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

GW572016 (Lapatinib) is a tyrosine kinase inhibitor in clinical development for cancer that is a potent dual inhibitor of epidermal growth factor receptor (EGFR,ErbB-1) and ErbB-2. We determined the crystal structure of EGFR bound to GW572016. The compound is bound to an inactive-like conformation of EGFR that is very different from the active-like structure bound by the selective EGFR inhibitor OSI-774 (Tarceva) described previously. Surprisingly,we found that GW572016 has a very slow off-rate from the purified intracellular domains of EGFR and ErbB-2 compared with OSI-774 and another EGFR selective inhibitor,ZD-1839 (Iressa). Treatment of tumor cells with these inhibitors results in down-regulation of receptor tyrosine phosphorylation. We evaluated the duration of the drug effect after washing away free compound and found that the rate of recovery of receptor phosphorylation in the tumor cells reflected the inhibitor off-rate from the purified intracellular domain. The slow off-rate of GW572016 correlates with a prolonged down-regulation of receptor tyrosine phosphorylation in tumor cells. The differences in the off-rates of these drugs and the ability of GW572016 to inhibit ErbB-2 can be explained by the enzyme-inhibitor structures. View Publication

The dynamic interplay between dendritic cells (DCs) and human immunodeficiency virus type-1 (HIV-1) is thought to result in viral dissemination and evasion of antiviral immunity. Although initial observations suggested that the C-type lectin receptor (CLR) DC-SIGN was responsible for the trans-infection function of the virus,subsequent studies demonstrated that trans-infection of CD4(+) T cells with HIV-1 can also occur through DC-SIGN-independent mechanisms. We demonstrate that a cell surface molecule designated DCIR (for DC immunoreceptor),a member of a recently described family of DC-expressing CLRs,can participate in the capture of HIV-1 and promote infection in trans and in cis of autologous CD4(+) T cells from human immature monocyte-derived DCs. The contribution of DCIR to these processes was revealed using DCIR-specific siRNAs and a polyclonal antibody specific for the carbohydrate recognition domain of DCIR. Data from transfection experiments indicated that DCIR acts as a ligand for HIV-1 and is involved in events leading to productive virus infection. Finally,we show that the neck domain of DCIR is important for the DCIR-mediated effect on virus binding and infection. These results point to a possible role for DCIR in HIV-1 pathogenesis by supporting the productive infection of DCs and promoting virus propagation. View Publication

Chimeric Antigen Receptor (CAR) T cell therapy is a pivotal treatment for hematological malignancies. However,CAR T cell products exhibit batch-to-batch variability in cell number,quality,and in vivo efficacy due to donor-to-donor heterogeneity,and pre/post-manufacturing processes,and the manufacturing of such products necessitates careful testing,both post-manufacturing and pre-infusion. Here,we introduce the Cell Trajectory Modulation (CTM) assay,a microfluidic,label-free approach for the rapid evaluation of the functional attributes of CAR T cells based on biophysical features (i.e.,size,deformability). CTM assay correlates with phenotypic metrics,including CD4:CD8 ratio,memory subtypes,and cytotoxic activity. Validated across multiple donors and culture platforms,the CTM assay requires fewer than 10,000 cells and delivers results within 10 minutes. Compared to labeled flow cytometry processing,the CTM assay offers real-time data to guide adaptive manufacturing workflows. Thus,the CTM assay offers an improvement over existing phenotypic assessments,marking a step forward in advancing CAR T cell therapy manufacturing. CAR T cell manufacturing faces significant challenges that impact cell quality and in vivo efficacy. This necessitates reliable cellular characterization methods. Here the authors present a real-time,label-free,microfluidic method that profiles cellular biophysical properties and correlates them to activation state and CAR T potency,facilitating the rapid phenotypic cell assessment during production. View Publication

The ability to differentiate human pluripotent stem cells into endothelial cells with properties of cord-blood endothelial colony-forming cells (CB-ECFCs) may enable the derivation of clinically relevant numbers of highly proliferative blood vessel-forming cells to restore endothelial function in patients with vascular disease. We describe a protocol to convert human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs) into cells similar to CB-ECFCs at an efficiency of textgreater10(8) ECFCs produced from each starting pluripotent stem cell. The CB-ECFC-like cells display a stable endothelial phenotype with high clonal proliferative potential and the capacity to form human vessels in mice and to repair the ischemic mouse retina and limb,and they lack teratoma formation potential. We identify Neuropilin-1 (NRP-1)-mediated activation of KDR signaling through VEGF165 as a critical mechanism for the emergence and maintenance of CB-ECFC-like cells. View Publication

Human embryonic stem cells (hESCs) exhibit unique cell cycle structure,self-renewal and pluripotency. The Forkhead box transcription factor M1 (FOXM1) is critically required for the maintenance of pluripotency in mouse embryonic stem cells and mouse embryonal carcinoma cells,but its role in hESCs remains unclear. Here,we show that FOXM1 expression was enriched in undifferentiated hESCs and was regulated in a cell cycle-dependent manner with peak levels detected at the G2/M phase. Expression of FOXM1 did not correlate with OCT4 and NANOG during in vitro differentiation of hESCs. Importantly,knockdown of FOXM1 expression led to aberrant cell cycle distribution with impairment in mitotic progression but showed no profound effect on the undifferentiated state. Interestingly,FOXM1 depletion sensitized hESCs to oxidative stress. Moreover,genome-wide analysis of FOXM1 targets by ChIP-seq identified genes important for M phase including CCNB1 and CDK1,which were subsequently confirmed by ChIP and RNA interference analyses. Further peak set comparison against a differentiating hESC line and a cancer cell line revealed a substantial difference in the genomic binding profile of FOXM1 in hESCs. Taken together,our findings provide the first evidence to support FOXM1 as an important regulator of cell cycle progression and defense against oxidative stress in hESCs. View Publication

The Mediator complex forms the bridge between transcriptional activators and the RNA polymerase II. Med1 (also known as PBP or TRAP220) is a key component of Mediator that interacts with nuclear hormone receptors and GATA transcription factors. Here,we show dynamic recruitment of GATA-1,TFIIB,Mediator,and RNA polymerase II to the β-globin locus in induced mouse erythroid leukemia cells and in an erythropoietin-inducible hematopoietic progenitor cell line. Using Med1 conditional knockout mice,we demonstrate a specific block in erythroid development but not in myeloid or lymphoid development,highlighted by the complete absence of β-globin gene expression. Thus,Mediator subunit Med1 plays a pivotal role in erythroid development and in β-globin gene activation. View Publication

Accurate characterization of hematopoietic stem cells (HSC) products is needed to better anticipate the hematopoietic reconstitution and the outcome in patients. Although CD34+ viable cells enumeration is a key predictor of time to correction of aplasia,it does not fully inform about functionality of cells contained in the graft. CFU assay is the gold standard in vitro potency assay to assess clonogenicity of HSC and consists on the count and identification of colonies several days after culture in a semi solid media. Manual count of colonies with optic microscope is the most commonly used method but its important variability and subjectivity hinders the universal implementation of this potency assay. The aim of this study is to validate a standardized method using the STEMvision™ system,the first semi-automated instrument for imaging and scoring hematopoietic colonies,according to French and European recommendations. Results obtained highlight better performance criteria with STEMvision™ system than the manual method. This semi-automatic device tends to reduce the coefficients of variation of repeatability,inter-operator variability and intermediate precision. This newly available platform could represent an interesting option,significantly improving performances of CFU assays used for the characterization of hematopoietic progenitors. View Publication

Long-term marrow cultures (LTMC) allow the proliferation and differentiation of primitive human hematopoietic progenitor cells to be maintained for many weeks in the absence of exogenously provided hematopoietic growth factors. Previous investigations focused on defining various types of cells that are present in this culture system and on measuring the cycling behavior of the different subpopulations of colony-forming cells maintained within it. These studies suggested that mesenchymal stromal elements derived from the input marrow play a key role in regulating the turnover of the most primitive,high-proliferative potential erythroid and granulopoietic colony-forming cells that are found almost exclusively in the adherent layer of LTMC. In this study we show that the re-entry into S-phase of these primitive hematopoietic progenitors that occurs after each weekly medium change is due to an as yet undefined constituent of horse serum,which is absent from fetal calf serum. However,this effect is not unique to the factor present in horse serum. It is also elicited by the addition to LTMC of several well-defined growth regulatory molecules,ie,platelet-derived growth factor (PDGF),interleukin-1 (IL-1),transforming growth factor alpha (TGF-alpha),and IL-2. None of these was able to stimulate hematopoietic colony-forming cells in methylcellulose assays,although all have known actions on mesenchymal cells including,in some cases,the ability to increase production of growth factors that can stimulate primitive high-proliferative potential hematopoietic progenitors in clonogenic assays. Interestingly,a stimulating effect was not obtained after addition of endotoxin to LTMC. TGF-beta,a direct-acting negative regulator that acts selectively on primitive hematopoietic progenitor cells if added to LTMC simultaneously with new medium or IL-1,blocked their stimulating activity. These results suggest a model in which indirect,local modulation of both positive and negative regulatory factors via effects on mesenchymal elements determines the rate of turnover of adjacent populations of very primitive hematopoietic cells that are normally maintained in a quiescent state in vivo. View Publication

AIM: The recently published REPAIR-AMI and ASTAMI trial showed differences in contractile recovery of left ventricular function after infusion of bone marrow-derived cells in acute myocardial infarction. Since the trials used different protocols for cell isolation and storage (REPAIR-AMI: Ficoll,storage in X-vivo 10 medium plus serum; ASTAMI: Lymphoprep,storage in NaCl plus plasma),we compared the functional activity of BMC isolated by the two different protocols. METHODS AND RESULTS: The recovery of total cell number,colony-forming units (CFU),and the number of mesenchymal stem cells were significantly reduced to 77 +/- 4%,83 +/- 16%,and 65 +/- 15%,respectively,when using the ASTAMI protocol compared with the REPAIR protocol. The capacity of the isolated BMC to migrate in response to stromal cell-derived factor 1 (SDF-1) was profoundly reduced when using the ASTAMI cell isolation procedure (42 +/- 8% and 78 +/- 3% reduction in healthy and CAD-patient cells,respectively). Finally,infusion of BMC into a hindlimb ischaemia model demonstrated a significantly blunted blood-flow-recovery by BMC isolated with the ASTAMI protocol (54 +/- 6% of the effect obtained by REPAIR cells). Comparison of the individual steps identified the use of NaCl and plasma for cell storage as major factors for functional impairment of the BMC. CONCLUSION: Cell isolation protocols have a major impact on the functional activity of bone marrow-derived progenitor cells. The assessment of cell number and viability may not entirely reflect the functional capacity of cells in vivo. Additional functional testing appears to be mandatory to assure proper cell function before embarking on clinical cell therapy trials. View Publication