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

The role of human NK cells in viral infections is poorly understood. We used a cytokine flow-cytometry assay to simultaneously investigate the IFN-gamma response of NK and T lymphocytes to influenza A virus (fluA). When PBMCs from fluA-immune adult donors were incubated with fluA,IFN-gamma was produced by both CD56(dim) and CD56(bright) subsets of NK cells,as well as by fluA-specific T cells. Purified NK cells did not produce IFN-gamma in response to fluA,while depletion of T lymphocytes reduced to background levels the fluA-induced IFN-gamma production by NK cells,which indicates that T cells are required for the IFN-gamma response of NK cells. The fluA-induced IFN-gamma production of NK cells was suppressed by anti-IL-2 Ab,while recombinant IL-2 replaced the helper function of T cells for IFN-gamma production by NK cells. This indicates that IL-2 produced by fluA-specific T cells is involved in the T cell-dependent IFN-gamma response of NK cells to fluA. Taken together,these results suggest that at an early stage of recurrent viral infection,NK-mediated innate immunity to the virus is enhanced by preexisting virus-specific T cells. View Publication

Kaposi's sarcoma (KS) is a common neoplasm in HIV-1-infected individuals causing significant morbidity and mortality. Despite recent advances,the pathogenesis of this potentially life-threatening neoplasm remains unclear,and there is currently no cure for KS. Notch proteins are known to play a fundamental role in cell fate decisions including proliferation,differentiation,and apoptosis. It is,therefore,not surprising that Notch proteins have been implicated in tumorigenesis and appear to function as either oncogenes or tumor suppressor proteins depending on cellular context. In this report,we demonstrate elevated levels of activated Notch-1,-2,and -4 in KS tumor cells in vivo and in vitro compared to endothelial cells,the precursor of the KS cell. Notch activation was confirmed through luciferase reporter assays and localization of Hes (Hairy/Enhancer of Split)-1 and Hey (Hairy/Enhancer of Split related with YRPW)1 (primary targets of the Notch pathway) in KS cell nuclei. Studies using gamma-secretase inhibitors (GSI and LY-411,575),which block Notch activation,resulted in apoptosis in primary and immortalized KS cells. Similar studies injecting GSI into established KS cell tumors on mice demonstrated growth inhibition or tumor regression that was characterized by apoptosis in treated,but not control tumors. The results indicate that KS cells overexpress activated Notch and interruption of Notch signaling inhibits KS cell growth. Thus,targeting Notch signaling may be of therapeutic value in KS patients. View Publication

Fanconi anemia (FA) is a rare inherited genomic instability syndrome representing one of the best examples of hematopoietic stem cell deficiency. Although FA might be an excellent candidate for bone marrow (BM) genetic correction ex vivo,knockout animal models are not sufficient to guide preclinical steps,and gene therapy attempts have proven disappointing so far. Contributing to these poor results is a characteristic and dramatic early BM-cells die-off when placed in culture. We show here that human primary FA BM cell survival can be ameliorated by using specific culture conditions that limit oxidative stress. When coupled with retrovirus-mediated transfer of the main complementation group FANCA-cDNA,we could achieve long-term reconstitution of the stem cell compartment both in vitro and in vivo. Gene-corrected BM cultures grew for textgreater120 days,and after cultured cell transplantation into NOD/SCID mice,clonogenic human cells carrying the FANCA transgene could be detected 6 months after transduction. By comparison,untransduced cells died in culture by 15 days. Of necessity for ethical reasons,experiments were conducted on a very limited number of primary BM cells. By using low cytokine regimen and conditions matching regulatory requirements,a contingent of gene-corrected cells slowly emerges with an unmet potential for in vivo engraftment. Future therapeutic applications of stem cells might be expanding from these data. In addition,we provide a model of gene-corrected human primary cell growth that carries the potential to better delineate the combined role of both DNA damage and oxidative stress in the pathogenesis of FA. View Publication

While all forms of tobacco exposure have negative health effects,the significance of exposure to electronic cigarettes (eCig) is not fully understood. Here,we studied the global effects of eCig on the micro RNA (miRNA) transcriptome in human lung epithelial cells. Primary human bronchial epithelial (NHBE) cells differentiated at air-liquid interface were exposed to eCig liquid. Exposure of NHBE to any eCig liquid resulted in the induction of oxidative stress-response genes including GCLM,GCLC,GPX2,NQO1 and HO-1. Vaporization of,and/or the presence of nicotine in,eCig liquid was associated with a greater response. We identified 578 miRNAs dysregulated by eCig exposure in NHBE,and 125 miRNA affected by vaporization of eCig liquid. Nicotine containing eCig vapor displayed the most profound effects upon miRNA expression. We selected 8 miRNAs (29A,140,126,374A,26A-2,147B,941 and 589) for further study. We validated increased expression of multiple miRNAs,including miR126,following eCig exposure. We also found significant reduction in the expression of two miR126 target genes,MYC and MRGPRX3,following exposure. These data demonstrated that eCig exposure has profound effects upon gene expression in human lung epithelial cells,some of which are epigenetically programmed at the level of miRNA regulation. View Publication

The tryptophan metabolite kynurenine has critical immunomodulatory properties and can function as an aryl hydrocarbon receptor (AHR) ligand. Here we show that the ability of T cells to transport kynurenine is restricted to cells activated by the T-cell antigen receptor or proinflammatory cytokines. Kynurenine is transported across the T-cell membrane by the System L transporter SLC7A5. Accordingly,the ability of kynurenine to activate the AHR is restricted to T cells that express SLC7A5. We use the fluorescence spectral properties of kynurenine to develop a flow cytometry-based assay for rapid,sensitive and quantitative measurement of the kynurenine transport capacity in a single cell. Our findings provide a method to assess the susceptibility of T cells to kynurenine,and a sensitive single cell assay to monitor System L amino acid transport. View Publication

Under defined differentiation conditions,human embryonic stem cells (hESCs) can be directed toward a mesendoderm (ME) or neuroectoderm (NE) fate,the first decision during hESC differentiation. Coupled with lineage-specific G1 lengthening,a divergent ciliation pattern emerged within the first 24 hr of induced lineage specification,and these changes heralded a neuroectoderm decision before any neural precursor markers were expressed. By day 2,increased ciliation in NE precursors induced autophagy that resulted in the inactivation of Nrf2 and thereby relieved transcriptional activation of OCT4 and NANOG. Nrf2 binds directly to upstream regions of these pluripotency genes to promote their expression and repress NE derivation. Nrf2 suppression was sufficient to rescue poorly neurogenic iPSC lines. Only after these events had been initiated did neural precursor markers get expressed at day 4. Thus,we have identified a primary cilium-autophagy-Nrf2 (PAN) control axis coupled to cell-cycle progression that directs hESCs toward NE. View Publication

Oct4 and Sox2 are pluripotent stem cell factors but the interplay between them in tumorigenesis is unclear. The aim of the present study was to investigate the roles of Oct4 and Sox2 in the reprogramming of oral cancer stem cells. One or both Oct4 and Sox2 were overexpressed in immortalized oral epithelial (hTERT+-OME) cells by lentivirus transduction. In addition,Oct4 and Sox2 proteins in two oral squamous cell carcinoma cell (OSCC) lines (Cal27 and primary cultured OSCC from a T2N2M0 patient) were individually or combinedly knocked down by shRNA. The results showed that the doubly transduced (Oct4+Sox2+) cells could trigger neoplasms in immunodeficient mice after lentivirus transduction,but single transduced (Oct4+ or Sox2+) cells had no tumor formation ability. The knockdown Sox2low and knockdown Oct4lowSox2low cells resulted in decreased tumor size in the immunodeficient mice but the single knockdown Oct4low cancer cells acquired more aggressive xenografts. Our findings suggest that Oct4+Sox2+ cells may be reprogrammed cancer stem cells inducing oral carcinogenesis. View Publication

Group 2 innate lymphoid cells (ILC2s) in the lung are stimulated by inhaled allergens. ILC2s do not directly recognize allergens but they are stimulated by cytokines including interleukin (IL)-33 released by damaged epithelium. In response to allergens,lung ILC2s produce T helper 2 cell type cytokines inducing T cell-independent allergic lung inflammation. Here we examined the fate of lung ILC2s upon allergen challenges. ILC2s proliferated and secreted cytokines upon initial stimulation with allergen or IL-33,and this phase was followed by a contraction phase as cytokine production ceased. Some ILC2s persisted long after the resolution of the inflammation as allergen-experienced ILC2s and responded to unrelated allergens more potently than naive ILC2s,mediating severe allergic inflammation. The allergen-experienced ILC2s exhibited a gene expression profile similar to that of memory T cells. The memory-like properties of allergen-experienced ILC2s may explain why asthma patients are often sensitized to multiple allergens. View Publication

AIMS/HYPOTHESIS Endothelial cells (ECs) play an essential role in pancreatic organogenesis. We hypothesise that effective in vitro interactions between human microvascular endothelial cells (HMECs) and human pluripotent stem cells (hPSCs) results in the generation of functional pancreatic beta cells. METHODS Embryoid bodies (EBs) derived from hPSCs were cultured alone (controls) or with ECs in collagen gels. Subsequently,cells were analysed for pancreatic beta cell markers,and then isolated and expanded. Insulin secretion in response to glucose was evaluated in vitro by static and dynamic (perifusion) assays,and in vivo by EB transplantation into immunodeficient mice. RESULTS Co-cultured EBs had a higher expression of mature beta cells markers and enhanced insulin secretion in vitro,compared with controls. In mice,transplanted EBs had higher levels of human C-peptide secretion with a significant reduction in hyperglycaemia after the selective destruction of native pancreatic beta cells. In addition,there was significant in vitro upregulation of bone morphogenetic proteins 2 and 4 (BMP-2,4) in co-cultured cells,compared with controls. CONCLUSIONS/INTERPRETATION ECs provide essential signalling in vitro,such as activation of the BMP pathway,for derivation of functional insulin-producing beta cells from hPSCs. View Publication

Success in the differentiating human embryonic stem cells (hESCs) into insulin-secreting β cells raises new hopes for diabetes treatment. In this work,we demonstrated the feasibility of developing islet organoids from hESCs within biomimetic 3D scaffolds. We showed that such a 3D microenvironment is critical to the generation of pancreatic endoderm and endocrine from hESCs. The organoids formed consisted of pancreatic α,β,δ,and pancreatic polypeptide (PP) cells. A high-level co-expression of PDX1,NKX6.1,and NGN3 in these cells suggests the characteristics of pancreatic β cells. More importantly,most insulin-secreting cells generated did not express glucagon,somatostatin,or PP. The expression of mature β cell marker genes such as Pdx1,Ngn3,Insulin,MafA,and Glut2 was detected in these 3D-induced cell clusters. A high-level expression of C-peptide confirmed the de novo endogenous insulin production in these 3D induced cells. Insulin-secretory granules,an indication of β cell maturity,were detected in these cells as well. Glucose challenging experiments suggested that these cells are sensitive to glucose levels due to their elevated maturity. Exposing the cells to a high concentration of glucose induced a sharp increase in insulin secretion. View Publication

BACKGROUND: Cancer stem cells are presumed to have virtually unlimited proliferative and self-renewal abilities and to be highly resistant to chemotherapy,a feature that is associated with overexpression of ATP-binding cassette transporters. We investigated whether prolonged continuous selection of cells for drug resistance enriches cultures for cancer stem-like cells. METHODS: Cancer stem cells were defined as CD44+/CD24? cells that could self-renew (ie,generate cells with the tumorigenic CD44+/CD24? phenotype),differentiate,invade,and form tumors in vivo. We used doxorubicin-selected MCF-7/ADR cells,weakly tumorigenic parental MCF-7 cells,and MCF-7/MDR,an MCF-7 subline with forced expression of ABCB1 protein. Cells were examined for cell surface markers and side-population fractions by microarray and flow cytometry,with in vitro invasion assays,and for ability to form mammospheres. Xenograft tumors were generated in mice to examine tumorigenicity (n = 52). The mRNA expression of multidrug resistance genes was examined in putative cancer stem cells and pathway analysis of statistically significantly differentially expressed genes was performed. All statistical tests were two-sided. RESULTS: Pathway analysis showed that MCF-7/ADR cells express mRNAs from ABCB1 and other genes also found in breast cancer stem cells (eg,CD44,TGFB1,and SNAI1). MCF-7/ADR cells were highly invasive,formed mammospheres,and were tumorigenic in mice. In contrast to parental MCF-7 cells,more than 30% of MCF-7/ADR cells had a CD44+/CD24? phenotype,could self-renew,and differentiate (ie,produce CD44+/CD24? and CD44+/CD24+ cells) and overexpressed various multidrug resistance-linked genes (including ABCB1,CCNE1,and MMP9). MCF-7/ADR cells were statistically significantly more invasive in Matrigel than parental MCF-7 cells (MCF-7 cells = 0.82 cell per field and MCF-7/ADR = 7.51 cells per field,difference = 6.69 cells per field,95% confidence interval = 4.82 to 8.55 cells per field,P textless .001). No enrichment in the CD44+/CD24? or CD133+ population was detected in MCF-7/MDR. CONCLUSION: The cell population with cancer stem cell characteristics increased after prolonged continuous selection for doxorubicin resistance. View Publication

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by ectopic expression of four transcription factors. However,the efficiency of human iPS cell generation is extremely low and therefore elucidating the mechanisms underlying cellular reprogramming is of prime importance. We demonstrate that 8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) improves the reprogramming efficiency of human neonatal foreskin fibroblast (HFF1) cells transduced with the four transcription factors by 2-fold. The combination of 8-Br-cAMP and VPA synergistically increases the efficiency to 6.5-fold. The effect of 8-Br-cAMP or VPA may in part be due to the up-regulation of cytokine-related and inflammatory pathways. Remarkably,the synergistic effect of 8-Br-cAMP and VPA on cellular reprogramming may be due to the transient decrease of p53 protein during the early stages of reprogramming. However,it could also be due to additional differentially regulated genes and pathways such as the up-regulation of cytokine-related,inflammatory pathways and self-renewal supporting gene,namely cyclin-encoding CCND2,and the associated genes CCNA1 and CCNE1. Conversely,we also see the down-regulation of the p53 (CCNB2,GTSE1,SERPINE1) and cell cycle (PLK1,CCNB2) pathways. Our data demonstrates that a cyclic AMP analog,8-Br-cAMP,enhances the efficiency of cellular reprogramming. In addition,8-Br-cAMP and VPA have a synergistic effect on cellular reprogramming,which may be in part due to the transient down-regulation of the p53 signaling pathway during the early stages of reprogramming. View Publication