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

Mast cells are tissue-resident immune cells that play a key role in inflammation and allergy. Here we show that interaction of mast cells with antibody-targeted cells induces the polarized exocytosis of their granules resulting in a sustained exposure of effector enzymes,such as tryptase and chymase,at the cell-cell contact site. This previously unidentified mast cell effector mechanism,which we name the antibody-dependent degranulatory synapse (ADDS),is triggered by both IgE- and IgG-targeted cells. ADDSs take place within an area of cortical actin cytoskeleton clearance in the absence of microtubule organizing centre and Golgi apparatus repositioning towards the stimulating cell. Remarkably,IgG-mediated degranulatory synapses also occur upon contact with opsonized Toxoplasma gondii tachyzoites resulting in tryptase-dependent parasite death. Our results broaden current views of mast cell degranulation by revealing that human mast cells form degranulatory synapses with antibody-targeted cells and pathogens for dedicated secretion and defence. View Publication

BACKGROUND This article is part of a series of publications providing formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks. We report the optimization and validation for fitness-for-purpose of automated and manual protocols for isolating peripheral blood mononuclear cells (PBMCs) from whole blood,and compare the two methods. METHODS The manual method was optimized for whole blood centrifugation speed,gradient type (Ficoll,Leucosep,CPT),and freezing method (Mr Frosty,Controlled Rate Freezing). Various parameters of the automated protocol using a CPT gradient on a Tecan liquid handler were optimized. Optimal protocols were validated in parallel for reproducibility and robustness. Optimization and validation were assessed in terms of cell yield,viability,recovery,white blood cell (WBC) subpopulation distribution,gene expression,and lymphoblastoid cell line (LCL) transformation. RESULTS An initial centrifugation of whole blood at 2000 g was considered optimal for further processing,allowing isolation of plasma and PBMCs from a single sample. The three gradients gave similar outcomes in terms of cell yield,viability,and WBC subpopulation distribution. Ficoll showed some advantages and was selected for further evaluations. Optimization of the automated protocol script using a CPT gradient gave 61% cell recovery. No significant differences in quality,quantity,and WBC subpopulation distribution were seen between the two freezing methods,and Mr. Frosty was selected. The manual and automated protocols were reproducible in terms of quantity,recovery,viability,WBC subpopulation distribution,gene expression,and LCL transformation. Most (75%-100%) of the 13 robustness parameters were accepted for both methods with an 8 h pre-centrifugation delay versus 38%-85% after 24 h. Differences identified between the automated and manual methods were not considered consequential. CONCLUSIONS We validated the first fully automated method for isolating viable PBMCs,including RNA analysis and generation of LCLs. We recommend processing within 8 h of blood collection. View Publication

We have developed a simple protocol for inducing the myocardial differentiation of human induced pluripotent stem (iPS) cells. Human iPS cell-derived embryonic bodies (EBs) were treated with a combination of activin-A,bone morphogenetic protein-4 and wnt-3a for one day in serum-free suspension culture,and were subsequently treated with noggin for three days. Thereafter,the EBs were subjected to adherent culture in media with 5% serum. All EBs were differentiated into spontaneously beating EBs,which were identified by the presence of striated muscles in transmission electron microscopy and the expression of the specific cardiomyocyte markers,NKX2-5 and TNNT2. The beating rate of the beating EBs was decreased by treatment with a rapidly activating delayed rectifier potassium current (Ikr) channel blocker,E-4031,an Ikr trafficking inhibitor,pentamidin,and a slowly activating delayed rectifier potassium current (Iks) channel blocker,chromanol 293B,and was increased by treatment with a beta-receptor agonist,isoproterenol. At a low concentration,verapamil,a calcium channel blocker,increased the beating rate of the beating EBs,while a high concentration decreased this rate. These findings suggest that the spontaneously beating EBs were myocardial cell clusters. This simple protocol for myocardial differentiation would be useful in providing a sufficient number of the beating myocardial cell clusters for studies requiring human myocardium. 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

Pluripotent stem cells are able to give rise to all cell types of the organism. There are two sources for human pluripotent stem cells: embryonic stem cells (ESCs) derived from surplus blastocysts created for in vitro fertilization and induced pluripotent stem cells (iPSCs) generated by reprogramming of somatic cells. ESCs have been an area of intense research during the past decade,and two clinical trials have been recently approved. iPSCs were created only recently,and most of the research has been focused on the iPSC generation protocols and investigation of mechanisms of direct reprogramming. The iPSC technology makes possible to derive pluripotent stem cells from any patient. However,there are a number of hurdles to be overcome before iPSCs will find a niche in practice. In this review,we discuss differences and similarities of the two pluripotent cell types and assess prospects for application of these cells in biomedicine. View Publication

Pluripotent cells represent a powerful tool for tissue regeneration,but their clinical utility is limited by their propensity to form teratomas. Little is known about their interaction with the surrounding niche following implantation and how this may be applied to promote survival and functional engraftment. In this study,we evaluated the ability of an osteogenic microniche consisting of a hydroxyapatite-coated,bone morphogenetic protein-2-releasing poly-L-lactic acid scaffold placed within the context of a macroenvironmental skeletal defect to guide in vivo differentiation of both embryonic and induced pluripotent stem cells. In this setting,we found de novo bone formation and participation by implanted cells in skeletal regeneration without the formation of a teratoma. This finding suggests that local cues from both the implanted scaffold/cell micro- and surrounding macroniche may act in concert to promote cellular survival and the in vivo acquisition of a terminal cell fate,thereby allowing for functional engraftment of pluripotent cells into regenerating tissue. View Publication

There is increasing evidence to suggest that embryonic stem cells (ESCs) are capable of differentiating into hepatocytes in vitro. In this study,we used a combination of cytokines and sodium butyrate in a novel three-step procedure to efficiently direct the differentiation of mouse ESCs into hepatocytes. Mouse ESCs were first differentiated into definitive endoderm cells by 3 days of treatment with Activin A. The definitive endoderm cells were then differentiated into hepatocytes by the addition of acidic fibroblast growth factor (aFGF) and sodium butyrate to the culture medium for 5 days. After 10 days of further in vitro maturation,the morphological and phenotypic markers of hepatocytes were characterized using immunohistochemistry,immunoblotting,and reverse transcription-polymerase chain reaction (RT-PCR). Furthermore,the cells were tested for functions associated with mature hepatocytes,including glycogen storage and indocyanine green uptake and release,and the ratio of hepatic differentiation was determined by counting the percentage of albumin-positive cells. In the presence of medium containing cytokines and sodium butyrate,numerous epithelial cells resembling hepatocytes were observed,and approximately 74% of the cells expressed the hepatic marker,albumin,after 18 days in culture. RT-PCR analysis and immunohistochemistry showed that these cells expressed adult liver cell markers,and had the abilities of glycogen storage and indocyanine green uptake and release. We have developed an efficient method for directing the differentiation of mouse ESCs into cells that exhibit the characteristics of mature hepatocytes. This technique will be useful for research into the molecular mechanisms underlying liver development,and could provide a source of hepatocytes for transplantation therapy and drug screening. View Publication

Targeted nucleases,including zinc-finger nucleases (ZFNs),transcription activator-like (TAL) effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9),have provided researchers with the ability to manipulate nearly any genomic sequence in human cells and model organisms. However,realizing the full potential of these genome-modifying technologies requires their safe and efficient delivery into relevant cell types. Unlike methods that rely on expression from nucleic acids,the direct delivery of nuclease proteins to cells provides rapid action and fast turnover,leading to fewer off-target effects while maintaining high rates of targeted modification. These features make nuclease protein delivery particularly well suited for precision genome engineering. Here we describe procedures for implementing protein-based genome editing in human embryonic stem cells and primary cells. Protocols for the expression,purification and delivery of ZFN proteins,which are intrinsically cell-permeable; TALEN proteins,which can be internalized via conjugation with cell-penetrating peptide moieties; and Cas9 ribonucleoprotein,whose nucleofection into cells facilitates rapid induction of multiplexed modifications,are described,along with procedures for evaluating nuclease protein activity. Once they are constructed,nuclease proteins can be expressed and purified within 6 d,and they can be used to induce genomic modifications in human cells within 2 d. View Publication

The atypical cannabinoid Abn-CBD improves the inflammatory status in preclinical models of several pathologies,including autoimmune diseases. However,its potential for modulating inflammation in autoimmune type 1 diabetes (T1D) is unknown. Herein we investigate whether Abn-CBD can modulate the inflammatory response during T1D onset using a mouse model of T1D (non-obese diabetic- (NOD)-mice) and of beta cell damage (streptozotocin (STZ)-injected mice). Six-week-old female NOD mice were treated with Abn-CBD (0.1-1 mg/kg) or vehicle during 12 weeks and then euthanized. Eight-to-ten-week-old male C57Bl6/J mice were pre-treated with Abn-CBD (1 mg/kg of body weight) or vehicle for 1 week,following STZ challenge,and euthanized 1 week later. Blood,pancreas,pancreatic lymph nodes (PLNs) and T cells were collected and processed for analysis. Glycemia was also monitored. In NOD mice,treatment with Abn-CBD significantly reduced the severity of insulitis and reduced the pro-inflammatory profile of CD4+ T cells compared to vehicle. Concomitantly,Abn-CBD significantly reduced islet cell apoptosis and improved glucose tolerance. In STZ-injected mice,Abn-CBD decreased circulating proinflammatory cytokines and ameliorated islet inflammation reducing intra-islet phospho-NF-$\kappa$B and TXNIP. Abn-CBD significantly reduced 2 folds intra-islet CD8+ T cells and reduced Th1/non-Th1 ratio in PLNs of STZ-injected mice. Islet cell apoptosis and intra-islet fibrosis were also significantly reduced in Abn-CBD pre-treated mice compared to vehicle. Altogether,Abn-CBD reduces circulating and intra-islet inflammation,preserving islets,thus delaying the progression of insulitis. Hence,Abn-CBD and related compounds emerge as new candidates to develop pharmacological strategies to treat the early stages of T1D. View Publication