搜索结果: 'gmp t cell activator'

BACKGROUND: Ewing's sarcoma cell lines may represent a good in vitro model for the understanding of tumor biology in this heterogeneous group of diseases. In the present study,we report the establishment and characterization of a primary Ewing's sarcoma cell line (LDS-Falck 01). MATERIALS AND METHODS: LDS-Falck 01 was generated from a malignant pleural effusion of a patient with metastatic peripheral primitive neuroectodermal tumor arising from the chest wall. Extensive characterization of the cells was accomplished using immunocytochemical,RT-PCR and cytogenetic studies. RESULTS: In vitro LDS-Falck 01 cells had both anchorage-dependent and -independent growth patterns. Immunocytochemical studies showed that cells were PAS-,vimentin-,CD99- and NSE-positive,EGFR- and CD117-negative. Cytogenetic analysis revealed a complex hyperdiploid karyotype with multiple chromosomal aberrations including an unbalanced translocation t(11;22)(q24;q12). The EWS/FLI1 chimeric transcript type 1 was detected. CONCLUSION: This cell line may represent a valid tool for investigating the biomolecular characteristics of this group of neoplasms and their sensitivity to therapeutic agents. View Publication

Neural stem and progenitor cells (NSCs/NPCs) are distinct groups of cells found in the mammalian central nervous system (CNS). Previously we determined that members of the High Mobility Group (HMG) B family of chromatin structural proteins modulate NSC proliferation and self-renewal. Among them HMGB2 was found to be dynamically expressed in proliferating and differentiating NSCs,suggesting that it may regulate NSC maintenance. We report now that Hmgb2(-/-) mice exhibit SVZ hyperproliferation,increased numbers of SVZ NSCs,and a trend towards aberrant increases in newly born neurons in the olfactory bulb (OB) granule cell layer. Increases in the levels of the transcription factor p21 and the Neural cell adhesion molecule (NCAM),along with down-regulation of the transcription/pluripotency factor Oct4 in the Hmgb2-/- SVZ point to a possible pathway for this increased proliferation/differentiation. Our findings suggest that HMGB2 functions as a modulator of neurogenesis in young adult mice through regulation of NSC proliferation,and identify a potential target via which CNS repair could be amplified following trauma or disease-based neuronal degeneration. View Publication

Zika virus (ZIKV) infection has been associated with severe complications both in the developing and adult nervous system. To investigate the deleterious effects of ZIKV infection,we used human neural progenitor cells (NPC),derived from induced pluripotent stem cells (iPSC). We found that NPC are highly susceptible to ZIKV and the infection results in cell death. ZIKV infection led to a marked reduction in cell proliferation,ultrastructural alterations and induction of autophagy. Induction of apoptosis of Sox2 + cells was demonstrated by activation of caspases 3/7,8 and 9,and by ultrastructural and flow cytometry analyses. ZIKV-induced death of Sox2 + cells was prevented by incubation with the pan-caspase inhibitor,Z-VAD-FMK. By confocal microscopy analysis we found an increased number of cells with supernumerary centrosomes. Live imaging showed a significant increase in mitosis abnormalities,including multipolar spindle,chromosome laggards,micronuclei and death of progeny after cell division. FISH analysis for chromosomes 12 and 17 showed increased frequency of aneuploidy,such as monosomy,trisomy and polyploidy. Our study reinforces the link between ZIKV and abnormalities in the developing human brain,including microcephaly. View Publication

The embryonic neuroepithelium gives rise to the entire central nervous system in vivo,making it an important tissue for developmental studies and a prospective cell source for regenerative applications. Current protocols for deriving homogenous neuroepithelial cultures from human pluripotent stem cells (hPSCs) consist of either embryoid body-mediated neuralization followed by a manual isolation step or adherent differentiation using small molecule inhibitors. Here,we report that hPSCs maintained under chemically defined,feeder-independent,and xeno-free conditions can be directly differentiated into pure neuroepithelial cultures ([mt]90% Pax6(+)/N-cadherin(+) with widespread rosette formation) within 6 days under adherent conditions,without small molecule inhibitors,and using only minimalistic medium consisting of Dulbecco's modified Eagle's medium/F-12,sodium bicarbonate,selenium,ascorbic acid,transferrin,and insulin (i.e.,E6 medium). Furthermore,we provide evidence that the defined culture conditions enable this high level of neural conversion in contrast to hPSCs maintained on mouse embryonic fibroblasts (MEFs). In addition,hPSCs previously maintained on MEFs could be rapidly converted to a neural compliant state upon transfer to these defined conditions while still maintaining their ability to generate all three germ layers. Overall,this fully defined and scalable protocol should be broadly useful for generating therapeutic neural cells for regenerative applications. View Publication

Utilizing human pluripotent stem cells (hPSCs) in cell-based therapy and drug discovery requires large-scale cell production. However,scaling up conventional adherent cultures presents challenges of maintaining a uniform high quality at low cost. In this regard,suspension cultures are a viable alternative,because they are scalable and do not require adhesion surfaces. 3D culture systems such as bioreactors can be exploited for large-scale production. However,the limitations of current suspension culture methods include spontaneous fusion between cell aggregates and suboptimal passaging methods by dissociation and reaggregation. 3D culture systems that dynamically stir carrier beads or cell aggregates should be refined to reduce shearing forces that damage hPSCs. Here,we report a simple 3D sphere culture system that incorporates mechanical passaging and functional polymers. This setup resolves major problems associated with suspension culture methods and dynamic stirring systems and may be optimal for applications involving large-scale hPSC production. ?? 2014 The Authors. View Publication

BACKGROUND: Endothelial cells line the luminal surface of blood vessels and form a barrier between the blood and other tissues of the body. Ets variant 2 (ETV2) is transiently expressed in both zebrafish and mice and is necessary and sufficient for vascular endothelial cell specification. Overexpression of this gene in early zebrafish and mouse embryos results in ectopic appearance of endothelial cells. Ectopic expression of ETV2 in later development results in only a subset of cells responding to the signal.backslashnbackslashnFINDINGS: We have examined the expression pattern of ETV2 in differentiating human embryonic stem cells (ESCs) to determine when the peak of ETV2 expression occurs. We show that overexpression of ETV2 in differentiating human ESC is able to increase the number of endothelial cells generated when administered during or after the endogenous peak of gene expression.backslashnbackslashnCONCLUSIONS: Addition of exogenous ETV2 to human ESCs significantly increased the number of cells expressing angioblast genes without arterial or venous specification. This may be a viable solution to generate in vitro endothelial cells for use in research and in the clinic. View Publication

For diseases of the brain,the pig (Sus scrofa) is increasingly being used as a model organism that shares many anatomical and biological similarities with humans. We report that pig induced pluripotent stem cells (iPSC) can recapitulate events in early mammalian neural development. Pig iPSC line (POU5F1(high)/SSEA4(low)) had a higher potential to form neural rosettes (NR) containing neuroepithelial cells than either POU5F1(low)/SSEA4(low) or POU5F1(low)/SSEA4(high) lines. Thus,POU5F1 and SSEA4 pluripotency marker profiles in starting porcine iPSC populations can predict their propensity to form more robust NR populations in culture. The NR were isolated and expanded in vitro,retaining their NR morphology and neuroepithelial molecular properties. These cells expressed anterior central nervous system fate markers OTX2 and GBX2 through at least seven passages,and responded to retinoic acid,promoting a more posterior fate (HOXB4+,OTX2-,and GBX2-). These findings offer insight into pig iPSC development,which parallels the human iPSC in both anterior and posterior neural cell fates. These in vitro similarities in early neural differentiation processes support the use of pig iPSC and differentiated neural cells as a cell therapy in allogeneic porcine neural injury and degeneration models,providing relevant translational data for eventual human neural cell therapies. View Publication

The neutralization of integrin α4 (Itga4) is currently used as treatment in multiple sclerosis. Although most studies have focused on its function on lymphocyte migration to the CNS,we have uncovered the importance of Itga4 for the generation of regulatory B cells in peripheral immune organs and their control of pathogenic T cell response and CNS pathology. Our study underscores the importance of looking at the dual role of B cells in CNS autoimmunity and provides important perspectives regarding the efficacy and side effects associated with Itga4 neutralization and other B cell-targeting therapies. View Publication

Lineage specific human embryonic stem cell (hESC) reporter cell line is a versatile tool for biological studies on real time monitoring of differentiation,physiological and biochemical features of special cell types and pathological mechanism of disease. Here we report the generation of ChAT-zsGreen reporter hESC line that express zsGreen under the control of the choline acetyltransferase (ChAT) promoter using CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats)/Cas9 system. We show that the ChAT-zsGreen hESC reporter cell lines retain the features of undifferentiated hESC. After cholinergic neuronal differentiation,cholinergic neurons were clearly labeled with green fluorescence protein (zsGreen). The ChAT-zsGreen reporter hESC lines are invaluable not only for the monitoring cholinergic neuronal differentiation but also for study physiological and biochemical hallmarks of cholinergic neurons. View Publication

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support,but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study,five separate laboratories,each with experience in human embryonic stem cell culture,used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods,with propagation in the presence of Knockout Serum Replacer,FGF-2,and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment,death,and differentiated morphology by phase contrast microscopy,for growth by serial cell counts,and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems,only the control and those based on two commercial media,mTeSR1 and STEMPRO,supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment,cell death,or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study,and the lack of success with other formulations from academic groups compared to previously published results,include: the complex combination of growth factors present in the commercial preparations; improved development,manufacture,and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories. View Publication

Endocannabinoids are arachidonic acid derivatives and part of a novel bioactive lipid signaling system,along with their G-coupled cannabinoid receptors (CB�? and CB₂) and the enzymes involved in their biosynthesis and degradation. However,their roles in hematopoiesis and hematopoietic stem and progenitor cell (HSPC) functions are not well characterized. Here,we show that bone marrow stromal cells express endocannabinoids (anandamide and 2-arachidonylglycerol),whereas CB₂ receptors are expressed in human and murine HSPCs. On ligand stimulation with CB₂ agonists,CB₂ receptors induced chemotaxis,migration,and enhanced colony formation of bone marrow cells,which were mediated via ERK,PI3-kinase,and Gαi-Rac1 pathways. In vivo,the CB₂ agonist AM1241 induced mobilization of murine HSPCs with short- and long-term repopulating abilities. In addition,granulocyte colony-stimulating factor -induced mobilization of HSPCs was significantly decreased by specific CB₂ antagonists and was impaired in Cnr2(-/-) cannabinoid type 2 receptor knockout mice. Taken together,these results demonstrate that the endocannabinoid system is involved in hematopoiesis and that CB₂/CB₂ agonist axis mediates repopulation of hematopoiesis and mobilization of HSPCs. Thus,CB₂ agonists may be therapeutically applied in clinical conditions,such as bone marrow transplantation. View Publication

PURPOSE To investigate the expression status and clinical implications of stem cell genes CD133 and CD44 in the colorectal cancers with early liver metastasis. MATERIALS AND METHODS The differential genes of early liver metastases in colorectal cancer were detected by RT(2) Profiler™ PCR Array. The expression and the relationship of stem cell gene CD133 and CD44 were analyzed by immunofluorescent tests. RESULTS CD133 and CD44 were significantly higher co-expressed in colorectal cancer with early liver metastases compared to those without early liver metastases,and the content of CD133 and CD44 proteins decreased following growth of the transplanted tumors. Of the 80 cases without early liver metastases,12 were observed CD133 and CD44 proteins co-expression,while 36 of the 40 cases with early liver metastases were found CD133 and CD44 proteins co-expression (15% vs. 90%,P textless 0.05). Survival analysis revealed CD133 and CD44 proteins co-expression was associated with poorest prognosis (57.14% vs. 87.41%,X(2) = 48.49,P = 0.001). After Cox regression,age,Duck's stage,lymph node metastasis,and CD133 and CD44 proteins co-expression were shown to be the independent prognostic factors of colorectal cancers. CONCLUSIONS CD133 and CD44 proteins were highly co-expressed in colorectal cancer with early liver metastases,and may be a potential biomarker for the early liver metastases. View Publication