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

Improving effector T-cell functions is highly desirable for preventive or therapeutic interventions of diverse diseases. Signal transducer and activator of transcription 3 (Stat3) in the myeloid compartment constrains Th1-type immunity,dampening natural and induced antitumor immune responses. We have recently developed an in vivo small interfering RNA (siRNA) delivery platform by conjugating a Toll-like receptor 9 agonist with siRNA that efficiently targets myeloid and B cells. Here,we show that either CpG triggering combined with the genetic Stat3 ablation in myeloid/B cell compartments or administration of the CpG-Stat3siRNA drastically augments effector functions of adoptively transferred CD8+ T cells. Specifically,we show that both approaches are capable of increasing dendritic cell and CD8(+) T-cell engagement in tumor-draining lymph nodes. Furthermore,both approaches can significantly activate the transferred CD8(+) T cells in vivo,upregulating effector molecules such as perforin,granzyme B,and IFN-γ. Intravital multiphoton microscopy reveals that Stat3 silencing combined with CpG triggering greatly increases killing activity and tumor infiltration of transferred T cells. These results suggest the use of CpG-Stat3siRNA,and possibly other Stat3 inhibitors,as a potent adjuvant to improve T-cell therapies. View Publication

On the basis of its inhibition by SB216763,we identified the multifunctional enzyme Glycogen Synthase Kinase 3beta (GSK3beta) as a central regulator for differentiation and cell survival of adult neural stem cells. Detected by proteomic approaches,members of the Wnt/beta-catenin signaling pathway appear to participate in enhanced neuronal differentiation and activated transcription of beta-catenin target genes during GSK3beta inhibition,associated with decreased apoptosis. View Publication

Rheumatoid Arthritis (RA) causes chronic inflammation of joints. The cytokines TNFalpha and IFNgamma are central players in RA,however their source has not been fully elucidated. Natural Killer (NK) cells are best known for their role in elimination of viral-infected and transformed cells,and they secrete pro-inflammatory cytokines. NK cells are present in the synovial fluids (SFs) of RA patients and are considered to be important in bone destruction. However,the phenotype and function of NK cells in the SFs of patients with erosive deformative RA (DRA) versus non-deformative RA (NDRA) is poorly characterized. Here we characterize the NK cell populations present in the blood and SFs of DRA and NDRA patients. We demonstrate that a distinct population of activated synovial fluid NK (sfNK) cells constitutes a large proportion of immune cells found in the SFs of DRA patients. We discovered that although sfNK cells in both DRA and NDRA patients have similar phenotypes,they function differently. The DRA sfNK secrete more TNFalpha and IFNgamma upon exposure to IL-2 and IL-15. Consequently,we suggest that sfNK cells may be a marker for more severely destructive RA disease. View Publication

The restoration of pluripotency circuits by the reactivation of endogenous stemness factors,such as SOX2,may provide a new paradigm in cancer development. The tumoral stem cell reprogramming hypothesis,i.e.,the ability of stemness factors to redirect normal and differentiated tumor cells toward a less-differentiated and stem-like state,adds new layers of complexity to cancer biology,because the effects of such reprogramming may remain dormant until engaged later in response to (epi)genetic and/or (micro)environmental events. To test this hypothesis,we utilized an in vitro model of a SOX2-overexpressing cancer stem cell (CSC)-like cellular state that was recently developed in our laboratory by employing Yamanaka's nuclear reprogramming technology in the estrogen receptor $$ (ER$$)-positive MCF-7 breast cancer cell line. Despite the acquisition of distinct molecular features that were compatible with a breast CSC-like cellular state,such as strong aldehyde dehydrogenase activity,as detected by ALDEFLUOR,and overexpression of the SSEA-4 and CD44 breast CSC markers,the tumor growth-initiating ability of SOX2-overexpressing CSC-like MCF-7 cells solely occurred in female nude mice supplemented with estradiol when compared with MCF-7 parental cells. Ser118 phosphorylation of estrogen receptor $$ (ER$$),which is a pivotal integrator of the genomic and nongenomic E 2/ER$$ signaling pathways,drastically accumulated in nuclear speckles in the interphase nuclei of SOX2-driven CSC-like cell populations. Moreover,SOX2-positive CSC-like cells accumulated significantly higher numbers of actively dividing cells,and the highest levels of phospho-Ser118-ER$$ occurred when chromosomes lined up on a metaphase plate. The previously unrecognized link between E 2/ER$$ signaling and SOX2-driven stem cell circuitry may significantly impact our current understanding of breast cancer initiation and progression,i.e.,SOX2 can promote non-genomic E 2 signaling that leads to nuclear phospho-Ser118-ER$$,which ultimately exacerbates genomic ER signaling in response to E 2. Because E 2 stimulation has been recently shown to enhance breast tumor-initiating cell survival by downregulating miR-140,which targets SOX2,the establishment of a bidirectional cross-talk interaction between the stem cell self-renewal regulator,SOX2,and the local and systemic ability of E 2 to increase breast CSC activity may have profound implications for the development of new CSC-directed strategies for breast cancer prevention and therapy. View Publication

A robust method for culturing human pluripotent stem (hPS) cells under chemically defined and xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine. Here,we describe a protocol for monolayer culturing of Oct-4-positive hPS cells on a specific laminin-521 (LN-521) isoform,under xeno-free and chemically defined conditions. The cells are dispersed into single-cell suspension and then plated on LN-521 isoform at densities higher than 5,000 cells per cm²,where they attach,migrate and survive by forming small monolayer cell groups. The cells avidly divide and expand horizontally until the entire dish is covered by a confluent monolayer. LN-521,in combination with E-cadherin,allows cloning of individual hPS cells in separate wells of 96-well plates without the presence of rho-associated protein kinase (ROCK) inhibitors or any other inhibitors of anoikis. Characterization of cells maintained for several months in culture reveals pluripotency with a minimal degree of genetic abnormalities. View Publication

BACKGROUND: Induced pluripotent stem cells (iPSCs) can be differentiated into potentially unlimited lineages of cell types for use in autologous cell therapy. However,the efficiency of the differentiation procedure and subsequent function of the iPSC-derived cells may be influenced by epigenetic factors that the iPSCs retain from their tissues of origin; thus,iPSC-derived cells may be more effective for treatment of myocardial injury if the iPSCs were engineered from cardiac-lineage cells,rather than dermal fibroblasts. METHODS AND RESULTS: We show that human cardiac iPSCs (hciPSCs) can be generated from cardiac fibroblasts and subsequently differentiated into exceptionally pure (textgreater92%) sheets of cardiomyocytes (CMs). The hciPSCs passed through all the normal stages of differentiation before assuming a CM identity. When using the fibrin gel-enhanced delivery of hciPSC-CM sheets at the site of injury in infarcted mouse hearts,the engraftment rate was 31.91%+/-5.75% at Day 28 post transplantation. The hciPSC-CM in the sheet also appeared to develop a more mature,structurally aligned phenotype 28 days after transplantation and was associated with significant improvements in cardiac function,vascularity,and reduction in apoptosis. CONCLUSIONS: These data strongly support the potential of hciPSC-CM sheet transplantation for the treatment of heart with acute myocardial infarction. View Publication

Regeneration of human cartilage is inherently inefficient; an abundant autologous source,such as human induced pluripotent stem cells (hiPSCs),is therefore attractive for engineering cartilage. We report a growth factor-based protocol for differentiating hiPSCs into articular-like chondrocytes (hiChondrocytes) within 2 weeks,with an overall efficiency textgreater90%. The hiChondrocytes are stable and comparable to adult articular chondrocytes in global gene expression,extracellular matrix production,and ability to generate cartilage tissue in vitro and in immune-deficient mice. Molecular characterization identified an early SRY (sex-determining region Y) box (Sox)9(low) cluster of differentiation (CD)44(low)CD140(low) prechondrogenic population during hiPSC differentiation. In addition,2 distinct Sox9-regulated gene networks were identified in the Sox9(low) and Sox9(high) populations providing novel molecular insights into chondrogenic fate commitment and differentiation. Our findings present a favorable method for generating hiPSC-derived articular-like chondrocytes. The hiChondrocytes are an attractive cell source for cartilage engineering because of their abundance,autologous nature,and potential to generate articular-like cartilage rather than fibrocartilage. In addition,hiChondrocytes can be excellent tools for modeling human musculoskeletal diseases in a dish and for rapid drug screening. View Publication

We examined the role of NFκB1 in the homeostasis and function of peripheral follicular (Fo) B cells. Aging mice lacking NFκB1 (Nfκb1(-/-)) develop lymphoproliferative and multiorgan autoimmune disease attributed in large part to the deregulated activity ofNfκb1(-/-)Fo B cells that produce excessive levels of the proinflammatory cytokine interleukin 6 (IL-6). Despite enhanced germinal center (GC) B cell differentiation,the formation of GC structures was severely disrupted in theNfκb1(-/-)mice. Bone marrow chimeric mice revealed that the Fo B cell-intrinsic loss of NFκB1 led to the spontaneous generation of GC B cells. This was primarily the result of an increase in IL-6 levels,which promotes the differentiation of Fo helper CD4(+)T cells and acts in an autocrine manner to reduce antigen receptor and toll-like receptor activation thresholds in a population of proliferating IgM(+)Nfκb1(-/-)Fo B cells. We demonstrate that p50-NFκB1 repressesIl-6transcription in Fo B cells,with the loss of NFκB1 also resulting in the uncontrolled RELA-driven transcription ofIl-6.Collectively,our findings identify a previously unrecognized role for NFκB1 in preventing multiorgan autoimmunity through its negative regulation ofIl-6gene expression in Fo B cells. View Publication

Human pluripotent stem cells (hPSCs),including embryonic stem cells and induced pluripotent stem cells,are potentially useful in regenerative therapies for heart disease. For medical applications,clinical-grade cardiac cells must be produced from hPSCs in a defined,cost-effective manner. Cell-based screening led to the discovery of KY02111,a small molecule that promotes differentiation of hPSCs to cardiomyocytes. Although the direct target of KY02111 remains unknown,results of the present study suggest that KY02111 promotes differentiation by inhibiting WNT signaling in hPSCs but in a manner that is distinct from that of previously studied WNT inhibitors. Combined use of KY02111 and WNT signaling modulators produced robust cardiac differentiation of hPSCs in a xeno-free,defined medium,devoid of serum and any kind of recombinant cytokines and hormones,such as BMP4,Activin A,or insulin. The methodology has potential as a means for the practical production of human cardiomyocytes for regeneration therapies. View Publication

PURPOSE The cancer stem cell theory postulates that tumors contain a subset of cells with stem cell properties of self-renewal,differentiation,and tumor initiation. The purpose of this study is to determine the role of Notch activity in identifying lung cancer stem cells. EXPERIMENTAL DESIGN We investigated the role of Notch activity in lung adenocarcinoma using a Notch GFP reporter construct and a $$-secretase inhibitor (GSI),which inhibits Notch pathway activity. RESULTS Transduction of lung cancer cells with Notch GFP reporter construct identified a subset of cells with high Notch activity (GFP-bright). GFP-bright cells had the ability to form more tumor spheres in serum-free media and were able to generate both GFP-bright and GFP-dim (lower Notch activity) cell populations. GFP-bright cells were resistant to chemotherapy and were tumorigenic in serial xenotransplantation assays. Tumor xenografts of mice treated with GSI had decreased expression of downstream effectors of Notch pathway and failed to regenerate tumors upon reimplantation in NOD/SCID mice. Using multivariate analysis,we detected a statistically significant correlation between poor clinical outcome and Notch activity (reflected in increased Notch ligand expression or decreased expression of the negative modulators),in a group of 443 patients with lung adenocarcinoma. This correlation was further confirmed in an independent group of 89 patients with adenocarcinoma in which Hes-1 overexpression correlated with poor overall survival. CONCLUSIONS Notch activity can identify lung cancer stem cell-like population and its inhibition may be an appropriate target for treating lung adenocarcinoma. View Publication

B1 cells differ in many ways from conventional B cells,most prominently in the production of natural immunoglobulin,which is vitally important for protection against pathogens. B1 cells have also been implicated in the pathogenesis of autoimmune dyscrasias and malignant diseases. It has been impossible to accurately study B1 cells during health and illness because the nature of human B1 cells has not been successfully defined. This has produced controversy regarding the existence of human B1 cells. Here,we determined the phenotype of human B1 cells by testing sort-purified B cell fractions for three fundamental B1 cell functions based on mouse studies: spontaneous IgM secretion,efficient T cell stimulation,and tonic intracellular signaling. We found that a small population of CD20(+)CD27(+)CD43(+) cells present in both umbilical cord and adult peripheral blood fulfilled these criteria and expressed a skewed B cell receptor repertoire. These B cells express little or no surface CD69 and CD70,both of which are markedly up-regulated after activation of CD20(+)CD27(-)CD43(-) (naive) and CD20(+)CD27(+)CD43(-) (memory) B cells. This work identifies human B1 cells as CD20(+)CD27(+)CD43(+)CD70(-). We determined that the proportion of B1 cells declines with age,which may contribute to disease susceptibility. Identification of human B1 cells provides a foundation for future studies on the nature and role of these cells in human disease. View Publication

CREB-binding protein (CREBBP) is important for the cell-autonomous regulation of hematopoiesis,including the stem cell compartment. In the present study,we show that CREBBP plays an equally pivotal role in microenvironment-mediated regulation of hematopoiesis. We found that the BM microenvironment of Crebbp(+/-) mice was unable to properly maintain the immature stem cell and progenitor cell pools. Instead,it stimulates myeloid differentiation,which progresses into a myeloproliferation phenotype. Alterations in the BM microenvironment resulting from haploinsufficiency of Crebbp included a marked decrease in trabecular bone that was predominantly caused by increased osteoclastogenesis. Although CFU-fibroblast (CFU-F) and total osteoblast numbers were decreased,the bone formation rate was similar to that found in wild-type mice. At the molecular level,we found that the known hematopoietic modulators matrix metallopeptidase-9 (MMP9) and kit ligand (KITL) were decreased with heterozygous levels of Crebbp. Lastly,potentially important regulatory proteins,endothelial cell adhesion molecule 1 (ESAM1) and cadherin 5 (CDH5),were increased on Crebbp(+/-) endothelial cells. Our findings reveal that a full dose of Crebbp is essential in the BM microenvironment to maintain proper hematopoiesis and to prevent excessive myeloproliferation. View Publication