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

Constitutive activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signaling pathway in human cancers is often associated with mutational activation of BRAF or RAS. MAPK/ERK kinase 1/2 kinases lie downstream of RAS and BRAF and are the only acknowledged activators of ERK1/2,making them attractive targets for therapeutic intervention. AZD6244 (ARRY-142886) is a potent,selective,and ATP-uncompetitive inhibitor of MAPK/ERK kinase 1/2. In vitro cell viability inhibition screening of a tumor cell line panel found that lines harboring BRAF or RAS mutations were more likely to be sensitive to AZD6244. The in vivo mechanisms by which AZD6244 inhibits tumor growth were investigated. Chronic dosing with 25 mg/kg AZD6244 bd resulted in suppression of growth of Colo-205,Calu-6,and SW-620 xenografts,whereas an acute dose resulted in significant inhibition of ERK1/2 phosphorylation. Increased cleaved caspase-3,a marker of apoptosis,was detected in Colo-205 and Calu-6 but not in SW-620 tumors where a significant decrease in cell proliferation was detected. Chronic dosing of AZD6244 induced a morphologic change in SW-620 tumors to a more differentiated phenotype. The potential of AZD6244 in combination with cytotoxic drugs was evaluated in mice bearing SW-620 xenografts. Treatment with tolerated doses of AZD6244 and either irinotecan or docetaxel resulted in significantly enhanced antitumor efficacy relative to that of either agent alone. These results indicate that AZD6244 has potential to inhibit proliferation and induce apoptosis and differentiation,but the response varies between different xenografts. Moreover,enhanced antitumor efficacy can be obtained by combining AZD6244 with the cytotoxic drugs irinotecan or docetaxel. View Publication

Phenotypic markers associated with human hematopoietic stem cells (HSCs) were developed and validated using uncultured cells. Because phenotype and function can be dissociated during culture,better markers to prospectively track and isolate HSCs in ex vivo cultures could be instrumental in advancing HSC-based therapies. Using an expansion system previously shown to increase hematopoietic progenitors and SCID-repopulating cells (SRCs),we demonstrated that the rhodamine-low phenotype was lost,whereas AC133 expression was retained throughout culture. Furthermore,the AC133(+)CD38(-) subpopulation was significantly enriched in long-term culture-initiating cells (LTC-IC) and SRCs after culture. Preculture and postculture analysis of total nucleated cell and LTC-IC number,and limiting dilution analysis in NOD/SCID mice,showed a 43-fold expansion of the AC133(+)CD38(-) subpopulation that corresponded to a 7.3-fold and 4.4-fold expansion of LTC-ICs and SRCs in this subpopulation,respectively. Thus,AC133(+)CD38(-) is an improved marker that tracks and enriches for LTC-IC and SRC in ex vivo cultures. View Publication

T cell dysfunction is crucial to the pathogenesis of systemic lupus erythematosus (SLE); however,the molecular mechanisms involved in the deficient expression of the T cell receptor-associated CD3zeta chain in SLE are not clear. SLE T cells express abnormally increased levels of an alternatively spliced isoform of CD3zeta that lacks a 562-bp region in its 3'-untranslated region (UTR). We showed previously that two adenosine/uridine-rich elements (ARE) in this splice-deleted region of CD3zeta transcript are critical for the mRNA stability and protein expression of CD3zeta. In this study we show for the first time that the mRNA-stabilizing protein HuR binds to these two ARE bearing regions of CD3zeta 3'-UTR. Knockdown of HuR resulted in decreased expression of the CD3zeta chain,whereas overexpression led to the increase of CD3zeta chain levels. Additionally,overexpression of HuR in human T cells resulted in increased mRNA stability of CD3zeta. Our results identify the 3'-UTR of CD3zeta as a novel target for the mRNA-stabilizing protein HuR. Thus,the absence of two critical AREs in the alternatively spliced CD3zeta 3'-UTR found in SLE T cells may result in decreased HuR binding,representing a possible molecular mechanism contributing to the reduced stability and expression of CD3zeta in SLE. View Publication

Interleukin (IL)-23 is thought to be critical in the pathogenesis of psoriasis,and anti-IL-23 monoclonal antibodies (mAbs) have been approved for the treatment of psoriasis. We speculated that an anti-IL-23 receptor mAb might have greater efficacy than an anti-IL-23 mAb in the treatment of local inflamed lesions with high IL-23 levels. We previously generated an anti-human IL-23 receptor mAb,AS2762900-00,which potently blocked IL-23-induced cell proliferation,regardless of the concentration of IL-23. Here,we evaluated the therapeutic potential of AS2762900-00 in the treatment of psoriasis. Compared with untreated control,AS2762900-00 significantly reduced the epidermal thickness of lesions in a clinically relevant psoriatic human skin xenograft model. The expression of inflammatory genes including genes downstream of IL-23 signaling in the lesion tended to be lower in the AS2762900-00 group than the untreated group,suggesting that the inhibitory effects of AS2762900-00 in the psoriatic human skin xenograft model might occur via blockade of IL-23 signaling pathways. Further,AS2762900-00 showed an inhibitory effect on signal transducer and activator of transcription 3 (STAT3) phosphorylation as a downstream signal of IL-23 receptor activation in whole blood from patients with psoriasis. We also confirmed that AS2762900-00 inhibited IL-23-induced STAT3 phosphorylation in a concentration-dependent manner using whole blood from healthy donors. These data suggest that AS2762900-00 is a promising drug candidate for the treatment of psoriasis. In addition,STAT3 phosphorylation in whole blood may be a useful biomarker for the evaluation of the pharmacodynamic effects of AS2762900-00 in healthy volunteers in clinical development. View Publication

Development of targeted cancer therapy requires a thorough understanding of mechanisms of tumorigenesis as well as mechanisms of action of therapeutics. This is challenging because by the time patients are diagnosed with cancer,early events of tumorigenesis have already taken place. Similarly,development of cancer immunotherapies is hampered by a lack of appropriate small animal models with autologous human tumor and immune system. In this article,we report the development of a mouse model of human acute myeloid leukemia (AML) with autologous immune system for studying early events of human leukemogenesis and testing the efficacy of immunotherapeutics. To develop such a model,human hematopoietic stem/progenitor cells (HSPC) are transduced with lentiviruses expressing a mutated form of nucleophosmin (NPM1),referred to as NPM1c. Following engraftment into immunodeficient mice,transduced HSPCs give rise to human myeloid leukemia,whereas untransduced HSPCs give rise to human immune cells in the same mice. The de novo AML,with CD123+ leukemic stem or initiating cells (LSC),resembles NPM1c+ AML from patients. Transcriptional analysis of LSC and leukemic cells confirms similarity of the de novo leukemia generated in mice with patient leukemia and suggests Myc as a co-operating factor in NPM1c-driven leukemogenesis. We show that a bispecific conjugate that binds both CD3 and CD123 eliminates CD123+ LSCs in a T cell-dependent manner both in vivo and in vitro. These results demonstrate the utility of the NPM1c+ AML model with an autologous immune system for studying early events of human leukemogenesis and for evaluating efficacy and mechanism of immunotherapeutics. View Publication

Efficient differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to a variety of lineages requires step-wise approaches replicating the key commitment stages found during embryonic development. Here we show that expression of PdgfR-α segregates mouse ESC-derived Flk-1 mesoderm into Flk-1(+)PdgfR-α(+) cardiac and Flk-1(+)PdgfR-α(-) hematopoietic subpopulations. By monitoring Flk-1 and PdgfR-α expression,we found that specification of cardiac mesoderm and cardiomyocytes is determined by remarkably small changes in levels of Activin/Nodal and BMP signaling. Translation to human ESCs and iPSCs revealed that the emergence of cardiac mesoderm could also be monitored by coexpression of KDR and PDGFR-α and that this process was similarly dependent on optimal levels of Activin/Nodal and BMP signaling. Importantly,we found that individual mouse and human pluripotent stem cell lines require optimization of these signaling pathways for efficient cardiac differentiation,illustrating a principle that may well apply in other contexts. View Publication

Cell Research 23,122 (2013). doi:10.1038/cr.2012.119 View Publication

Human induced pluripotent stem cells and their differentiation into cardiac myocytes (hiPSC-CMs) provides a unique and valuable platform for studies of cardiac muscle structure–function. This includes studies centered on disease etiology,drug development,and for potential clinical applications in heart regeneration/repair. Ultimately,for these applications to achieve success,a thorough assessment and physiological advancement of the structure and function of hiPSC-CMs is required. HiPSC-CMs are well noted for their immature and sub-physiological cardiac muscle state,and this represents a major hurdle for the field. To address this roadblock,we have developed a hiPSC-CMs (?-MHC dominant) experimental platform focused on directed physiological enhancement of the sarcomere,the functional unit of cardiac muscle. We focus here on the myosin heavy chain (MyHC) protein isoform profile,the molecular motor of the heart,which is essential to cardiac physiological performance. We hypothesized that inducing increased expression of ?-MyHC in ?-MyHC dominant hiPSC-CMs would enhance contractile performance of hiPSC-CMs. To test this hypothesis,we used gene editing with an inducible ?-MyHC expression cassette into isogeneic hiPSC-CMs,and separately by gene transfer,and then investigated the direct effects of increased ?-MyHC expression on hiPSC-CMs contractility and relaxation function. Data show improved cardiac functional parameters in hiPSC-CMs induced with ?-MyHC. Positive inotropy and relaxation was evident in comparison to ?-MyHC dominant isogenic controls both at baseline and during pacing induced stress. This approach should facilitate studies of hiPSC-CMs disease modeling and drug screening,as well as advancing fundamental aspects of cardiac function parameters for the optimization of future cardiac regeneration,repair and re-muscularization applications. View Publication

Human regulatory T cells (Treg) suppress other immune cells. Their dysfunction contributes to the pathophysiology of autoimmune diseases,including type 1 diabetes (T1D). Infusion of Tregs is being clinically evaluated as a novel way to prevent or treat T1D. Genetic modification of Tregs,most notably through the introduction of a chimeric antigen receptor (CAR) targeting Tregs to pancreatic islets,may improve their efficacy. We evaluated CAR targeting of human Tregs to monocytes,a human β cell line and human islet β cells in vitro. Targeting of HLA-A2-CAR (A2-CAR) bulk Tregs to HLA-A2+ cells resulted in dichotomous cytotoxic killing of human monocytes and islet β cells. In exploring subsets and mechanisms that may explain this pattern,we found that CD39 expression segregated CAR Treg cytotoxicity. CAR Tregs from individuals with more CD39low/- Tregs and from individuals with genetic polymorphism associated with lower CD39 expression (rs10748643) had more cytotoxicity. Isolated CD39− CAR Tregs had elevated granzyme B expression and cytotoxicity compared to the CD39+ CAR Treg subset. Genetic overexpression of CD39 in CD39low CAR Tregs reduced their cytotoxicity. Importantly,β cells upregulated protein surface expression of PD-L1 and PD-L2 in response to A2-CAR Tregs. Blockade of PD-L1/PD-L2 increased β cell death in A2-CAR Treg co-cultures suggesting that the PD-1/PD-L1 pathway is important in protecting islet β cells in the setting of CAR immunotherapy. In summary,introduction of CAR can enhance biological differences in subsets of Tregs. CD39+ Tregs represent a safer choice for CAR Treg therapies targeting tissues for tolerance induction. View Publication

CD8 + cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide–MHC-I complex by the T-cell receptor (TCR) and co-stimulation. This cognate interaction promotes the organisation of intimate cell–cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell–cell contact,promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux,providing the necessary energy through oxidative phosphorylation,and its own protein translation on 55S ribosomes. However,the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Here,we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton,mitochondria,and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 55S by puromycin. We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs. View Publication

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease leading to motor neuron loss. Currently mutations in > 40 genes have been linked to ALS,but the contribution of many genes and genetic mutations to the ALS pathogenic process remains poorly understood. Therefore,we first performed comparative interactome analyses of five recently discovered ALS-associated proteins (C21ORF2,KIF5A,NEK1,TBK1,and TUBA4A) which highlighted many novel binding partners,and both unique and shared interactors. The analysis further identified C21ORF2 as a strongly connected protein. The role of C21ORF2 in neurons and in the nervous system,and of ALS-associated C21ORF2 variants is largely unknown. Therefore,we combined human iPSC-derived motor neurons with other models and different molecular cell biological approaches to characterize the potential pathogenic effects of C21ORF2 mutations in ALS. First,our data show C21ORF2 expression in ALS-relevant mouse and human neurons,such as spinal and cortical motor neurons. Further,the prominent ALS-associated variant C21ORF2-V58L caused increased apoptosis in mouse neurons and movement defects in zebrafish embryos. iPSC-derived motor neurons from C21ORF2-V58L-ALS patients,but not isogenic controls,show increased apoptosis,and changes in DNA damage response,mitochondria and neuronal excitability. In addition,C21ORF2-V58L induced post-transcriptional downregulation of NEK1,an ALS-associated protein implicated in apoptosis and DDR. In all,our study defines the pathogenic molecular and cellular effects of ALS-associated C21ORF2 mutations and implicates impaired post-transcriptional regulation of NEK1 downstream of mutant C21ORF72 in ALS. The online version contains supplementary material available at 10.1186/s40478-024-01852-6. View Publication