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

Background Proteoglycans are found on the cell surface and in the extracellular matrix,and serve as prime sites for interaction with signaling molecules. Proteoglycans help regulate pathways that control stem cell fate,and therefore represent an excellent tool to manipulate these pathways. Despite their importance,there is a dearth of data linking glycosaminoglycan structure within proteoglycans with stem cell differentiation. Methods Human embryonic stem cell line WA09 (H9) was differentiated into early mesoderm and endoderm lineages,and the glycosaminoglycanomic changes accompanying these transitions were studied using transcript analysis,immunoblotting,immunofluorescence and disaccharide analysis. Results Pluripotent H9 cell lumican had no glycosaminoglycan chains whereas in splanchnic mesoderm lumican was glycosaminoglycanated. H9 cells have primarily non-sulfated heparan sulfate chains. On differentiation towards splanchnic mesoderm and hepatic lineages N-sulfo group content increases. Differences in transcript expression of NDST1,HS6ST2 and HS6ST3,three heparan sulfate biosynthetic enzymes,within splanchnic mesoderm cells compared to H9 cells correlate to changes in glycosaminoglycan structure. Conclusions Differentiation of embryonic stem cells markedly changes the proteoglycanome. General significance The glycosaminoglycan biosynthetic pathway is complex and highly regulated,and therefore,understanding the details of this pathway should enable better control with the aim of directing stem cell differentiation. ?? 2014 Elsevier B.V. All rights reserved. View Publication

Mesenchymal stem cells (MSCs) are being tested in a wide range of human diseases; however,loss of potency and inconsistent quality severely limit their use. To overcome these issues,we have utilized a developmental precursor called the hemangioblast as an intermediate cell type in the derivation of a highly potent and replenishable population of MSCs from human embryonic stem cells (hESCs). This method circumvents the need for labor-intensive hand-picking,scraping,and sorting that other hESC-MSC derivation methods require. Moreover,unlike previous reports on hESC-MSCs,we have systematically evaluated their immunomodulatory properties and in vivo potency. As expected,they dynamically secrete a range of bioactive factors,display enzymatic activity,and suppress T-cell proliferation that is induced by either allogeneic cells or mitogenic stimuli. However,they also display unique immunophenotypic properties,as well as a smaller size and textgreater30,000-fold proliferative capacity than bone marrow-derived MSCs. In addition,this is the first report which demonstrates that hESC-MSCs can inhibit CD83 up-regulation and IL-12p70 secretion from dendritic cells and enhance regulatory T-cell populations induced by interleukin 2 (IL-2). This is also the first report which shows that hESC-MSCs have therapeutic efficacy in two different autoimmune disorder models,including a marked increase in survival of lupus-prone mice and a reduction of symptoms in an autoimmune model of uveitis. Our data suggest that this novel and therapeutically active population of MSCs could overcome many of the obstacles that plague the use of MSCs in regenerative medicine and serve as a scalable alternative to current MSC sources. View Publication

There is an urgent need for new and advanced approaches to modeling the pathological mechanisms of complex human neurological disorders. This is underscored by the decline in pharmaceutical research and development efficiency resulting in a relative decrease in new drug launches in the last several decades. Induced pluripotent stem cells represent a new tool to overcome many of the shortcomings of conventional methods,enabling live human neural cell modeling of complex conditions relating to aberrant neurodevelopment,such as schizophrenia,epilepsy and autism as well as age-associated neurodegeneration. This review considers the current status of induced pluripotent stem cell-based modeling of neurological disorders,canvassing proven and putative advantages,current constraints,and future prospects of next-generation culture systems for biomedical research and translation. View Publication

3D suspension culture is generally considered a promising method to achieve efficient expansion and controlled differentiation of human pluripotent stem cells (hPSCs). In this work,we focused on developing an integrated culture platform for expansion and neural commitment of hPSCs into neural precursors using 3D suspension conditions and chemically-defined culture media. We evaluated different inoculation methodologies for hPSC expansion as 3D aggregates and characterized the resulting cultures in terms of aggregate size distribution. It was demonstrated that upon single-cell inoculation,after four days of culture,3D aggregates were composed of homogenous populations of hPSC and were characterized by an average diameter of 139 ± 26 μm,which was determined to be the optimal size to initiate neural commitment. Temporal analysis revealed that upon neural specification it is possible to maximize the percentage of neural precursor cells expressing the neural markers Sox1 and Pax6 after nine days of culture. These results highlight our ability to define a robust method for production of hPSC-derived neural precursors that minimizes processing steps and that constitutes a promising alternative to the traditional planar adherent culture system due to a high potential for scaling-up. View Publication

Staphylococcus aureus (S. aureus) is a human pathogen as well as a frequent colonizer of skin and mucosa. This bacterium potently activates conventional T-cells through superantigens and it is suggested to induce T-cell cytokine-production as well as to promote a regulatory phenotype in T-cells in order to avoid clearance. This study aimed to investigate how S. aureus impacts the production of regulatory and pro-inflammatory cytokines and the expression of CD161 and HELIOS by peripheral CD4(+)FOXP3(+) T-cells. Stimulation of PBMC with S. aureus 161:2-cell free supernatant (CFS) induced expression of IL-10,IFN-γ and IL-17A in FOXP3(+) cells. Further,CD161 and HELIOS separated the FOXP3(+) cells into four distinct populations regarding cytokine-expression. Monocyte-depletion decreased S. aureus 161:2-induced activation of FOXP3(+) cells while pre-stimulation of purified monocytes with S. aureus 161:2-CFS and subsequent co-culture with autologous monocyte-depleted PBMC was sufficient to mediate activation of FOXP3(+) cells. Together,these data show that S. aureus potently induces FOXP3(+) cells and promotes a diverse phenotype with expression of regulatory and pro-inflammatory cytokines connected to increased CD161-expression. This could indicate potent regulation or a contribution of FOXP3(+) cells to inflammation and repression of immune-suppression upon encounter with S. aureus. View Publication

Human pluripotent stem cells (hPSCs) offer a renewable source of cells that can be expanded indefinitely and differentiated into virtually any type of cell in the human body,including neurons. This opens up unprecedented possibilities to study neuronal cell and developmental biology and cellular pathology of the nervous system,provides a platform for the screening of chemical libraries that affect these processes,and offers a potential source of transplantable cells for regenerative approaches to neurological disease. However,defining protocols that permit a large number and high yield of neurons has proved difficult. We present differentiation protocols for the generation of distinct subtypes of neurons in a highly reproducible manner,with minimal experiment-to-experiment variation. These neurons form synapses with neighboring cells,exhibit spontaneous electrical activity,and respond appropriately to depolarization. hPSC-derived neurons exhibit a high degree of maturation and survive in culture for up to 4-5 months,even without astrocyte feeder layers. View Publication

Current neural induction protocols for human embryonic stem (hES) cells rely on embryoid body formation,stromal feeder co-culture or selective survival conditions. Each strategy has considerable drawbacks,such as poorly defined culture conditions,protracted differentiation and low yield. Here we report that the synergistic action of two inhibitors of SMAD signaling,Noggin and SB431542,is sufficient to induce rapid and complete neural conversion of textgreater80% of hES cells under adherent culture conditions. Temporal fate analysis reveals the appearance of a transient FGF5(+) epiblast-like stage followed by PAX6(+) neural cells competent to form rosettes. Initial cell density determines the ratio of central nervous system and neural crest progeny. Directed differentiation of human induced pluripotent stem (hiPS) cells into midbrain dopamine and spinal motoneurons confirms the robustness and general applicability of the induction protocol. Noggin/SB431542-based neural induction should facilitate the use of hES and hiPS cells in regenerative medicine and disease modeling and obviate the need for protocols based on stromal feeders or embryoid bodies. View Publication

Bacillus anthracis secretes two bipartite toxins,edema toxin (ET) and lethal toxin (LT),which impair immune responses and contribute directly to the pathology associated with the disease anthrax. Edema factor,the catalytic subunit of ET,is an adenylate cyclase that impairs host defenses by raising cellular cyclic AMP (cAMP) levels. Synthetic cAMP analogues and compounds that raise intracellular cAMP levels lead to phenotypic and functional changes in dendritic cells (DCs). Here,we demonstrate that ET induces a maturation state in human monocyte-derived DCs (MDDCs) similar to that induced by lipopolysaccharide (LPS). ET treatment results in downregulation of DC-SIGN,a marker of immature DCs,and upregulation of DC maturation markers CD83 and CD86. Maturation of DCs by ET is accompanied by an increased ability to migrate toward the lymph node-homing chemokine macrophage inflammatory protein 3beta,like LPS-matured DCs. Interestingly,cotreating with LT differentially affects the ET-induced maturation of MDDCs while not inhibiting ET-induced migration. These findings reveal a mechanism by which ET impairs normal innate immune function and may explain the reported adjuvant effect of ET. View Publication

BACKGROUND: Human tonsils are a rich source of B lymphocytes exhibiting a variety of phenotypes and activation states. Existing methods of purification are time consuming or costly. The aim of the present study was to optimize conditions to isolate large numbers of highly purified primary B lymphocytes from tonsils in a short and cost-effective single step,using a commercially available reagent designed for purifying cells from whole blood (RosetteSep). This technique relies on the presence of the large excess of red blood cells in whole blood for the formation of immunorosettes,whereas single cell suspensions from tonsils contain relatively few red blood cells. RESULTS: B cell enrichment from tonsils was achieved using RosetteSep with no modification to the whole blood procedure; however,the degree of purity depended on the extent of red blood cell contamination of the starting tonsil cell suspension. Addition of a 50-fold excess of allogeneic human red blood cells,but not sheep red blood cells,reproducibly resulted in high levels of purity. Depletion of mononuclear cells from the donor red blood cells eliminated potential contamination with allogeneic B cells. CONCLUSION: RosetteSep reagent can be used in combination with allogeneic human red blood cells to reproducibly isolate tonsil B lymphocytes to high levels of purity with no change in phenotype or loss of cells. This method provides considerable time and cost savings compared to other methods. View Publication

Bone marrow-derived mesenchymal stromal cells (MSC) possess an immune plasticity manifested by either an immunosuppressive or,when activated with IFN-gamma,an APC phenotype. Herein,TLR expression by MSC and their immune regulatory role were investigated. We observed that human MSC and macrophages expressed TLR3 and TLR4 at comparable levels and TLR-mediated activation of MSC resulted in the production of inflammatory mediators such as IL-1beta,IL-6,IL-8/CXCL8,and CCL5. IFN-alpha or IFN-gamma priming up-regulated production of these inflammatory mediators and expression of IFNB,inducible NO synthase (iNOS),and TRAIL upon TLR activation in MSC and macrophages,but failed to induce IL-12 and TNF-alpha production in MSC. Nonetheless,TLR activation in MSC resulted in the formation of an inflammatory site attracting innate immune cells,as evaluated by human neutrophil chemotaxis assays and by the analysis of immune effectors retrieved from Matrigel-embedded MSC injected into mice after in vitro preactivation with cytokines and/or TLR ligands. Hence,TLR-activated MSC are capable of recruiting immune inflammatory cells. In addition,IFN priming combined with TLR activation may increase immune responses induced by Ag-presenting MSC through presentation of Ag in an inflammatory context,a mechanism that could be applied in a cell-based vaccine. View Publication

Previous reports have shown that antidepressants increase neuronal cell proliferation and enhance neuroplasticity both in vivo and in vitro. This study investigated the direct effects of one such antidepressant,fluoxetine,on cell proliferation and on the production of neurotrophic factors in neuronal precursors derived from human embryonic stem cells (hESCs; H9). Fluoxetine induced the differentiation of neuronal precursors,strongly enhancing neuronal characteristics. The rate of proliferation was higher in fluoxetine -treated cells than in control cells,as determined by MTT [3(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide] assay. The CPDL (cumulative population doubling level) of the fluoxetine-treated cells was significantly increased in comparison to that of control cells (ptextless.001). Bromodeoxyuridine incorporation and staurosporine-induced apoptosis assays were elevated in fluoxetine-treated cells. Quantitative RT-PCR analysis revealed no significant differences in the expression of neurotrophic factors,brain-derived neurotrophic factor (BDNF);glial-derived neurotrophic factor (GDNF) and cAMP-responsive element-binding protein (CREB) between cells treated with fluoxetine for two weeks and their untreated counterparts. These results may help elucidate the mechanism of action of fluoxetine as a therapeutic drug for the treatment of depression. Data presented herein provide more evidence that,in addition to having a direct chemical effect on serotonin levels,fluoxetine can influence hESC-derived neuronal cells by increasing cell proliferation,while allowing them to maintain their neuronal characteristics. View Publication

hESCs (human embryonic stem cells) have enormous potential for use in pharmaceutical development and therapeutics; however,to realize this potential,there is a requirement for simple and reproducible cell culture methods that provide adequate numbers of cells of suitable quality. We have discovered a novel way of blocking the spontaneous differentiation of hESCs in the absence of exogenous cytokines by supplementing feeder-free conditions with EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine],an established inhibitor of ADA (adenosine deaminase) and cyclic nucleotide PDE2 (phosphodiesterase 2). hESCs maintained in feeder-free conditions with EHNA for more than ten passages showed no reduction in hESC-associated markers including NANOG,POU5F1 (POU domain class 5 transcription factor 1,also known as Oct-4) and SSEA4 (stage-specific embryonic antigen 4) compared with cells maintained in feeder-free conditions containing bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA,but,upon removing EHNA,hESC populations underwent efficient spontaneous,multi-lineage and directed differentiation. EHNA also acts as a strong blocker of directed neuronal differentiation. Chemically distinct inhibitors of ADA and PDE2 lacked the capacity of EHNA to suppress hESC differentiation,suggesting that the effect is not driven by inhibition of either ADA or PDE2. Preliminary structure-activity relationship analysis found the differentiation-blocking properties of EHNA to reside in a pharmacophore comprising a close adenine mimetic with an extended hydrophobic substituent in the 8- or 9-position. We conclude that EHNA and simple 9-alkyladenines can block directed neuronal and spontaneous differentiation in the absence of exogenous cytokine addition,and may provide a useful replacement for bFGF in large-scale or cGMP-compliant processes. View Publication