搜索结果: 'gmp t cell activator'

BACKGROUND: The enhancement of circulating endothelial progenitor cells (EPCs) obtained by exercise training can be beneficial to patients with cardiac disease. Changes in the levels and differentiation of CD34(pos)/KDR(pos) EPCs,as well as the plasma concentration of vascular endothelial growth factor (VEGF) and stromal cell-derived factor (SDF)-1 EPC-mobilizing cytokines,were evaluated in patients with chronic heart failure after 8 weeks of supervised aerobic training (SAT) and 8 weeks of subsequent discontinued SAT (DSAT). METHODS AND RESULTS: The levels of circulating EPC and EPC differentiation potential of 22 patients who underwent SAT were studied by fluorescence-activated cell sorter analysis and colony forming-unit assay,respectively. The plasma levels of VEGF and SDF-1 were measured by enzyme-linked immunosorbent assay. In response to SAT,the levels of both EPC and VEGF/SDF-1 markedly increased (P textless .001 vs baseline) but returned to the baseline levels after DSAT. A similar change was observed with the EPC clonogenic potential,but on DSAT the baseline level was incompletely attained. CONCLUSIONS: In response to SAT,patients with chronic heart failure show enhanced EPC levels and clonogenic potential that is mirrored by increased plasma VEGF and SDF-1 levels. DSAT can interfere with the maintenance of training-acquired VEGF/SDF-1-related EPC levels and clonogenic potential. View Publication

Human embryonic stem cells (hESC) can differentiate to cardiomyocytes in vitro but with generally poor efficiency. Here,we describe a novel method for the efficient generation of cardiomyocytes from hESC in a scalable suspension culture process. Differentiation in serum-free medium conditioned by the cell line END2 (END2-CM) readily resulted in differentiated cell populations with more than 10% cardiomyocytes without further enrichment. By screening candidate molecules,we have identified SB203580,a specific p38 MAP kinase inhibitor,as a potent promoter of hESC-cardiogenesis. SB203580 at concentrations textless10 microM,induced more than 20% of differentiated cells to become cardiomyocytes and increased total cell numbers,so that the overall cardiomyocyte yield was approximately 2.5-fold higher than controls. Gene expression indicated that early mesoderm formation was favored in the presence of SB203580. Accordingly,transient addition of the inhibitor at the onset of differentiation only was sufficient to determine the hESC fate. Patch clamp electrophysiology showed that the distribution of cardiomyocyte phenotypes in the population was unchanged by the compound. Interestingly,cardiomyogenesis was strongly inhibited at SB203580 concentrations textgreater or =15 microM. Thus,modulation of the p38MAP kinase pathway,in combination with factors released by END2 cells,plays an essential role in early lineage determination in hESC and the efficiency of cardiomyogenesis. Our findings contribute to transforming human cardiomyocyte generation from hESC into a robust and scalable process. View Publication

Implanted materials,such as medical devices,provoke the body to initiate an inflammatory reaction,known as the foreign body reaction (FBR),which causes several complications for example in hip prostheses,silicone implants,peritoneal dialysis catheters and left ventricular assist devices. FBR is initiated by macrophage adherence and results in granulation tissue formation. The early immunobiology and development of this tissue is not completely understood,but there are indications from related myofibroblast-forming diseases such as vascular repair and fibrosis that primitive stem cells also play a role in the formation of FBR-tissue. To investigate this,acellular photo-oxidized bovine pericardium patches were implanted intraperitoneally in rats and retrieved at time-points ranging from 6h to 7 days. A significant fraction of Sca-1(+) (6h-2 days),c-kit(+),CD34(+) and CD271(+) (2-3 days) stem/progenitor cells were detected. Colony-forming and differentiation capacity of the primitive stem cells into adipo-,osteo-,and myofibroblasts were shown. The presence of these primitive cells and their myofibroblastic differentiation potential were also confirmed at RNA level. The identification of specific primitive cells during FBR may have important implications for the inflammatory responses to inert materials and their use in tissue prostheses. View Publication

OBJECTIVES: Our objective was to develop and assess a novel endogenous progenitor cell (EPC) assay based on aldehyde dehydrogenase (ALDH) activity,and to define the relationship of ALDH-bright (ALDH(br)) cells with previously defined EPCs,patient age,and extent of coronary artery disease. BACKGROUND: Accurate assessment of circulating EPCs is of significant interest,yet current assays have limitations. Progenitor cells display high levels of ALDH activity. An assay based on ALDH activity may offer a simple means for enumerating EPCs. METHODS: We simultaneously determined the numbers of EPCs based on ALDH activity and cell surface expression of CD133,CD34,and vascular endothelial growth factor receptor-2 in 110 patients undergoing cardiac catheterization. We assessed the reproducibility of these estimates,correlation among EPC assays,and the association of ALDH(br) numbers with age and disease severity. RESULTS: Aldehyde dehydrogenase-bright cells were easily identified in nonmobilized peripheral blood with median and mean frequencies of 0.041% and 0.074%,respectively. Aldehyde dehydrogenase-bright cells expressed CD34 or CD133 cell surface markers (57.0% and 27.1%,respectively),correlated closely with CD133+CD34+ cells (r = 0.72; p textless 0.001),and differentiated into endothelial cells with greater efficiency than CD133+CD34+ cells. Aldehyde dehydrogenase-bright cell numbers were inversely associated with patient age and coronary disease severity. CONCLUSIONS: Aldehyde dehydrogenase activity represents a novel simplified method for quantifying EPCs. The correlation of ALDH(br) cells with clinical factors and outcomes warrants further study. View Publication

With increasing worldwide demand for safe blood,there is much interest in generating red blood cells in vitro as an alternative clinical product. However,available methods for in vitro generation of red cells from adult and cord blood progenitors do not yet provide a sustainable supply,and current systems using pluripotent stem cells as progenitors do not generate viable red cells. We have taken an alternative approach,immortalizing early adult erythroblasts generating a stable line,which provides a continuous supply of red cells. The immortalized cells differentiate efficiently into mature,functional reticulocytes that can be isolated by filtration. Extensive characterization has not revealed any differences between these reticulocytes and in vitro-cultured adult reticulocytes functionally or at the molecular level,and importantly no aberrant protein expression. We demonstrate a feasible approach to the manufacture of red cells for clinical use from in vitro culture. View Publication

Recently,many hurdles and limitations for production of clinically applicable iPSC derivatives have been overcome. Transgene-free iPSCs can be efficiently derived from easily accessible cell sources such as blood. Here we describe the generation of transgene-free hiPS cells from cord blood derived CD34+ cells,reprogrammed using CytoTune™ Sendai reprogramming vectors. CD34+ cell isolation,cultivation,reprogramming and establishment of resulting hiPSC lines were performed under the exclusive usage of animal-derived component-free (ADCF) materials and components. View Publication

The derivation of astrocytes from human pluripotent stem cells is currently slow and inefficient. We demonstrate that overexpression of the transcription factors SOX9 and NFIB in human pluripotent stem cells rapidly and efficiently yields homogeneous populations of induced astrocytes. In our study these cells exhibited molecular and functional properties resembling those of adult human astrocytes and were deemed suitable for disease modeling. Our method provides new possibilities for the study of human astrocytes in health and disease. View Publication

The human brain is composed of a complex assembly of about 171 billion heterogeneous cellular units (86 billion neurons and 85 billion non-neuronal glia cells). A comprehensive description of brain cells is necessary to understand the nervous system in health and disease. Recently,advances in genomics have permitted the accurate analysis of the full transcriptome of single cells (scRNA-seq). We have built upon such technical progress to combine scRNA-seq with patch-clamping electrophysiological recording and morphological analysis of single human neurons in vitro. This new powerful method,referred to as Patch-seq,enables a thorough,multimodal profiling of neurons and permits us to expose the links between functional properties,morphology,and gene expression. Here,we present a detailed Patch-seq protocol for isolating single neurons from in vitro neuronal cultures. We have validated the Patch-seq whole-transcriptome profiling method with human neurons generated from embryonic and induced pluripotent stem cells (ESCs/iPSCs) derived from healthy subjects,but the procedure may be applied to any kind of cell type in vitro. Patch-seq may be used on neurons in vitro to profile cell types and states in depth to unravel the human molecular basis of neuronal diversity and investigate the cellular mechanisms underlying brain disorders. View Publication

We examined the signaling pathways and cell type-specific responses of IFN regulatory factor (IRF) 5,an immune-regulatory transcription factor. We show that the protein kinases IKK$\alpha$,IKK$\beta$,IKK$\epsilon$,and TANK-binding kinase 1 each confer IRF5 phosphorylation/dimerization,thus extending the family of IRF5 activator kinases. Among primary human immune cell subsets,we found that IRF5 is most abundant in plasmacytoid dendritic cells (pDCs). Flow cytometric cell imaging revealed that IRF5 is specifically activated by endosomal TLR signaling. Comparative analyses revealed that IRF3 is activated in pDCs uniquely through RIG-I-like receptor (RLR) signaling. Transcriptomic analyses of pDCs show that the partitioning of TLR7/IRF5 and RLR/IRF3 pathways confers differential gene expression and immune cytokine production in pDCs,linking IRF5 with immune regulatory and proinflammatory gene expression. Thus,TLR7/IRF5 and RLR-IRF3 partitioning serves to polarize pDC response outcome. Strategies to differentially engage IRF signaling pathways should be considered in the design of immunotherapeutic approaches to modulate or polarize the immune response for specific outcome. View Publication

CRISPR pools are being widely employed to identify gene functions. However,current technology,which utilizes DNA as barcodes,permits limited phenotyping and bulk-cell resolution. To enable novel screening capabilities,we developed a barcoding system operating at the protein level. We synthesized modules encoding triplet combinations of linear epitopes to generate {\textgreater}100 unique protein barcodes (Pro-Codes). Pro-Code-expressing vectors were introduced into cells and analyzed by CyTOF mass cytometry. Using just 14 antibodies,we detected 364 Pro-Code populations; establishing the largest set of protein-based reporters. By pairing each Pro-Code with a different CRISPR,we simultaneously analyzed multiple phenotypic markers,including phospho-signaling,on dozens of knockouts. Pro-Code/CRISPR screens found two interferon-stimulated genes,the immunoproteasome component Psmb8 and a chaperone Rtp4,are important for antigen-dependent immune editing of cancer cells and identified Socs1 as a negative regulator of Pd-l1. The Pro-Code technology enables simultaneous high-dimensional protein-level phenotyping of 100s of genes with single-cell resolution. View Publication

PURPOSE Tumor-associated macrophages (TAMs) and the hyperactivation of phosphoinositide 3-kinase(PI3K)/AKT pathway are involved in the pathogenesis of Hodgkin lymphoma (HL) and affect disease outcome. Since the $\delta$ and $\gamma$ isoforms of PI3K are overexpressed in Hodgkin/Reed-Sternberg (HRS) cells and the tumor microenvironment (TME),we propose that the PI3K$\delta$/$\gamma$ inhibitor RP6530 might affect both HRS cells and TME,ultimately leading to an enhanced antitumor response. EXPERIMENTAL DESIGN HL cell lines (L-540,KM-H2 and L-428) and primary human macrophages were used to investigate the activity of RP6530 in vitro and in vivo in HL cell line xenografts. RESULTS In vitro,RP6530 besides killing and inhibiting the proliferation of HL cells,downregulated lactic acid metabolism,switching the activation of macrophages from an immunosuppressive M2-like phenotype to a more inflammatory M1-like state. By RNA sequencing,we define tumor glycolysis as a specific PI3K$\delta$/$\gamma$-dependent pathway implicated in the metabolic reprogramming of cancer cells. We identify the metabolic regulator Pyruvate Kinase M2 (PKM2) as the main mediator of tumor-induced immunosuppressive phenotype of macrophages. Furthermore,we show in human tumor xenografts that RP6530 repolarizes TAMs into pro-inflammatory macrophages and inhibits tumor vasculature,leading to tumor regression. Interestingly,HL patients experiencing objective responses (CR and PR) in a phase 1 trial using RP6530 showed a significant inhibition of circulating MDSCs and an average mean reduction in serum TARC levels of 40{\%} (range,4-76{\%}). CONCLUSIONS Our results support PI3K$\delta$/$\gamma$ inhibition as a novel therapeutic strategy that targets both malignant cells and the TME to treat HL patients. View Publication

During chronic viral infection,the inflammatory function of CD4 T-cells becomes gradually attenuated. Concurrently,Th1 cells progressively acquire the capacity to secrete the cytokine IL-10,a potent suppressor of antiviral T cell responses. To determine the transcriptional changes that underlie this adaption process,we applied a single-cell RNA-sequencing approach and assessed the heterogeneity of IL-10-expressing CD4 T-cells during chronic infection. Here we show an IL-10-producing population with a robust Tfh-signature. Using IL-10 and IL-21 double-reporter mice,we further demonstrate that IL-10+IL-21+co-producing Tfh cells arise predominantly during chronic but not acute LCMV infection. Importantly,depletion of IL-10+IL-21+co-producing CD4 T-cells or deletion of Il10 specifically in Tfh cells results in impaired humoral immunity and viral control. Mechanistically,B cell-intrinsic IL-10 signaling is required for sustaining germinal center reactions. Thus,our findings elucidate a critical role for Tfh-derived IL-10 in promoting humoral immunity during persistent viral infection. View Publication