwig et al. and commercially available as mTeSR1 [mTeSR1 is a trade mark of WiCell (Madison,WI) licensed to STEMCELL Technologies (Vancouver,Canada)]. This medium,together with adjustments to the culturing protocol,facilitates reproducible labeling that is easily scalable to the protein amounts required by proteomic work flows. It greatly enhances the usability of quantitative proteomics as a tool for the study of mechanisms underlying hESCs differentiation and self-renewal. Associated data have been deposited to the ProteomeXchange with the identifier PXD000151. View Publication

Peripheral blood was collected from a clinically characterized female Kleefstra syndrome patient with a heterozygous,de novo,premature termination codon (PTC) mutation (NM024757.4(EHMT1):c.3413GtextgreaterA; p.Trp1138Ter). Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the human OSKM transcription factors using the Sendai-virus (SeV) delivery system. The pluripotency of transgene-free iPSC line was verified by the expression of pluripotency-associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. Furthermore,the iPSC line showed normal karyotype. Our model might offer a good platform to study the pathomechanism of Kleefstra syndrome,also for drug testing,early biomarker discovery and gene therapy studies. View Publication

Human bone marrow (BM) or mobilized peripheral blood (mPB) CD34(+) cells have been shown to loose their stem cell quality during culture period more easily than those from cord blood (CB). We previously reported that human umbilical CB stem cells could effectively be expanded in the presence of human recombinant cytokines and a newly established murine bone marrow stromal cell line HESS-5. In this study we assessed the efficacy of this xenogeneic coculture system using human BM and mPB CD34(+) cells as materials. We measured the generation of CD34(+)CD38(-) cells and colony-forming units,and assessed severe-combined immunodeficient mouse-repopulating cell (SRC) activity using cells five days after serum-free cytokine-containing culture in the presence or the absence of a direct contact with HESS-5 cells. As compared with the stroma-free culture,the xenogeneic coculture was significantly superior on expansion of CD34(+)CD38(-) cells and colony-forming cells and on maintenance of SRC activity. The PKH26 study demonstrated that cell division was promoted faster in cells cocultured with HESS-5 cells than in cells cultured without HESS-5 cells. These results indicate that HESS-5 supports rapid generation of primitive progenitor cells (PPC) and maintains reconstituting ability of newly generated stem cells during ex vivo culture irrespective of the source of samples. This xenogeneic coculture system will be useful for ex vivo manipulation such as gene transduction to promote cell division and the generation of PPC and to prevent loss of stem cell quality. View Publication