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

AbstractThe generation of chimeric antigen receptor?modified natural killer (CAR?NK) cells using induced pluripotent stem cells (iPSCs) has emerged as one of the paradigms for manufacturing off?the?shelf universal immunotherapy. However,there are still some challenges in enhancing the potency,safety,and multiple actions of CAR?NK cells. Here,iPSCs were site?specifically integrated at the ribosomal DNA (rDNA) locus with interleukin 24 (IL24) and CD19?specific chimeric antigen receptor (CAR19),and successfully differentiated into iPSC?derived NK (iNK) cells,followed by expansion using magnetic beads in vitro. Compared with the CAR19?iNK cells,IL24 armored CAR19?iNK (CAR19?IL24?iNK) cells showed higher cytotoxic capacity and amplification ability in vitro and inhibited tumor progression more effectively with better survival in a B?cell acute lymphoblastic leukaemia (B?ALL) (Nalm?6 (Luc1))?bearing mouse model. Interestingly,RNA?sequencing analysis showed that IL24 may enhance iNK cell function through nuclear factor kappa B (NF?B) pathway?related genes while exerting a direct effect on tumor cells. This study proved the feasibility and potential of combining IL24 with CAR?iNK cell therapy,suggesting a novel and promising off?the?shelf immunotherapy strategy. Zhang et al. successfully regenerated iNK cells from human iPSCs with rDNA locus gene editing. IL24 enhances the antitumor activity and proliferation of armored CAR?iNK cells,which may be involved in cellular?positive upregulation and adhesion pathways. View Publication

OBJECTIVE As an inhibitor of the AhR signalling pathway,StemRegenin 1 (SR1) not only promotes the expansion of CD34+ cells but also increases CD34- cell numbers. These CD34- cells influenced the ex vivo expansion of CD34+ cells. In this work,the effects of periodically removing CD34- cells combined with SR1 addition on the ex vivo expansion and biological functions of HSCs were investigated. MATERIALS AND METHODS CD34- cells were removed periodically with SR1 addition to investigate cell subpopulations,cell expansion,biological functions,expanded cell division mode and supernatant TGF-$\beta$1 contents. RESULTS After 10-day culture,the expansion of CD34+ cells in the CD34- cell removal plus SR1 group was significantly higher than that in the control group and the SR1 group. Moreover,periodically removing CD34- cells with SR1 addition improved the biological function of expanded CD34+ cells and significantly increased the percentage of self-renewal symmetric division of CD34+ cells. In addition,the concentration of total TGF-$\beta$1 and activated TGF-$\beta$1 in the supernatant was significantly lower than those in the control group and the SR1 group. RT-qPCR results showed that the periodic removal of CD34- cells with cooperation from SR1 further reduced the expression of AhR-related genes. CONCLUSIONS Periodic removal of CD34- cells plus cooperation with SR1 improved the expansion of CD34+ cells,maintained better biological function of expanded CD34+ cells and reduced the TGF-$\beta$1 contents by downregulating AhR signalling. View Publication

PURPOSE: The purpose of this study was to determine whether histone deacetylase (HDAC) inhibitors (HDACI) such as vorinostat or entinostat (SNDX-275) could increase the lethality of the dual Bcr/Abl-Aurora kinase inhibitor KW-2449 in various Bcr/Abl(+) human leukemia cells,including those resistant to imatinib mesylate (IM). EXPERIMENTAL DESIGN: Bcr/Abl(+) chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) cells,including those resistant to IM (T315I,E255K),were exposed to KW-2449 in the presence or absence of vorinostat or SNDX-275,after which apoptosis and effects on signaling pathways were examined. In vivo studies combining HDACIs and KW2449 were carried out by using a systemic IM-resistant ALL xenograft model. RESULTS: Coadministration of HDACIs synergistically increased KW-2449 lethality in vitro in multiple CML and Ph(+) ALL cell types including human IM resistant cells (e.g.,BV-173/E255K and Adult/T315I). Combined treatment resulted in inactivation of Bcr/Abl and downstream targets (e.g.,STAT5 and CRKL),as well as increased reactive oxygen species (ROS) generation and DNA damage (γH2A.X). The latter events and cell death were significantly attenuated by free radical scavengers (TBAP). Increased lethality was also observed in primary CD34(+) cells from patients with CML,but not in normal CD34(+) cells. Finally,minimally active vorinostat or SNDX275 doses markedly increased KW2449 antitumor effects and significantly prolonged the survival of murine xenografts bearing IM-resistant ALL cells (BV173/E255K). CONCLUSIONS: HDACIs increase KW-2449 lethality in Bcr/Abl(+) cells in association with inhibition of Bcr/Abl,generation of ROS,and induction of DNA damage. This strategy preferentially targets primary Bcr/Abl(+) hematopoietic cells and exhibits enhanced in vivo activity. Combining KW-2449 with HDACIs warrants attention in IM-resistant Bcr/Abl(+) leukemias. 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 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

Background:A primary barrier to curing HIV is the HIV reservoir. The leading infectious cause of death worldwide for people living with HIV is tuberculosis (TB),but we do not know how TB impacts the HIV reservoir.Methods:Participants in identification and validation cohorts were selected from previously enrolled studies at Groupe Haïtien d'Étude du Sarcome de Kaposi et des Infections Opportunistes (GHESKIO) in Port au Prince,Haiti. Intact and non-intact proviral DNA were quantified using droplet digital PCR of peripheral blood mononuclear cell (PBMC)-derived CD4+ T cells. Kruskal-Wallis tests were used to compare medians with tobit regression for censoring.Results:In the identification cohort,we found that people living with HIV with a history of active pulmonary TB (n=19) had higher levels of intact provirus than people living with HIV without a history of active TB (n=47) (median 762; IQR,183-1173 vs 117; IQR,24-279 intact provirus per million CD4,respectively; P=0.0001). This difference also was seen in the validation cohort (n=31),(median 102; IQR,0-737 vs 0; IQR,0-24.5 intact provirus per million CD4,P=0.03) for TB vs no-TB history groups,respectively. The frequencies of CD4+ T cells with any detectable proviral fragment was directly proportional to the levels of interleukin-1 beta (r=0.524,P= 0.0025) and interleukin-2 (r=0.622,P=0.0002).Conclusions:People living with HIV with a history of active pulmonary TB have more HIV pro-virus in their circulating CD4+ T cells,even years after TB cure. We need to characterize which CD4+ T cells are harboring intact provirus to consider the impact of T cell-targeting HIV cure interventions for people living in TB-endemic areas. View Publication

Selumetinib (AZD6244; ARRY-142886) is a tight-binding,uncompetitive inhibitor of mitogen-activated protein kinase kinases (MEK) 1 and 2 currently in clinical development. We evaluated the effects of selumetinib in 31 human breast cancer cell lines and 43 human non-small cell lung cancer (NSCLC) cell lines to identify characteristics correlating with in vitro sensitivity to MEK inhibition. IC(50) textless1 micromol/L (considered sensitive) was seen in 5 of 31 breast cancer cell lines and 15 of 43 NSCLC cell lines,with a correlation between sensitivity and raf mutations in breast cancer cell lines (P = 0.022) and ras mutations in NSCLC cell lines (P = 0.045). Evaluation of 27 of the NSCLC cell lines with Western blots showed no clear association between MEK and phosphoinositide 3-kinase pathway activation and sensitivity to MEK inhibition. Baseline gene expression profiles were generated for each cell line using Agilent gene expression arrays to identify additional predictive markers. Genes associated with differential sensitivity to selumetinib were seen in both histologies,including a small number of genes in which differential expression was common to both histologies. In total,these results suggest that clinical trials of selumetinib in breast cancer and NSCLC might select patients whose tumors harbor raf and ras mutations,respectively. View Publication

Treatment with arsenic trioxide (As(2)O(3)) by inducing apoptosis and partial differentiation of acute promyelocytic leukemia (APL) cells results in clinical remission in APL patients resistant to chemotherapy and all-trans-retinoic acid. As(2)O(3) (iAs(III)) is methylated in the liver to mono- and dimethylated metabolites,including methylarsonic acid,methylarsonous acid,dimethylarsinic acid,and dimethylarsinous acid. Methylated trivalent metabolites that are potent cytotoxins,genotoxins,and enzyme inhibitors may contribute to the in vivo therapeutic effect of iAs(III). Therefore,we compared the potency of iAs(III) and trivalent metabolites using chemical precursors of methylarsonous acid and dimethylarsinous acid to induce differentiation,growth inhibition,and apoptosis. Methylarsine oxide (MAs(III)O) and to a lesser extent iododimethylarsine were more potent growth inhibitors and apoptotic inducers than iAs(III) in NB4 cells,an APL cell line. This was also observed in K562 human leukemia,lymphoma cell lines,and in primary culture of chronic lymphocytic leukemia cells,but not human bone marrow progenitor cells. Apoptosis was associated with greater hydrogen peroxide accumulation and inhibition of glutathione peroxidase activity. MAs(III)O,in contrast to iAs(III),did not induce PML-retinoic acid receptor alpha degradation,or restore PML nuclear bodies or differentiation in NB4 cells. In a cocultivation experiment,hepatoma-derived HepG2 cells,but not NB4 cells,methylate radiolabeled iAs(III). Methylated metabolites released from HepG2 cells are preferentially accumulated by NB4 cells. This experimental model suggests that in vivo hepatic methylation of iAs(III) may contribute to As(2)O(3)-induced apoptosis but not differentiation of APL cells. MAs(III)O as an apoptotic inducer should be considered in the treatment of other hematologic malignancies like lymphoma. View Publication

In response to inflammatory stimuli,monocytes/macrophages secrete greater quantities of the proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha),interleukin-1beta (IL-1beta) and IL-6. The inflammatory process and the innate immune response are related to the activation of several transcription factors,such as nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1). The proteasome is a multimeric protease complex,which plays a vital role in several cellular functions,including the regulation of transcription factors like NF-kappaB. In this study,we used the human monocyte cell line U937 stimulated with lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) as a model to investigate the in vitro effects of MG132,a proteasome inhibitor,on the release of TNF-alpha,IL-1beta and IL-6 and on the expression of their membrane and soluble receptors TNF-R1,IL-1R1 and IL-6R. We also analysed the effects of MG132 on the activation of NF-kappaB and AP-1 and on the IkappaB molecule. MG132 significantly inhibited the secretion of those proinflammatory cytokines. MG132 increased the release of the soluble receptors TNF-R1 and IL-1R1 from U937 cells and decreased their cell-surface expression. MG132 also increased IL-6R cell-surface expression and decreased its release. Proteasome inhibition also led to an increase in LPS+PMA-induced AP-1 activation and the attenuation of LPS+PMA-induced IkappaB degradation,resulting in the abolition of NF-kappaB activation. Our experiments strongly suggest that the proteasome is an important factor in the regulation of proinflammatory cytokines and their receptors. View Publication

Adult neural stem and progenitor cells (NSPCs) are usually defined retrospectively by their ability to proliferate in vivo (bromodeoxyuridine uptake) or to form neurospheres and to differentiate into neurons,astrocytes and oligodendrocytes in vitro. Additional strategies to identify and to isolate NSPCs are of great importance for the investigation of cell differentiation and fate specification. Using the cell surface molecules Prominin-1 and Lewis X and a metabolic marker,the aldehyde dehydrogenase activity,we isolated and characterized five main populations of NSPCs in the neurogenic subventricular zone (SVZ) and the non-neurogenic spinal cord (SC). We used clonal analysis to assess neurosphere formation and multipotency,BrdU retention to investigate in vivo proliferation activity and quantified the expression of NSPC associated genes. Surprisingly,we found many similarities in NSPC subpopulations derived from the SVZ and SC suggesting that subtypes with similar intrinsic potential exist in both regions. The marker defined classification of NSPCs will help to distinguish subpopulations of NSPCs and allows their prospective isolation using fluorescence activated cell sorting. View Publication

Neural rosettes derived from human induced pluripotent stem cells (iPSCs) have been claimed to be a highly robust in vitro cellular model for biomedical application. They are able to propagate in vitro in the presence of mitogens,including basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). However,these two mitogens are also involved in anterior-posterior patterning in a gradient dependent manner along the neural tube axis. Here,we compared the regional identity of neural rosette cells and specific neural subtypes of their progeny propagated with low and high concentrations of bFGF and EGF. We observed that low concentrations of bFGF and EGF in the culturing system were able to induce forebrain identity of the neural rosettes and promote subsequent cortical neuronal differentiation. On the contrary,high concentrations of these mitogens stimulate a mid-hindbrain fate of the neural rosettes,resulting in subsequent cholinergic neuron differentiation. Thus,our results indicate that different concentrations of bFGF and EGF supplemented during propagation of neural rosettes are involved in altering the identity of the resultant neural cells. View Publication

PURPOSE: We have previously demonstrated that in pancreatic ductal adenocarcinoma (PDAC)-derived cell lines,the common Src family kinase inhibitors PP2 and PP1 effectively inhibited morphologic alterations associated with TGFβ1-mediated epithelial-to-mesenchymal transition (EMT) by blocking the kinase activity of the TGF-β type I receptor ALK5 rather than Src (Ungefroren et al. in Curr Cancer Drug Targets 11:524,2011). In this report,the ability of PP2 and PP1,the more specific Src inhibitor SU6656,and the ALK5 inhibitor SB431542 to functionally block TGF-β1-dependent EMT and cell motility in established PDAC (Panc-1,Colo 357) and primary NSCLC (Tu459) cell lines were investigated. METHODS: The effects of PP2,PP1,SU6656,and SB431542 on TGF-β1-dependent cell scattering/EMT,cell migration/invasion,and expression of invasion-associated genes were measured by using the real-time cell analysis assay on the xCELLigence system and quantitative real-time RT-PCR,respectively. RESULTS: In all three cell lines tested,PP1,PP2,and SB431542 effectively blocked TGF-β1-induced cell scattering/EMT,migration,and invasion and in Colo 357 cells inhibited the induction of the invasion-associated MMP2 and MMP9 genes. In contrast,SU6656 only blocked TGF-β1-induced invasion in Panc-1 and Tu459 but not Colo 357 cells. PP1,and to a greater extent PP2,also inhibited the high spontaneous migratory activity of Panc-1 cells expressing a kinase-active ALK5 mutant. CONCLUSIONS: These data provide evidence that PP2 and PP1 are powerful inhibitors of TGF-β-induced cell migration and invasion in vitro and directly target ALK5. Both agents may be useful as dual TGF-β/Src inhibitors in experimental therapeutics to prevent metastatic spread in late-stage PDAC and NSCLC. View Publication