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

Autoimmune diseases are a physiological state wherein immune responses are directed against and damage the body's own tissues. Cytokines secreted by infiltrated inflammatory cells contribute to the pathogenesis of autoimmune diseases. TIPE2,one of the four family members of Tumor necrosis factor-$\alpha$ induced protein-8 (TNFAIP8),is a negative regulator of innate and adaptive immunity and plays essential roles in the maintenance of immune tolerance. However,studies on the role of TIPE2 during the development of autoimmune diseases have generated contradictory results. In the current study,we sought to determine the role of TIPE2 during the development of IMQ-induced psoriasis and Experimental Autoimmune Uveitis (EAU) in mice. Our study revealed that,while TIPE2-deficiency alleviates psoriasis,it exacerbates the development of EAU. Further studies demonstrated that,although TIPE2-deficient T cells produced more IL-17A,they do not migrate efficiently to the local inflammatory site,i.e.,the skin. This in turn led to the decreased IL-17A production in the skin and consequently reduced the severity of psoriasis in TIPE2-deficient mice. However,although TIPE2-deficient T cells still produced more IL-17A in EAU model,they migrate into the inflamed eye as efficient as TIPE2-sufficient T cells,and consequently exacerbates the development of EAU in TIPE2-deficient mice. Taken together,these results indicate that TIPE2 may either promote or suppress autoimmunity depending on the specific inflammatory microenvironment in different types of autoimmune diseases. View Publication

The maturation process of natural killer (NK) cells,which is regulated by multiple transcription factors,determines their functionality,but few checkpoints specifically targeting this process have been thoroughly studied. Here we show that NK-specific deficiency of glucose-regulated protein 94 (gp96) leads to decreased maturation of NK cells in mice. These gp96-deficient NK cells exhibit undermined activation,cytotoxicity and IFN-γ production upon stimulation,as well as weakened responses to IL-15 for NK cell maturation,in vitro. In vivo,NK-specific gp96-deficient mice show increased tumor growth. Mechanistically,we identify Eomes as the downstream transcription factor,with gp96 binding to Trim28 to prevent Trim28-mediated ubiquitination and degradation of Eomes. Our study thus suggests the gp96-Trim28-Eomes axis to be an important regulator for NK cell maturation and cancer surveillance in mice. Natural killer (NK) cell maturation and function are regulated by multiple transcription factors (TF),but detailed molecular insights are scarce. Here the authors show that a TF,Eomes,is important for NK cell responses and cancer surveillance,in which Eomes expression is regulated by gp96 and Trim28 via the ubiquitination and degradation pathways. View Publication

To search for new indicators of self-renewing hematopoietic stem cells (HSCs),highly purified populations were isolated from adult mouse marrow,micromanipulated into a specially designed microscopic array,and cultured for 4 days in 300 ng/ml Steel factor,20 ng/ml IL-11,and 1 ng/ml flt3-ligand. During this period,each cell and its progeny were imaged at 3-min intervals by using digital time-lapse photography. Individual clones were then harvested and assayed for HSCs in mice by using a 4-month multilineage repopulation endpoint (textgreater1% contribution to lymphoid and myeloid lineages). In a first experiment,6 of 14 initial cells (43%) and 17 of 61 clones (28%) had HSC activity,demonstrating that HSC self-renewal divisions had occurred in vitro. Characteristics associated with HSC activity included longer cell-cycle times and the absence of uropodia on a majority of cells within the clone during the final 12 h of culture. Combining these criteria maximized the distinction of clones with HSC activity from those without and identified a subset of 27 of the 61 clones. These 27 clones included all 17 clones that had HSC activity; a detection efficiency of 63% (2.26 times more frequently than in the original group). The utility of these characteristics for discriminating HSC-containing clones was confirmed in two independent experiments where all HSC-containing clones were identified at a similar 2- to 3-fold-greater efficiency. These studies illustrate the potential of this monitoring system to detect new features of proliferating HSCs that are predictive of self-renewal divisions. View Publication

BACKGROUND: Human cord blood (hCB) is the main source of hematopoietic stem and progenitor cells (HSCs/PCs) for transplantation. Efforts to overcome relative shortages of HSCs/PCs have led to technologies to expand HSCs/PCs ex vivo. However,methods suitable for clinical practice have yet to be fully established. METHODOLOGY/PRINCIPAL FINDINGS: In this study,we screened biologically active natural products for activity to promote expansion of hCB HSCs/PCs ex vivo,and identified Garcinol,a plant-derived histone acetyltransferase (HAT) inhibitor,as a novel stimulator of hCB HSC/PC expansion. During a 7-day culture of CD34(+)CD38(-) HSCs supplemented with stem cell factor and thrombopoietin,Garcinol increased numbers of CD34(+)CD38(-) HSCs/PCs more than 4.5-fold and Isogarcinol,a derivative of Garcinol,7.4-fold. Furthermore,during a 7-day culture of CD34(+) HSCs/PCs,Garcinol expanded the number of SCID-repopulating cells (SRCs) 2.5-fold. We also demonstrated that the capacity of Garcinol and its derivatives to expand HSCs/PCs was closely correlated with their inhibitory effect on HAT. The Garcinol derivatives which expanded HSCs/PCs inhibited the HAT activity and acetylation of histones,while inactive derivatives did not. CONCLUSIONS/SIGNIFICANCE: Our findings identify Garcinol as the first natural product acting on HSCs/PCs and suggest the inhibition of HAT to be an alternative approach for manipulating HSCs/PCs. View Publication

Hoechst-effluxing cells (side population cells) are a rare subset of cells found in adult tissues that are highly enriched for stem and progenitor cell activity. To identify potential stem and progenitor cells during lung development,we generated gene expression profiles for CD45- and CD45+ side population cells in the embryonic day 17.5 lung. We found that side population cells comprise 1% of total embryonic day 17.5 lung cells (55% CD45+,45% CD45-). Gene profiling data demonstrated an overrepresentation of endothelial genes within the CD45- side population. We used expression of several distinct genes to identify two types of CD45- side population cells: 1) von Willebrand factor+/smooth muscle actin+ cells that reside in the muscular layer of select large vessels and 2) von Willebrand factor+/intercellular adhesion molecule+ cells that reside within the endothelial layer of select small vessels. Gene profiling of the CD45+ side population indicated an overrepresentation of genes associated with myeloid cell differentiation. Consistent with this,culturing CD45+ side population cells was associated with induction of mature dendritic markers (CD86). The microarray results suggested that expression of myeloperoxidase and proteinase-3 might be used to identify CD45+ side population cells. By immunohistochemistry,we found that myeloperoxidase+/proteinase-3+ cells represent a small subset of total CD45+ cells in the embryonic day 17.5 lung and that they reside in the mesenchyme and perivascular regions. This is the first detailed information regarding the phenotype and localization of side population cells in a developing organ. View Publication

BACKGROUND Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. METHODS Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10{\%} DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry,as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel,each sample was flow sorted into fibroblast,T-cell,B-cell,and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. RESULTS Upon dissociation,cryopreserved synovial tissue fragments yielded a high frequency of viable cells,comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with {\~{}} 30 arthroplasty and {\~{}} 20 biopsy samples yielded a consensus digestion protocol using 100 mu$g/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes,distinct populations of memory B cells and antibody-secreting cells,and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes,fibroblasts,and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell,including transcripts encoding characteristic lineage markers identified. CONCLUSIONS We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers. View Publication

We analyzed 112 patients with high-risk acute myeloid leukemia (61 in complete remission [CR]; 51 in relapse),who received human leukocyte-antigen (HLA)-haploidentical transplants from natural killer (NK) alloreactive (n = 51) or non-NK alloreactive donors (n = 61). NK alloreactive donors possessed HLA class I,killer-cell immunoglobulin-like receptor (KIR) ligand(s) which were missing in the recipients,KIR gene(s) for missing self recognition on recipient targets,and alloreactive NK clones against recipient targets. Transplantation from NK-alloreactive donors was associated with a significantly lower relapse rate in patients transplanted in CR (3% versus 47%) (P textgreater .003),better event-free survival in patients transplanted in relapse (34% versus 6%,P = .04) and in remission (67% versus 18%,P = .02),and reduced risk of relapse or death (relative risk versus non-NK-alloreactive donor,0.48; 95% CI,0.29-0.78; P textgreater .001). In all patients we tested the missing ligand" model which pools KIR ligand mismatched transplants and KIR ligand-matched transplants from donors possessing KIR(s) for which neither donor nor recipient have HLA ligand(s). Only transplantation from NK-alloreactive donors is associated with a survival advantage." View Publication

Animal models of hematopoietic stem cell transplantation have been used to analyze the turnover of bone marrow-derived cells and to demonstrate the critical role of recipient antigen-presenting cells (APC) in graft versus host disease (GVHD). In humans,the phenotype and lineage relationships of myeloid-derived tissue APC remain incompletely understood. It has also been proposed that the risk of acute GVHD,which extends over many months,is related to the protracted survival of certain recipient APC. Human dermis contains three principal subsets of CD45(+)HLA-DR(+) cells: CD1a(+)CD14(-) DC,CD1a(-)CD14(+) DC,and CD1a(-)CD14(+)FXIIIa(+) macrophages. In vitro,each subset has characteristic properties. After transplantation,both CD1a(+) and CD14(+) DC are rapidly depleted and replaced by donor cells,but recipient macrophages can be found in GVHD lesions and may persist for many months. Macrophages isolated from normal dermis secrete proinflammatory cytokines. Although they stimulate little proliferation of naive or memory CD4(+) T cells,macrophages induce cytokine expression in memory CD4(+) T cells and activation and proliferation of CD8(+) T cells. These observations suggest that dermal macrophages and DC are from distinct lineages and that persistent recipient macrophages,although unlikely to initiate alloreactivity,may contribute to GVHD by sustaining the responses of previously activated T cells. View Publication

Various combinations of antibodies directed to cell surface markers have been used to isolate human and rhesus macaque hematopoietic stem cells (HSCs). These protocols result in poor enrichment or require multiple complex steps. Recently,a simple phenotype for HSCs based on cell surface markers from the signaling lymphocyte activation molecule (SLAM) family of receptors has been reported in the mouse. We examined the possibility of using the SLAM markers to facilitate the isolation of highly enriched populations of HSCs in humans and rhesus macaques. We isolated SLAM (CD150(+)CD48(-)) and non-SLAM (not CD150(+)CD48(-)) cells from human umbilical cord blood CD34(+) cells as well as from human and rhesus macaque mobilized peripheral blood CD34(+) cells and compared their ability to form colonies in vitro and reconstitute immune-deficient (nonobese diabetic/severe combined immunodeficiency/interleukin-2 γc receptor(null),NSG) mice. We found that the CD34(+) SLAM population contributed equally or less to colony formation in vitro and to long-term reconstitution in NSG mice compared with the CD34(+) non-SLAM population. Thus,SLAM family markers do not permit the same degree of HSC enrichment in humans and rhesus macaques as in mice. View Publication

Recent population studies provide clues that the use of metformin may be associated with reduced incidence and improved prognosis of certain cancers. This drug is widely used in the treatment of type 2 diabetes,where it is often referred to as an insulin sensitizer" because it not only lowers blood glucose but also reduces the hyperinsulinemia associated with insulin resistance. As insulin and insulin-like growth factors stimulate proliferation of many normal and transformed cell types� View Publication

OBJECTIVES: Tolerogenic dendritic cells (tolDCs) constitute a promising experimental treatment for targeting autoreactive T cells in autoimmune diseases,including rheumatoid arthritis (RA). The authors' goal is to bring tolDC therapy for RA to the clinic. Here the authors address key translational issues related to the manufacturing of tolDCs from RA patients with current good manufacturing practice (cGMP)-compliant reagents,the stability of tolDCs,and the selection of suitable quality control markers. METHODS: Human monocyte-derived tolDCs were established from RA patients and healthy controls (HCs) using the immunosuppressive drugs dexamethasone and vitamin D₃,and the cGMP-grade immunomodulator,monophosphoryl lipid A,in the cGMP-compliant medium,CellGroDC. The functionality of tolDCs and tolDC-modulated autologous CD4 T cells was determined by flow cytometry,[³H]thymidine incorporation and ELISA. RESULTS: Clinical-grade tolDCs established from patients with RA exhibit a typical tolerogenic phenotype of reduced costimulatory molecules,low production of proinflammatory cytokines and impaired stimulation of autologous antigen-specific T cells,comparable to HC tolDCs. Toll-like receptor 2 (TLR-2) was highly expressed by tolDCs but not mature DCs. Furthermore,tolDCs suppressed mature DC-induced T cell proliferation,interferon γ and interleukin 17 production,and rendered T cells hyporesponsive to further stimulation. Importantly,tolDCs were phenotypically stable in the absence of immunosuppressive drugs and were refractory to further challenge with proinflammatory mediators. CONCLUSIONS: tolDCs established from patients with RA are comparable to those derived from healthy donors. TLR-2 was identified as an ideal marker for quality control of tolDCs. Potently tolerogenic and highly stable,these tolDCs are a promising cellular therapeutic for tailored immunomodulation in the treatment of RA. View Publication