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

The propagation of pluripotent mouse embryonic stem (ES) cells depends on signals transduced through the cytokine receptor subunit gp130. Signalling molecules activated downstream of gp130 in ES cells include STAT3,the protein tyrosine phosphatase SHP-2,and the mitogen-activated protein kinases,ERK1 and ERK2. A chimaeric receptor in which tyrosine 118 in the gp130 cytoplasmic domain was mutated did not engage SHP-2 and failed to activate ERKs. However,this receptor did support ES cell self-renewal. In fact,stem cell colonies formed at 100-fold lower concentrations of cytokine than the unmodified receptor. Moreover,altered ES cell morphology and growth were observed at high cytokine concentrations. These indications of deregulated signalling in the absence of tyrosine 118 were substantiated by sustained activation of STAT3. Confirmation that ERK activation is not required for self-renewal was obtained by propagation of pluripotent ES cells in the presence of the MEK inhibitor PD098059. In fact,the growth of undifferentiated ES cells was enhanced by culture in PD098059. Thus activation of ERKs appears actively to impair self-renewal. These data imply that the self-renewal signal from gp130 is a finely tuned balance of positive and negative effectors. View Publication

Chemokine stromal derived factor 1 (SDF-1) is involved in trafficking of hematopoietic stem cells (HSCs) from the bone marrow (BM) to peripheral blood (PB) and has been found to enhance postischemia angiogenesis. This study was aimed at investigating whether SDF-1 plays a role in differentiation of BM-derived c-kit(+) stem cells into endothelial progenitor cells (EPCs) and in ischemia-induced trafficking of stem cells from PB to ischemic tissues. We found that SDF-1 enhanced EPC number by promoting alpha(2),alpha(4),and alpha(5) integrin-mediated adhesion to fibronectin and collagen I. EPC differentiation was reduced in mitogen-stimulated c-kit(+) cells,while cytokine withdrawal or the overexpression of the cyclin-dependent kinase (CDK) inhibitor p16(INK4) restored such differentiation,suggesting a link between control of cell cycle and EPC differentiation. We also analyzed the time course of SDF-1 expression in a mouse model of hind-limb ischemia. Shortly after femoral artery dissection,plasma SDF-1 levels were up-regulated,while SDF-1 expression in the bone marrow was down-regulated in a timely fashion with the increase in the percentage of PB progenitor cells. An increase in ischemic tissue expression of SDF-1 at RNA and protein level was also observed. Finally,using an in vivo assay such as injection of matrigel plugs,we found that SDF-1 improves formation of tubulelike structures by coinjected c-kit(+) cells. Our findings unravel a function for SDF-1 in increase of EPC number and formation of vascular structures by bone marrow progenitor cells. View Publication

This protocol describes detailed procedures for the fluorescent high-throughput screening of small molecules that induce neurogenesis in cultures of skeletal muscle cells. The detection of neurogenesis relies on a fluorescent dye,FM 1-43,which is used to study the neuronal property of depolarization-induced synaptic vesicle recycling. Thus,small molecules with neurogenesis-inducing activity in skeletal muscle cells can be rapidly identified by measuring the fluorescence intensity of the treated cells using a fluorescent microplate reader. This protocol uses murine myoblast C2C12 cells for screening,which are readily available and relatively easy to culture. Neurogenesis of PC12 cells induced by nerve growth factor is employed as a positive control for this screening. The screening time for this protocol is 8 d,which also includes the procedure to detect depolarization-induced synaptic vesicle recycling using FM 1-43. View Publication

Differentiation of embryonic and adult myogenic progenitors undergoes a complex series of cell rearrangements and specification events which are controlled by distinct gene regulatory networks. Delineation of the molecular mechanisms that regulate skeletal muscle specification and formation should be important for understanding congenital myopathies and muscular degenerative diseases. Retinoic acid (RA) signaling plays an important role in development. However,the role of RA signaling in adult myogenic progenitors is poorly understood. Here,we investigate the role of RA signaling in regulating myogenic differentiation of myoblastic progenitor cells. Using the mouse myoblast progenitor C2C12 line as a model,we have found that the endogenous expression of most RAR and RXR isotypes is readily detected. While the nuclear receptor co-repressors are highly expressed,two of the three nuclear receptor co-activators and the enzymes involved in RA synthesis are expressed at low level or undetectable,suggesting that the RA signaling pathway may be repressed in myogenic progenitors. Using the alpha-myosin heavy chain promoter-driven reporter (MyHC-GLuc),we have demonstrated that either ATRA or 9CRA is able to effectively induce myogenic differentiation,which can be synergistically enhanced when both ATRA and 9CRA are used. Upon ATRA and 9CRA treatment of C2C12 cells the expression of late myogenic markers significantly increases. We have further shown that adenovirus-mediated exogenous expression of RARalpha and/or RXRalpha is able to effectively induce myogenic differentiation in a ligand-independent fashion. Morphologically,ATRA- and 9CRA-treated C2C12 cells exhibit elongated cell body and become multi-nucleated myoblasts,and even form myoblast fusion. Ultrastructural analysis under transmission electron microscope reveals that RA-treated myogenic progenitor cells exhibit an abundant presence of muscle fibers. Therefore,our results strongly suggest that RA signaling may play an important role in regulating myogenic differentiation. View Publication

To date,studies on T cells in acute myeloid leukemia (AML) have been limited to flow cytometric analysis of whole peripheral blood mononuclear cell (PBMC) specimens or functional work looking at the impact of AML myeloblasts on normal or remission T cells. This lack of information on T cells at the time of presentation with disease is due in part to the difficulty in isolating sufficiently pure T cells from these specimens for further study. Negative immunomagnetic selection has been the method of choice for isolating immune cells for functional studies due to concerns that binding antibodies to the cell surface may induce cellular activation,block ligand-receptor interactions or result in immune clearance. In order specifically to study T cells in presentation AML specimens,we set out to develop a method of isolating highly pure CD4 and CD8 T cells by negative selection from the peripheral blood (PB) of newly diagnosed AML patients. This technique,unlike T cell selection from PB from normal individuals or from patients with chronic lymphocytic leukaemia,was extremely problematic due to properties of the leukaemic myeloblasts. A successful method was eventually optimized requiring the use of a custom antibody cocktail consisting of CD33,CD34,CD123,CD11c and CD36,to deplete myeloblasts. View Publication

Interleukin 17 (IL-17)-producing CD4(+) helper T cells (T(H)-17 cells) share a developmental relationship with Foxp3(+) regulatory T cells (T(reg) cells). Here we show that a T(H)-17 population differentiates in the thymus in a manner influenced by recognition of self antigen and by the cytokines IL-6 and transforming growth factor-beta (TGF-beta). Like previously described T(H)-17 cells,the T(H)-17 cells that developed in the thymus expressed the transcription factor RORgamma t and the IL-23 receptor. These cells also expressed alpha(4)beta(1) integrins and the chemokine receptor CCR6 and were recruited to the lung,gut and liver. In the liver,these cells secreted IL-22 in response to self antigen and mediated host protection during inflammation. Thus,T(H)-17 cells,like T(reg) cells,can be selected by self antigens in the thymus. View Publication

A hallmark feature of glioblastoma is its strong self-renewal potential and immature differentiation state,which contributes to its plasticity and therapeutic resistance. Here,integrated genomic and biological analyses identified PLAGL2 as a potent protooncogene targeted for amplification/gain in malignant gliomas. Enhanced PLAGL2 expression strongly suppresses neural stem cell (NSC) and glioma-initiating cell differentiation while promoting their self-renewal capacity upon differentiation induction. Transcriptome analysis revealed that these differentiation-suppressive activities are attributable in part to PLAGL2 modulation of Wnt/beta-catenin signaling. Inhibition of Wnt signaling partially restores PLAGL2-expressing NSC differentiation capacity. The identification of PLAGL2 as a glioma oncogene highlights the importance of a growing class of cancer genes functioning to impart stem cell-like characteristics in malignant cells. View Publication

Mesenchymal stromal cells (MSCs) may be derived from a variety of tissues,with human umbilical cord (UC) providing an abundant and noninvasive source. Human UC-MSCs share similar in vitro immunosuppressive properties as MSCs obtained from bone marrow and cord blood. However,the mechanisms and cellular interactions used by MSCs to control immune responses remain to be fully elucidated. In this paper,we report that suppression of mitogen-induced T cell proliferation by human UC-,bone marrow-,and cord blood-MSCs required monocytes. Removal of monocytes but not B cells from human adult PBMCs (PBMNCs) reduced the immunosuppressive effects of MSCs on T cell proliferation. There was rapid modulation of a number of cell surface molecules on monocytes when PBMCs or alloantigen-activated PBMNCs were cultured with UC-MSCs. Indomethacin treatment significantly inhibited the ability of UC-MSCs to suppress T cell proliferation,indicating an important role for PGE(2). Monocytes purified from UC-MSC coculture had significantly reduced accessory cell and allostimulatory function when tested in subsequent T cell proliferation assays,an effect mediated in part by UC-MSC PGE(2) production and enhanced by PBMNC alloactivation. Therefore,we identify monocytes as an essential intermediary through which UC-MSCs mediate their suppressive effects on T cell proliferation. View Publication

The differentiation of patient-derived induced pluripotent stem cells (iPSCs) to committed fates such as neurons,muscle and liver is a powerful approach for understanding key parameters of human development and disease. Whether undifferentiated iPSCs themselves can be used to probe disease mechanisms is uncertain. Dyskeratosis congenita is characterized by defective maintenance of blood,pulmonary tissue and epidermal tissues and is caused by mutations in genes controlling telomere homeostasis. Short telomeres,a hallmark of dyskeratosis congenita,impair tissue stem cell function in mouse models,indicating that a tissue stem cell defect may underlie the pathophysiology of dyskeratosis congenita. Here we show that even in the undifferentiated state,iPSCs from dyskeratosis congenita patients harbour the precise biochemical defects characteristic of each form of the disease and that the magnitude of the telomere maintenance defect in iPSCs correlates with clinical severity. In iPSCs from patients with heterozygous mutations in TERT,the telomerase reverse transcriptase,a 50% reduction in telomerase levels blunts the natural telomere elongation that accompanies reprogramming. In contrast,mutation of dyskerin (DKC1) in X-linked dyskeratosis congenita severely impairs telomerase activity by blocking telomerase assembly and disrupts telomere elongation during reprogramming. In iPSCs from a form of dyskeratosis congenita caused by mutations in TCAB1 (also known as WRAP53),telomerase catalytic activity is unperturbed,yet the ability of telomerase to lengthen telomeres is abrogated,because telomerase mislocalizes from Cajal bodies to nucleoli within the iPSCs. Extended culture of DKC1-mutant iPSCs leads to progressive telomere shortening and eventual loss of self-renewal,indicating that a similar process occurs in tissue stem cells in dyskeratosis congenita patients. These findings in iPSCs from dyskeratosis congenita patients reveal that undifferentiated iPSCs accurately recapitulate features of a human stem cell disease and may serve as a cell-culture-based system for the development of targeted therapeutics. View Publication