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

TLR-mediated activation of dendritic cells (DCs) is associated with a metabolic transition in which mitochondrial oxidative phosphorylation is inhibited by endogenously synthesized NO and the cells become committed to glucose and aerobic glycolysis for survival. We show that inhibition of mechanistic target of rapamycin (mTOR) extends the lifespan of TLR-activated DCs by inhibiting the induction of NO production,thereby allowing the cells to continue to use their mitochondria to generate ATP,and allowing them the flexibility to use fatty acids or glucose as nutrients to fuel core metabolism. These data provide novel mechanistic insights into how mTOR modulates DC metabolism and cellular longevity following TLR activation and provide an explanation for previous findings that mTOR inhibition enhances the efficacy of DCs in autologous vaccination. View Publication

Butein has been reported to exert anti-inflammatory effect but the possible mechanism involved is still unclear. Here,we report the inhibitory effect of butein on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression. Butein also inhibited the induction of tumor necrosis factor-alpha and cyclooxygenase 2 by LPS. To further investigate the mechanism responsible for the inhibition of iNOS gene expression by butein,we examined the effect of butein on LPS-induced nuclear factor-kappaB (NF-kappaB) activation. The LPS-induced DNA binding activity of NF-kappaB was significantly inhibited by butein,and this effect was mediated through inhibition of the degradation of inhibitory factor-kappaB and phosphorylation of Erk1/2 MAP kinase. Furthermore,increased binding of the osteopontin alphavbeta3 integrin receptor by butein may explain its inhibitory effect on LPS-mediated NO production. Taken together,these results suggest that butein inhibits iNOS gene expression,providing possible mechanisms for its anti-inflammatory action. View Publication

Several hematopoietic growth factors,including interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-beta1),promote the differentiation of tolerogenic dendritic cells (DCs). Hepatocyte growth factor (HGF) is a pleiotropic cytokine whose effects on human DC differentiation and function have not been investigated. Monocytes cultured with HGF (HGFMo) differentiated into accessory cells with DC-like morphology,released low amounts of IL-12p70 and up-regulated IL-10 both at the mRNA and at the protein level. Upon activation with HGFMo,allogeneic CD4+CD25- T cells expressed the T regulatory (Treg)-associated transcription factor FoxP3,proliferated poorly,and released high levels of IL-10. Interestingly,blockade of surface immunoglobulin-like transcript 3 (ILT3) on HGFMo or neutralization of secreted IL-10 translated into partial restoration of T-cell proliferation. Secondary stimulation of HGFMo-primed CD4+ T cells with immunogenic DCs differentiated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 from monocytes of the same donor resulted in measurable T-cell proliferation. HGFMo-primed CD4+ T cells significantly inhibited the proliferation of naive CD4+CD25- T cells in a cell-contact-dependent manner. Finally,DNA microarray analysis revealed a unique gene-expression profile of HGF-activated monocytes. Collectively,our findings point to a novel role for HGF in the regulation of monocyte/DC functions that might be exploited therapeutically. View Publication

Imatinib mesylate (IM) induces clinical remissions in chronic-phase chronic myeloid leukemia (CML) patients but IM resistance remains a problem. We recently identified several features of CML CD34(+) stem/progenitor cells expected to confer resistance to BCR-ABL-targeted therapeutics. From a study of 25 initially chronic-phase patients,we now demonstrate that some,but not all,of these parameters correlate with subsequent clinical response to IM therapy. CD34(+) cells from the 14 IM nonresponders demonstrated greater resistance to IM than the 11 IM responders in colony-forming cell assays in vitro (P textless .001) and direct sequencing of cloned transcripts from CD34(+) cells further revealed a higher incidence of BCR-ABL kinase domain mutations in the IM nonresponders (10%-40% vs 0%-20% in IM responders,P textless .003). In contrast,CD34(+) cells from IM nonresponders and IM responders were not distinguished by differences in BCR-ABL or transporter gene expression. Interestingly,one BCR-ABL mutation (V304D),predicted to destabilize the interaction between p210(BCR-ABL) and IM,was detectable in 14 of 20 patients. T315I mutant CD34(+) cells found before IM treatment in 2 of 20 patients examined were preferentially amplified after IM treatment. Thus,2 properties of pretreatment CML stem/progenitor cells correlate with subsequent response to IM therapy. Prospective assessment of these properties may allow improved patient management. View Publication

BACKGROUND Accumulating evidence suggests that breast cancer involves tumour-initiating cells (TICs),which play a role in initiation,metastasis,therapeutic resistance and relapse of the disease. Emerging drugs that target TICs are becoming a focus of contemporary research. Mitocans,a group of compounds that induce apoptosis of cancer cells by destabilising their mitochondria,are showing their potential in killing TICs. In this project,we investigated mitochondrially targeted vitamin E succinate (MitoVES),a recently developed mitocan,for its in vitro and in vivo efficacy against TICs. METHODS The mammosphere model of breast TICs was established by culturing murine NeuTL and human MCF7 cells as spheres. This model was verified by stem cell marker expression,tumour initiation capacity and chemotherapeutic resistance. Cell susceptibility to MitoVES was assessed and the cell death pathway investigated. In vivo efficacy was studied by grafting NeuTL TICs to form syngeneic tumours. RESULTS Mammospheres derived from NeuTL and MCF7 breast cancer cells were enriched in the level of stemness,and the sphere cells featured altered mitochondrial function. Sphere cultures were resistant to several established anti-cancer agents while they were susceptible to MitoVES. Killing of mammospheres was suppressed when the mitochondrial complex II,the molecular target of MitoVES,was knocked down. Importantly,MitoVES inhibited progression of syngeneic HER2(high) tumours derived from breast TICs by inducing apoptosis in tumour cells. CONCLUSIONS These results demonstrate that using mammospheres,a plausible model for studying TICs,drugs that target mitochondria efficiently kill breast tumour-initiating cells. View Publication

Induced pluripotent stem cells (iPSCs) were generated from peripheral blood mononuclear cells (PBMCs) obtained from a 62-year-old female with familial hypertrophic cardiomyopathy (HCM). PBMCs were reprogrammed to a pluripotent state following transfection with non-integrative episomal vectors carrying reprogramming factors OCT4,SOX2,LIN28,KLF4 and L-MYC. iPSCs were shown to express pluripotency markers,possess trilineage differentiation potential,carry rare variants identified in DNA isolated directly from the patient's whole blood,have a normal karyotype and no longer carry episomal vectors for reprogramming. This line is a useful resource for identifying unknown genetic causes of HCM. View Publication

Recent studies using defined transcription factors to convert skin fibroblasts into chondrocytes have raised the question of whether osteo-chondroprogenitors expressing SOX9 and RUNX2 could also be generated during the course of the reprogramming process. Here,we demonstrated that doxycycline-inducible expression of reprogramming factors (KLF4 [K] and c-MYC [M]) for 6 days were sufficient to convert murine fibroblasts into SOX9(+)/RUNX2(+) cellular aggregates and together with SOX9 (S) promoted the conversion efficiency when cultured in a defined stem cell medium,mTeSR. KMS-reprogrammed cells possess gene expression profiles akin to those of native osteo-chondroprogenitors with elevated osteogenic properties and can differentiate into osteoblasts and chondrocytes in vitro,but form bone tissue upon transplantation under the skin and in the fracture site of mouse tibia. Altogether,we provide a reprogramming strategy to enable efficient derivation of osteo-chondrogenic cells that may hold promise for cell replacement therapy not limited to cartilage but also for bone tissues. View Publication

The β-hemoglobinopathies sickle cell disease and β-thalassemia are among the most common human genetic disorders worldwide. Hemoglobin A2 (HbA2,α₂δ₂) and fetal hemoglobin (HbF,α₂γ₂) both inhibit the polymerization of hemoglobin S,which results in erythrocyte sickling. Expression of erythroid Kruppel-like factor (EKLF) and GATA1 is critical for transitioning hemoglobin from HbF to hemoglobin A (HbA,α₂β₂) and HbA2. The lower levels of δ-globin expression compared with β-globin expression seen in adulthood are likely due to the absence of an EKLF-binding motif in the δ-globin proximal promoter. In an effort to up-regulate δ-globin to increase HbA2 expression,we created a series of EKLF-GATA1 fusion constructs composed of the transactivation domain of EKLF and the DNA-binding domain of GATA1,and then tested their effects on hemoglobin expression. EKLF-GATA1 fusion proteins activated δ-,γ-,and β-globin promoters in K562 cells,and significantly up-regulated δ- and γ-globin RNA transcript and protein expression in K562 and/or CD34(+) cells. The binding of EKLF-GATA1 fusion proteins at the GATA1 consensus site in the δ-globin promoter was confirmed by chromatin immunoprecipitation assay. Our studies demonstrate that EKLF-GATA1 fusion proteins can enhance δ-globin expression through interaction with the δ-globin promoter,and may represent a new genetic therapeutic approach to β-hemoglobinopathies. View Publication

The intricate molecular mechanisms that regulate ESC pluripotency are incompletely understood. Prior research indicated that activation of the Janus kinase-signal transducer and activator of transcription (STAT3) pathway or inhibition of extracellular signal-regulated kinase/glycogen synthase kinase 3 (ERK/GSK3) signaling maintains mouse ESC (mESC) pluripotency. Here,we demonstrate that inhibition of protein kinase C (PKC) isoforms maintains mESC pluripotency without the activation of STAT3 or inhibition of ERK/GSK3 signaling pathways. Our analyses revealed that the atypical PKC isoform,PKCζ plays an important role in inducing lineage commitment in mESCs through a PKCζ-nuclear factor kappa-light-chain-enhancer of activated B cells signaling axis. Furthermore,inhibition of PKC isoforms permits derivation of germline-competent ESCs from mouse blastocysts and also facilitates reprogramming of mouse embryonic fibroblasts toward induced pluripotent stem cells. Our results indicate that PKC signaling is critical to balancing ESC self-renewal and lineage commitment. View Publication

Diminishing homeostatic proliferation of memory T cells is essential for improving the efficacy of lymphoablation in transplant recipients. Our previous studies in a mouse heart transplantation model established that B lymphocytes secreting proinflammatory cytokines are critical for T cell recovery after lymphoablation. The goal of the current study was to identify mediators of B cell activation following lymphoablation in allograft recipients. Transcriptome analysis revealed that macrophage-inducible C-type lectin (Mincle,Clec4e) expression is up-regulated in B cells from heart allograft recipients treated with murine anti-thymocyte globulin (mATG). Recipient Mincle deficiency diminishes B cell production of pro-inflammatory cytokines and impairs T lymphocyte reconstitution. Mixed bone marrow chimeras lacking Mincle only in B lymphocytes have similar defects in T cell recovery. Conversely,treatment with a synthetic Mincle ligand enhances T cell reconstitution after lymphoablation in non-transplanted mice. Treatment with agonistic CD40 mAb facilitates T cell reconstitution in CD4 T cell-depleted,but not in Mincle-deficient,recipients indicating that CD40 signaling induces T cell proliferation via a Mincle-dependent pathway. These findings are the first to identify an important function of B cell Mincle as a sensor of damage-associated molecular patterns released by the graft and demonstrate its role in clinically relevant settings of organ transplantation. View Publication

Huntington’s disease (HD) causes selective degeneration of striatal and cortical neurons,resulting in cell mosaicism of coexisting still functional and dysfunctional cells. The impact of non-cell autonomous mechanisms between these cellular states is poorly understood. Here we generated telencephalic organoids with healthy or HD cells,grown separately or as mosaics of the two genotypes. Single-cell RNA sequencing revealed neurodevelopmental abnormalities in the ventral fate acquisition of HD organoids,confirmed by cytoarchitectural and transcriptional defects leading to fewer GABAergic neurons,while dorsal populations showed milder phenotypes mainly in maturation trajectory. Healthy cells in mosaic organoids restored HD cell identity,trajectories,synaptic density,and communication pathways upon cell-cell contact,while showing no significant alterations when grown with HD cells. These findings highlight cell-type-specific alterations in HD and beneficial non-cell autonomous effects of healthy cells,emphasizing the therapeutic potential of modulating cell-cell communication in disease progression and treatment. Mosaic organoids where pathological and healthy cells are grown together,reveal the rescue of phenotypes in pathological cells due to communication with healthy cells without harming them,as demonstrated by single-cell RNA-sequencing data. View Publication

Although bone marrow-derived cells with high aldehyde dehydrogenase activity (ALDH br ) have shown therapeutic potential against various diseases in animal studies,clinical trials have failed to show concurrent findings. We aimed to clarify the optimal conditions for the efficacy of ALDH br cells by using a murine bleomycin-induced pulmonary fibrosis model. We intravenously transferred male or female donor C57BL/6 mice-derived ALDH br cells into recipient C57BL/6 mice under various conditions,and used mCherry-expressing mice as a donor to trace the transferred ALDH br cells. Pulmonary fibrosis improved significantly when (1) female-derived,not male-derived,and (2) lineage (Lin)-negative,not lineage-positive,ALDH br cells were transferred during the (3) fibrotic,not inflammatory,phase. Consistent with the RNA-sequencing results,female-derived Lin − /ALDH br cells were more resistant to oxidative stress than male-derived cells in vitro,and transferred female-derived Lin − /ALDH br cells were more viable than male-derived cells in the fibrotic lung. The mechanism underlying the antifibrotic effects of Lin − /ALDH br cells was strongly associated with reduction of oxidative stress. Our results indicated that Lin − /ALDH br cell therapy could ameliorate pulmonary fibrosis by reducing oxidative stress and suggested that their efficacy was mediated by sex-related differences. Thus,sex-awareness strategies may be important for clinical application of bone marrow ALDH br cells as a therapeutic tool. The online version contains supplementary material available at 10.1186/s13287-024-03933-8. View Publication