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

Bone marrow (BM) failure syndrome encompasses a group of disorders characterized by BM stem cell dysfunction,resulting in varying degrees of hypoplasia and blood pancytopenia,and in many patients is autoimmune and inflammatory in nature. The important role of T helper 1 (Th1) polarized CD4+ T cells in driving BM failure has been clearly established in several models. However,animal model data demonstrating a functional role for CD8+ T cells in BM dysfunction is largely lacking and our objective was to test the hypothesis that CD8+ T cells play a non-redundant role in driving BM failure. Clinical evidence implicates a detrimental role for CD8+ T cells in BM failure and a beneficial role for Foxp3+ regulatory T cells (Tregs) in maintaining immune tolerance in the BM. We demonstrate that IL-2-deficient mice,which have a deficit in functional Tregs,develop spontaneous BM failure. Furthermore,we demonstrate a critical role for CD8+ T cells in the development of BM failure,which is dependent on the cytokine,IFNgamma$. CD8+ T cells promote hematopoietic stem cell dysfunction and depletion of myeloid lineage progenitor cells,resulting in anemia. Adoptive transfer experiments demonstrate that CD8+ T cells dramatically expedite disease progression and promote CD4+ T cell accumulation in the BM. Thus,BM dysregulation in IL-2-deficient mice is mediated by a Th1 and IFNgamma$-producing CD8+ T cell (Tc1) response. View Publication

Human embryonic stem cells (hESCs) offer exciting potential in regenerative medicine for the treatment of a host of diseases including cancer,Alzheimer's and Parkinson's disease. They also provide insight into human development and disease and can be used as models for drug discovery and toxicity analyses. The key properties of hESCs that make them so promising for medical use are that they have the ability to self-renew indefinitely in culture and they are pluripotent,which means that they can differentiate into any of more than 200 human cell types. Since proteins are the effectors of cellular processes,it is important to investigate hESC expression at the protein level as well as at the transcript level. In addition,post-translational modifications,such as phosphorylation,may influence the activity of pivotal proteins in hESCs,and this information can only be determined by studying the proteome. In this review,we summarize the results obtained from several proteomics analyses of hESCs that have been reported in the last few years. View Publication

Cryopreservation is the use of low temperatures to preserve structurally intact living cells. The cells that survive the thermodynamic journey from the 37 °C incubator to the -196 °C liquid nitrogen storage tank are free from the influences of time. Thus,cryopreservation is a critical component of cell culture and cell manufacturing protocols. Successful cryopreservation of human cells requires that the cells be derived from patient samples that are collected in a standardized manner,and carefully handled from blood draw through cell isolation. Furthermore,proper equipment must be in place to ensure consistency,reproducibility,and sterility. In addition,the correct choice and amount of cryoprotectant agent must be added at the correct temperature,and a controlled rate of freezing (most commonly 1 °C/min) must be applied prior to a standardized method of cryogenic storage. This appendix describes how human primary cells can be frozen for long-term storage and thawed for growth in a tissue culture vessel. View Publication

Characterization of pluripotent stem cells is required for the registration of stem cell lines and allows for an impartial and objective comparison of the results obtained when generating multiple lines. It is therefore crucial to establish specific,fast and reliable protocols to detect the hallmarks of pluripotency. Such protocols should include immunocytochemistry (takes 2 d),identification of the three germ layers in in vitro-derived embryoid bodies by immunocytochemistry (immunodetection takes 3 d) and detection of differentiation markers in in vivo-generated teratomas by immunohistochemistry (differentiation marker detection takes 4 d). Standardization of the immunodetection protocols used ensures minimum variations owing to the source,the animal species,the endogenous fluorescence or the inability to collect large amounts of cells,thereby yielding results as fast as possible without loss of quality. This protocol provides a description of all the immunodetection procedures necessary to characterize mouse and human stem cell lines in different circumstances.

Epithelial-to-mesenchymal transition (EMT) is strongly associated with cancer progression,but its potential role during premalignant development has not been studied. Here,we show that a 4-week exposure of immortalized human bronchial epithelial cells (HBEC) to tobacco carcinogens can induce a persistent,irreversible,and multifaceted dedifferentiation program marked by EMT and the emergence of stem cell-like properties. EMT induction was epigenetically driven,initially by chromatin remodeling through H3K27me3 enrichment and later by ensuing DNA methylation to sustain silencing of tumor-suppressive microRNAs (miRNA),miR-200b,miR-200c,and miR-205,which were implicated in the dedifferentiation program in HBECs and also in primary lung tumors. Carcinogen-treated HBECs acquired stem cell-like features characterized by their ability to form spheroids with branching tubules and enrichment of the CD44(high)/CD24(low),CD133,and ALDH1 stem cell-like markers. miRNA overexpression studies indicated that regulation of the EMT,stem-like,and transformed phenotypes in HBECs were distinct events. Our findings extend present concepts of how EMT participates in cancer pathophysiology by showing that EMT induction can participate in cancer initiation to promote the clonal expansion of premalignant lung epithelial cells. View Publication

NK cells express different TLRs,such as TLR3,TLR7,and TLR9,but little is known about their role in NK cell stimulation. In this study,we used specific agonists (poly(I:C),loxoribine,and synthetic oligonucleotides containing unmethylated CpG sequences to stimulate human NK cells without or with suboptimal doses of IL-12,IL-15,or IFN-alpha,and investigated the secretion of IFN-gamma,cytotoxicity,and expression of the activating receptor NKG2D. Poly(I:C) and loxoribine,in conjunction with IL-12,but not IL-15,triggered secretion of IFN-gamma. Inhibition of IFN-gamma secretion by chloroquine suggested that internalization of the TLR agonists was necessary. Also,secretion of IFN-gamma was dependent on MEK1/ERK,p38 MAPK,p70(S6) kinase,and NF-kappaB,but not on calcineurin. IFN-alpha induced a similar effect,but promoted lesser IFN-gamma secretion. However,cytotoxicity (51Cr release assays) against MHC class I-chain related A (MICA)- and MICA+ tumor targets remained unchanged,as well as the expression of the NKG2D receptor. Excitingly,IFN-gamma secretion was significantly increased when NK cells were stimulated with poly(I:C) or loxoribine and IL-12,and NKG2D engagement was induced by coculture with MICA+ tumor cells in a PI3K-dependent manner. We conclude that resting NK cells secrete high levels of IFN-gamma in response to agonists of TLR3 or TLR7 and IL-12,and this effect can be further enhanced by costimulation through NKG2D. Hence,integration of the signaling cascades that involve TLR3,TLR7,IL-12,and NKG2D emerges as a critical step to promote IFN-gamma-dependent NK cell-mediated effector functions,which could be a strategy to promote Th1-biased immune responses in pathological situations such as cancer. View Publication

Most polyreactive and antinuclear antibodies are removed from the human antibody repertoire during B cell development. To elucidate how B cell receptor (BCR) signaling may regulate human B cell tolerance,we tested the specificity of recombinant antibodies from single peripheral B cells isolated from patients suffering from X-linked agammaglobulinemia (XLA). These patients carry mutations in the Bruton's tyrosine kinase (BTK) gene that encode an essential BCR signaling component. We find that in the absence of Btk,peripheral B cells show a distinct antibody repertoire consistent with extensive secondary V(D)J recombination. Nevertheless,XLA B cells are enriched in autoreactive clones. Our results demonstrate that Btk is essential in regulating thresholds for human B cell tolerance. View Publication

NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5(eGFP/w) hESCs facilitate quantification of cardiac differentiation,purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP(+) cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages. View Publication

BACKGROUND: Hematopoietic progenitors are generated in the yolk sac and aorta-gonad-mesonephros region during early mouse development. At embryonic day 10.5 the first hematopoietic stem cells emerge in the aorta-gonad-mesonephros. Subsequently,hematopoietic stem cells and progenitors are found in the fetal liver. The fetal liver is a potent hematopoietic site,playing an important role in the expansion and differentiation of hematopoietic progenitors and hematopoietic stem cells. However,little is known concerning the regulation of fetal liver hematopoietic stem cells. In particular,the role of cytokines such as interleukin-1 in the regulation of hematopoietic stem cells in the embryo has been largely unexplored. Recently,we observed that the adult pro-inflammatory cytokine interleukin-1 is involved in regulating aorta-gonad-mesonephros hematopoietic progenitor and hematopoietic stem cell activity. Therefore,we set out to investigate whether interleukin-1 also plays a role in regulating fetal liver progenitor cells and hematopoietic stem cells. DESIGN AND METHODS: We examined the interleukin-1 ligand and receptor expression pattern in the fetal liver. The effects of interleukin-1 on hematopoietic progenitor cells and hematopoietic stem cells were studied by FACS and transplantation analyses of fetal liver explants,and in vivo effects on hematopoietic stem cell and progenitors were studied in Il1r1(-/-) embryos. RESULTS: We show that fetal liver hematopoietic progenitor cells express the IL-1RI and that interleukin-1 increases fetal liver hematopoiesis,progenitor cell activity and promotes hematopoietic cell survival. Moreover,we show that in Il1r1(-/-) embryos,hematopoietic stem cell activity is impaired and myeloid progenitor activity is increased. CONCLUSIONS: The IL-1 ligand and receptor are expressed in the midgestation liver and act in the physiological regulation of fetal liver hematopoietic progenitor cells and hematopoietic stem cells. View Publication