搜索结果: 'gmp t cell activator'

In the three decades since pluripotent mouse embryonic stem (ES) cells were first described they have been derived and maintained by using various empirical combinations of feeder cells,conditioned media,cytokines,growth factors,hormones,fetal calf serum,and serum extracts. Consequently ES-cell self-renewal is generally considered to be dependent on multifactorial stimulation of dedicated transcriptional circuitries,pre-eminent among which is the activation of STAT3 by cytokines (ref. 8). Here we show,however,that extrinsic stimuli are dispensable for the derivation,propagation and pluripotency of ES cells. Self-renewal is enabled by the elimination of differentiation-inducing signalling from mitogen-activated protein kinase. Additional inhibition of glycogen synthase kinase 3 consolidates biosynthetic capacity and suppresses residual differentiation. Complete bypass of cytokine signalling is confirmed by isolating ES cells genetically devoid of STAT3. These findings reveal that ES cells have an innate programme for self-replication that does not require extrinsic instruction. This property may account for their latent tumorigenicity. The delineation of minimal requirements for self-renewal now provides a defined platform for the precise description and dissection of the pluripotent state. View Publication

Endothelial progenitor cells (EPCs) play a critical role in postnatal and tumor vasculogenesis. Vascular endothelial growth inhibitor (VEGI; TNFSF15) has been shown to inhibit endothelial cell proliferation by inducing apoptosis. We report here that VEGI inhibits the differentiation of EPCs from mouse bone marrow-derived Sca1(+) mononuclear cells. Analysis of EPC markers indicates a significant decline of the expression of endothelial cell markers,but not stem cell markers,on VEGI-treated cells. Consistently,the VEGI-treated cells exhibit a decreased capability to adhere,migrate,and form capillary-like structures on Matrigel. In addition,VEGI induces apoptosis of differentiated EPCs but not early-stage EPCs. When treated with VEGI,an increase of phospho-Erk and a decrease of phospho-Akt are detected in early-stage EPCs,whereas activation of nuclear factor-kappaB,jun N-terminal kinase,and caspase-3 is seen in differentiated EPCs. Furthermore,VEGI-induced apoptosis of differentiated EPC is,at least partly,mediated by death receptor-3 (DR3),which is detected on differentiated EPC only. VEGI-induced apoptosis signals can be inhibited by neutralizing antibodies against DR3 or recombinant extracellular domain of DR3. These findings indicate that VEGI may participate in the modulation of postnatal vasculogenesis by inhibiting EPC differentiation. View Publication

Realizing the potential that human embryonic stem cells (hESCs) hold,both for the advancement of biomedical science and the development of new treatments for many human disorders,will be greatly facilitated by the introduction of standardized methods for assessing and altering the biological properties of these cells. The 7-day in vitro alkaline phosphatase colony-forming cell (AP(+)-CFC) assay currently offers the most sensitive and specific method to quantify the frequency of undifferentiated cells present in a culture. In this regard,it is superior to any phenotypic assessment protocol. The AP(+)-CFC assay,thus,provides a valuable tool for monitoring the quality of hESC cultures,and also for evaluating quantitative changes in pluripotent cell numbers following manipulations that may affect the self-renewal and differentiation properties of the treated cells. Two other methods routinely used to evaluate hESC pluripotency involve either culturing the cells under conditions that promote the formation of nonadherent differentiating cell aggregates (termed embryoid bodies),or transplanting the cells into immunodeficient mice to obtain teratomas containing differentiated cells representative of endoderm,mesoderm,and ectoderm lineages. View Publication

Routine techniques for the isolation of human peripheral blood mononuclear cells (PBMCs) include density centrifugation with Ficoll-Paque and isolation by cell preparation tubes (CPTs) and SepMate tubes with Lymphoprep. In a series of experiments,these three PBMC isolation techniques were compared for cell recovery and viability,PBMC population composition,and cell functionality,aiming to provide a starting basis for the selection of the most appropriate method of PBMC isolation for a specific downstream application. PBMCs were freshly isolated from venous blood of healthy male donors,applying the different techniques in parallel. Cell recovery and viability were assessed using a hemacytometer and trypan blue. Immunophenotyping was performed by flow cytometry. Cell functionality was assessed in stimulated (100 ng/mL staphylococcal enterotoxin B [SEB]) and unstimulated 24 hours PBMC cultures,with cytokine production and lactate dehydrogenase (LDH) release as readout measures. PBMC isolation by SepMate and CPT resulted in a 70% higher recovery than Ficoll isolation. CPT-isolated populations contained more erythrocyte contamination. Cell viability,assessed by trypan blue exclusion,was 100% for all three isolation techniques. SepMate and CPT isolation gave higher SEB-induced cytokine responses in cell cultures,for IFNγ and for secondary cytokines. IL-6 and IL-8 release in unstimulated cultures was higher for CPT-isolated PBMCs compared to Ficoll- and SepMate-isolated PBMCs. LDH release did not differ between cell isolation techniques. In addition to criteria such as cost and application practicalities,these data may support selection of a specific PBMC isolation technique for downstream analysis. View Publication

The tumor microenvironment and healing wounds both contain extremely high concentrations of adenosine triphosphate (ATP) compared to normal tissue. The P2Y2 receptor,an ATP-activated purinergic receptor,is typically associated with pulmonary,endothelial,and neurological cell signaling. Here,we examine ATP-dependent signaling in breast epithelial cells and how it is altered in metastatic breast cancer. Using rapid imaging techniques,we show how ATP-activated P2Y2 signaling causes an increase in intracellular Ca 2+ in non-tumorigenic breast epithelial cells,approximately 3-fold higher than their tumorigenic and metastatic counterparts. The non-tumorigenic cells respond to increased Ca 2+ with actin polymerization and localization to the cell edges after phalloidin staining,while the metastatic cells remain unaffected. The increase in intracellular Ca 2+ after ATP stimulation was blunted to control levels using a P2Y2 antagonist,which also prevented actin mobilization and significantly increased cell dissemination from spheroids in non-tumorigenic cells. Furthermore,the lack of Ca 2+ changes and actin mobilization in metastatic breast cancer cells could be due to the reduced P2Y2 expression,which correlates with poorer overall survival in breast cancer patients. This study elucidates the rapid changes that occur after elevated intracellular Ca 2+ in breast epithelial cells and how metastatic cancer cells have adapted to evade this cellular response. View Publication