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

Fibroblast growth factor receptor 1 (FGFR1) is overexpressed in multiple types of solid tumors,including head and neck squamous cell carcinoma (HNSCC). Being associated with poor prognosis,FGFR1 is a potential therapeutic target for aggressive tumors. T cell-based cancer immunotherapy has played a central role in novel cancer treatments. However,the potential of antitumor immunotherapy targeting FGFR1 has not been investigated. Here,we showed that FGFR-tyrosine kinase inhibitors (TKIs) augmented antitumor effects of immune checkpoint inhibitors in an HNSCC mouse model and upregulated tumoral MHC class I and MHC class II expression in vivo and in vitro. This upregulation was associated with the mitogen-activated protein kinase signaling pathway,which is a crucial pathway for cancer development through FGFR signaling. Moreover,we identified an FGFR1-derived peptide epitope (FGFR1305-319) that could elicit antigen-reactive and multiple HLA-restricted CD4+ T cell responses. These T cells showed direct cytotoxicity against tumor cells that expressed FGFR1. Notably,FGFR-TKIs augmented antitumor effects of FGFR1-reactive T cells against human HNSCC cells. These results indicate that the combination of FGFR-TKIs with immunotherapy,such as an FGFR1-targeting peptide vaccine or immune checkpoint inhibitor,could be a novel and robust immunologic approach for treating patients with FGFR1-expressing cancer cells. View Publication

BACKGROUND Triple-negative breast cancer (TNBC) has significantly worse prognosis. Acquired chemoresistance remains the major cause of therapeutic failure of TNBC. In clinic,the relapsed TNBC is commonly pan-resistant to various drugs with completely different resistant mechanisms. Investigation of the mechanisms and development of new drugs to target pan-chemoresistance will potentially improve the therapeutic outcomes of TNBC patients. METHODS In this study,1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT),combination index (CI)-isobologram,western blot,ALDEFLUOR analysis,clonogenic assay and immunocytochemistry were used. RESULTS The chemoresistant MDA-MB-231PAC10 cells are highly cross-resistant to paclitaxel (PAC),cisplatin (CDDP),docetaxel and doxorubicin. The MDA-MB-231PAC10 cells are quiescent with significantly longer doubling time (64.9 vs 31.7 h). This may be caused by high expression of p21(Waf1). The MDA-MB-231PAC10 cells express high aldehyde dehydrogenase (ALDH) activity and a panel of embryonic stem cell-related proteins,for example,Oct4,Sox2,Nanog and nuclealisation of HIF2$$ and NF-$$Bp65. We have previously reported that disulfiram (DS),an antialcoholism drug,targets cancer stem cells (CSCs) and enhances cytotoxicity of anticancer drugs. Disulfiram abolished CSC characters and completely reversed PAC and CDDP resistance in MDA-MB-231PAC10 cells. CONCLUSION Cancer stem cells may be responsible for acquired pan-chemoresistance. As a drug used in clinic,DS may be repurposed as a CSC inhibitor to reverse the acquired pan-chemoresistance. View Publication

Vocal fold epithelial cells are very difficult to study as the vocal fold epithelial cell lines do not exist and they cannot be removed from the healthy larynx without engendering a significant and unacceptable risk to vocal fold function. Here,we describe the procedure to create an engineered vocal fold tissue construct consisting of the scaffold composed of the collagen 1 gel seeded with human fibroblasts and simple epithelial progenitors seeded on the scaffold and cultivated at air-liquid interface for 19-21 days to derive the stratified squamous epithelium. This model of vocal fold mucosa is very similar in morphology,gene expression,and phenotypic characteristics to native vocal fold epithelial cells and the underlying lamina propria and,therefore,offers a promising approach to studying vocal fold biology and biomechanics in health and disease. View Publication

Endothelial cells (ECs) exist in different microenvironments in vivo,including under different levels of shear stress in arteries versus veins. Standard stem cell differentiation protocols to derive ECs and EC-subtypes from human induced pluripotent stem cells (hiPSCs) generally use growth factors or other soluble factors in an effort to specify cell fate. In this study,a biomimetic flow bioreactor was used to subject hiPSC-derived ECs (hiPSC-ECs) to shear stress to determine the impacts on phenotype and upregulation of markers associated with an anti-thrombotic,anti-inflammatory,arterial-like phenotype. The in vitro bioreactor system was able to efficiently mature hiPSC-ECs into arterial-like cells in 24 h,as demonstrated by qRT-PCR for arterial markers EphrinB2,CXCR4,Conexin40 and Notch1,as well protein-level expression of Notch1 intracellular domain (NICD). Furthermore,the exogenous addition of soluble factors was not able to fully recapitulate this phenotype that was imparted by shear stress exposure. The induction of these phenotypic changes was biomechanically mediated in the shear stress bioreactor. This biomimetic flow bioreactor is an effective means for the differentiation of hiPSC-ECs toward an arterial-like phenotype,and is amenable to scale-up for culturing large quantities of cells for tissue engineering applications. View Publication

RATIONALE: Tolerogenic dendritic cells and natural regulatory T cells have been implicated in the process of infectious tolerance in human allergic asthma. However,the significance of the influence of natural regulatory T cells on tolerogenic dendritic cells in the disease has not been investigated. OBJECTIVES: We aimed to characterize the mechanism of induction of the tolerogenic phenotype in circulating blood dendritic cells by allergic asthmatic natural regulatory T cells. METHODS: The study was performed in a cohort of 21 subjects with allergic asthma,21 healthy control subjects,and 21 subjects with nonallergic asthma. We cultured blood dendritic cells with natural regulatory T cells to study the induction of tolerogenic dendritic cells. Flow cytometry and proliferation assays were employed to analyze phenotype and function of dendritic cells as well as IL-10 production from natural regulatory T cells. MEASUREMENTS AND MAIN RESULTS: Dendritic cells cultured with natural regulatory T cells up-regulated IL-10,down-regulated costimulatory molecules,and stimulated the proliferation of CD4(+)CD25(-) effector T cells less potently. Allergic asthmatic natural regulatory T cells were significantly less efficient in inducing this tolerogenic phenotype of dendritic cells compared with healthy control and nonallergic asthmatic counterparts. Furthermore,this defective function of natural regulatory T cells was associated with their decreased IL-10 expression,disease severity,and could be reversed by oral corticosteroid therapy. CONCLUSIONS: These results provided the first evidences of impaired induction of tolerogenic dendritic cells mediated by natural regulatory T cells in human allergic asthma. View Publication

The ability of somatic cells to reprogram their ATP-generating machinery into a Warburg-like glycolytic metabotype while overexpressing stemness genes facilitates their conversion into either induced pluripotent stem cells (iPSCs) or tumor-propagating cells. AMP-activated protein kinase (AMPK) is a metabolic master switch that senses and decodes intracellular changes in energy status; thus,we have evaluated the impact of AMPK activation in regulating the generation of iPSCs from nonstem cells of somatic origin. The indirect and direct activation of AMPK with the antidiabetic biguanide metformin and the thienopyridone A-769662,respectively,impeded the reprogramming of mouse embryonic and human diploid fibroblasts into iPSCs. The AMPK activators established a metabolic barrier to reprogramming that could not be bypassed,even through p53 deficiency,a fundamental mechanism to greatly improve the efficiency of stem-cell production. Treatment with metformin or A-769662 before the generation of iPSC colonies was sufficient to drastically decrease iPSC generation,suggesting that AMPK activation impedes early stem cell genetic reprogramming. Monitoring the transcriptional activation status of each individual reprogramming factor (i.e.,Oct4,Sox2,Klf4 and c-Myc) revealed that AMPK activation notably prevented the transcriptional activation of Oct4,the master regulator of the pluripotent state. AMPK activation appears to impose a normalized metabolic flow away from the required pro-immortalizing glycolysis that fuels the induction of stemness and pluripotency,endowing somatic cells with an energetic infrastructure that is protected against reprogramming. AMPK-activating anti-reprogramming strategies may provide a roadmap for the generation of novel cancer therapies that metabolically target tumor-propagating cells. View Publication

Human endothelial cells (ECs) and pericytes are of great interest for research on vascular development and disease,as well as for future therapy. This protocol describes the efficient generation of ECs and pericytes from human pluripotent stem cells (hPSCs) under defined conditions. Essential steps for hPSC culture,differentiation,isolation and functional characterization of ECs and pericytes are described. Substantial numbers of both cell types can be derived in only 2-3 weeks: this involves differentiation (10 d),isolation (1 d) and 4 or 10 d of expansion of ECs and pericytes,respectively. We also describe two assays for functional evaluation of hPSC-derived ECs: (i) primary vascular plexus formation upon coculture with hPSC-derived pericytes and (ii) incorporation in the vasculature of zebrafish xenografts in vivo. These assays can be used to test the quality and drug sensitivity of hPSC-derived ECs and model vascular diseases with patient-derived hPSCs. View Publication

APOBEC3G (A3G) is an intrinsic antiviral factor that inhibits the replication of human immunodeficiency virus (HIV) by deaminating cytidine residues to uridine. This causes guanosine-to-adenosine hypermutation in the opposite strand and results in inactivation of the virus. HIV counteracts A3G through the activity of viral infectivity factor (Vif),which promotes degradation of A3G. We report that viral protein R (Vpr),which interacts with a uracil glycosylase,also counteracted A3G by diminishing the incorporation of uridine. However,this process resulted in activation of the DNA-damage–response pathway and the expression of natural killer (NK) cell–activating ligands. Our results show that pathogen-induced deamination of cytidine and the DNA-damage response to virus-mediated repair of the incorporation of uridine enhance the recognition of HIV-infected cells by NK cells. View Publication

Rationale: Mesenchymal stem cells (MSC) hold great promise in the treatment of various diseases including autoimmune diseases,inflammatory diseases,etc.,due to their pleiotropic properties. However,largely incongruent data were obtained from different MSC-based clinical trials,which may be partially due to functional heterogeneity among MSC. Here,we attempt to derive homogeneous mesenchymal stem cells with neuromesodermal origin from human pluripotent stem cells (hPSC) and evaluate their functional properties. Methods: Growth factors and/or small molecules were used for the differentiation of human pluripotent stem cells (hPSC) into neuromesodermal progenitors (NMP),which were then cultured in animal component-free and serum-free induction medium for the derivation and long-term expansion of MSC. The resulted NMP-MSC were detailed characterized by analyzing their surface marker expression,proliferation,migration,multipotency,immunomodulatory activity and global gene expression profile. Moreover,the in vivo therapeutic potential of NMP-MSC was detected in a mouse model of contact hypersensitivity (CHS). Results: We demonstrate that NMP-MSC express posterior HOX genes and exhibit characteristics similar to those of bone marrow MSC (BMSC),and NMP-MSC derived from different hPSC lines show high level of similarity in global gene expression profiles. More importantly,NMP-MSC display much stronger immunomodulatory activity than BMSC in vitro and in vivo,as revealed by decreased inflammatory cell infiltration and diminished production of pro-inflammatory cytokines in inflamed tissue of CHS models. Conclusion: Our results identify NMP as a new source of MSC and suggest that functional and homogeneous NMP-MSC could serve as a candidate for MSC-based therapies. View Publication

In mice there are two families of MHC class I-specific receptors,namely the Ly49 and CD94/NKG2 receptors. The latter receptors recognize the nonclassical MHC class I Qa-1(b) and are thought to be responsible for the recognition of missing-self and the maintenance of self-tolerance of fetal and neonatal NK cells that do not express Ly49. Currently,how NK cells acquire individual CD94/NKG2 receptors during their development is not known. In this study,we have established a multistep culture method to induce differentiation of embryonic stem (ES) cells into the NK cell lineage and examined the acquisition of CD94/NKG2 by NK cells as they differentiate from ES cells in vitro. ES-derived NK (ES-NK) cells express NK cell-associated proteins and they kill certain tumor cell lines as well as MHC class I-deficient lymphoblasts. They express CD94/NKG2 heterodimers,but not Ly49 molecules,and their cytotoxicity is inhibited by Qa-1(b) on target cells. Using RT-PCR analysis,we also report that the acquisition of these individual receptor gene expressions during different stages of differentiation from ES cells to NK cells follows a predetermined order,with their order of acquisition being first CD94; subsequently NKG2D,NKG2A,and NKG2E; and finally,NKG2C. Single-cell RT-PCR showed coexpression of CD94 and NKG2 genes in most ES-NK cells,and flow cytometric analysis also detected CD94/NKG2 on most ES-NK cells,suggesting that the acquisition of these receptors by ES-NK cells in vitro is nonstochastic,orderly,and cumulative. View Publication

We used primary peripheral blood T cells,a population that exists in G(0) and can be stimulated to enter the cell cycle synchronously,to define more precisely the effects of nicotine on pathways that control cell cycle entry and progression. Our data show that nicotine decreased the ability of T cells to transit through the G(0)/G(1) boundary (acquire competence) and respond to progression signals. These effects were due to nuclear factor of activated T cells c2 (NFATc2)-dependent repression of cyclin-dependent kinase 4 (CDK4) expression. Growth arrest at the G(0)/G(1) boundary was further enforced by inhibition of cyclin D2 expression and by increased expression and stabilization of p27Kip1. Intriguingly,T cells from habitual users of tobacco products and from NFATc2-deficient mice constitutively expressed CDK4 and were resistant to the antiproliferative effects of nicotine. These results indicate that nicotine impairs T cell cycle entry through NFATc2-dependent mechanisms and suggest that,in the face of chronic nicotine exposure,selection may favor cells that can evade these effects. We postulate that cross talk between nicotinic acetylcholine receptors and growth factor receptor-activated pathways offers a novel mechanism by which nicotine may directly impinge on cell cycle progression. This offers insight into possible reasons that underlie the unique effects of nicotine on distinct cell types and identifies new targets that may be useful control tobacco-related diseases. View Publication

The lack of natural killer (NK) cell-specific markers,as well as the overlap among several common surface antigens and functional properties,has obscured the delineation between NK cells and dendritic cells. Here,novel subsets of peripheral blood CD3/14/19(neg) NK cells and monocyte/dendritic cell (DC)-like cells were identified on the basis of CD7 and CD4 expression. Coexpression of CD7 and CD56 differentiates NK cells from CD56+ monocyte/DC-like cells,which lack CD7. In contrast to CD7+CD56+ NK cells,CD7(neg)CD56+ cells lack expression of NK cell-associated markers,but share commonalities in their expression of various monocyte/DC-associated markers. Using CD7,we observed approximately 60% of CD4+CD56+ cells were CD7(neg) cells,indicating the actual frequency of activated CD4+ NK cells is much lower in the blood than previously recognized. Functionally,only CD7+ NK cells secrete gamma interferon (IFNgamma) and degranulate after interleukin-12 (IL-12) plus IL-18 or K562 target cell stimulation. Furthermore,using CD7 to separate CD56+ NK cells and CD56+ myeloid cells,we demonstrate that unlike resting CD7+CD56+ NK cells,the CD7(neg)CD56+ myeloid cells stimulate a potent allogeneic response. Our data indicate that CD7 and CD56 coexpression discriminates NK cells from CD7(neg)CD56+ monocyte/DC-like cells,thereby improving our ability to study the intricacies of NK-cell subset phenotypes and functions in vivo. View Publication