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

Evolutionary innovations can be driven by changes in the rates of RNA translation and the emergence of new genes and small open reading frames (sORFs). In this study,we characterized the transcriptional and translational landscape of the hearts of four primate and two rodent species through integrative ribosome and transcriptomic profiling,including adult left ventricle tissues and induced pluripotent stem cell-derived cardiomyocyte cell cultures. We show here that the translational efficiencies of subunits of the mitochondrial oxidative phosphorylation chain complexes IV and V evolved rapidly across mammalian evolution. Moreover,we discovered hundreds of species-specific and lineage-specific genomic innovations that emerged during primate evolution in the heart,including 551 genes,504 sORFs and 76 evolutionarily conserved genes displaying human-specific cardiac-enriched expression. Overall,our work describes the evolutionary processes and mechanisms that have shaped cardiac transcription and translation in recent primate evolution and sheds light on how these can contribute to cardiac development and disease. Ruiz-Orera et al. used comparative transcriptomics and translatomics to analyze the cardiac evolution in primates and discovered species-specific and lineage-specific genomic innovations that might contribute to cardiac development and disease. View Publication

To study the role of different cytokine combinations on the proliferation and differentiation of highly purified primitive progenitor cells,a serum-free liquid culture system was used in combination with phenotypic and functional analysis of the cells produced in culture. CD34+ CD45RAlo CD71lo cells,purified from umbilical cord blood by flow cytometry and cell sorting,were selected for this study because of their high content of clonogenic cells (34%),particularly multipotent progenitors (CFU-MIX,12% of all cells). Four cytokine combinations were tested: (1) mast cell growth factor (MGF; a c-kit ligand) and interleukin-6 (IL-6); (2) MGF,IL-6,IL-3,and erythropoietin (Epo); (3) MGF,IL-6,granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-3 fusion protein (FP),macrophage colony-stimulating factor (M-CSF),and granulocyte-CSF (G-CSF); and (4) MGF,IL-6,FP,M-CSF,G-CSF,and Epo. Maximum numbers of erythroid progenitors (BFU-E,up to 55-fold increase) and mature erythroid cells were observed in the presence of MGF,IL-6,IL-3,and Epo,whereas maximum levels of myeloid progenitors (CFU-C,up to 70-fold increase) and mature myeloid cells were found in cultures supplemented with MGF,IL-6,FP,M-CSF,and G-CSF. When MGF,IL-6,FP,M-CSF,G-CSF,and Epo were present,maximum levels of both erythroid and myeloid progenitors and their progeny were observed. These results indicate that specific cytokine combinations can act directly on primitive hematopoietic cells resulting in significant expansion of progenitor cell numbers and influencing their overall patterns of proliferation and differentiation. Furthermore,the observations presented in this study suggest that the cytokine combinations used were unable to bias lineage commitment of multipotent progenitors,but rather had a permissive effect on the development of lineage-restricted clonogenic cells. View Publication

Dietary flavonoid intake has been reported to be inversely related to mortality from coronary heart disease,and the anti-atherosclerotic effect of flavonoids is considered to be due probably to their antioxidant properties. Oxidation of low density lipoprotein (LDL) has been reported to be induced by the constituent cells of the arterial wall. Accordingly,we examined the effect of pretreatment with tea flavonoids,such as theaflavin digallate,on the ability of cells to oxidize LDL. Theaflavin digallate pretreatment of macrophages or endothelial cells reduced cell-mediated LDL oxidation in a concentration- (0-400 microM) and time- (0-4 hr) dependent manner. This inhibitory effect of flavonoids on cell-mediated LDL oxidation was in the order of theaflavin digallate textgreater theaflavin textgreater or = epigallocatechin gallate textgreater epigallocatechin textgreater gallic acid. Further,we investigated the mechanisms by which flavonoids inhibited cell-mediated LDL oxidation using macrophages and theaflavin digallate. Theaflavin digallate pretreatment decreased superoxide production of macrophages and chelated iron ions significantly. These results suggest that tea flavonoids attenuate the ability of the cell to oxidize LDL,probably by reducing superoxide production in cells and chelating iron ions. View Publication

Various epidemiological studies show a positive correlation between high intake of dietary FAs and metastatic prostate cancer (CaP). Moreover,CaP metastasizes to the bone marrow,which harbors a rich source of lipids stored within adipocytes. Here,we use Fourier transform infrared (FTIR) microspectroscopy to study adipocyte biochemistry and to demonstrate that PC-3 cells uptake isotopically labeled FA [deuterated palmitic acid (D(31)-PA)] from an adipocyte. Using this vibrational spectroscopic technique,we detected subcellular locations in a single adipocyte enriched with D(31)-PA using the upsilon(as+s)(C-D)(2+3) (D(31)-PA): upsilon(as+s)(C-H)(2+3) (lipid hydrocarbon) signal. In addition,larger adipocytes were found to consist of a higher percentage of D(31)-PA of the total lipid found within the adipocyte. Following background subtraction,the upsilon(as)(C-D)(2+3) signal illuminated starved PC-3 cells cocultured with D(31)-PA-loaded adipocytes,indicating translocation of the labeled FA. This study demonstrates lipid-specific translocation between adipocytes and tumor cells and the use of FTIR microspectroscopy to characterize various biomolecular features of a single adipocyte without the requirement for cell isolation and lipid extraction. View Publication

Cell-matrix interactions are paramount for the successful repair and regeneration of damaged and diseased tissue. Since many tissues have an anisotropic architecture,it has been proposed that aligned extracellular matrix (ECM) structures in particular could guide and support the differentiation of resident mesenchymal stem and progenitor cells (MSCs). We therefore created aligned collagen type I structures using a microfluidic set-up with the aim to assess their impact on MSC growth and differentiation. In addition,we refined our aligned collagen matrices by incorporating the glycosaminoglycan (GAG) heparin to demonstrate the versatility of the applied methodology to study multiple ECM components in a single system. Our reconstituted,aligned ECM structures maintained and allowed multilineage (osteogenic/adipogenic/chondrogenic) differentiation of MSCs. Most noticeable was the observation that during osteogenesis,aligned collagen substrates choreographed ordered matrix mineralization. Likewise,myotube assembly of C2C12 cells was profoundly influenced by aligned topographic features resulting in enhanced myotube organization and length. Our results shed light on the regulation of MSCs through directional ECM structures and demonstrate the versatility of these cell culture platforms for guiding the morphogenesis of tissue types with anisotropic structures. View Publication

Triple-negative breast cancer (TNBC) has poor prognostic outcome compared with other types of breast cancer. The molecular and cellular mechanisms underlying TNBC pathology are not fully understood. Here,we report that the transcription factor BCL11A is overexpressed in TNBC including basal-like breast cancer (BLBC) and that its genomic locus is amplified in up to 38% of BLBC tumours. Exogenous BCL11A overexpression promotes tumour formation,whereas its knockdown in TNBC cell lines suppresses their tumourigenic potential in xenograft models. In the DMBA-induced tumour model,Bcl11a deletion substantially decreases tumour formation,even in p53-null cells and inactivation of Bcl11a in established tumours causes their regression. At the cellular level,Bcl11a deletion causes a reduction in the number of mammary epithelial stem and progenitor cells. Thus,BCL11A has an important role in TNBC and normal mammary epithelial cells. This study highlights the importance of further investigation of BCL11A in TNBC-targeted therapies. View Publication

Stem cells,including cancer stem cells (CSCs),require niches to maintain stemness,yet it is unclear how CSCs maintain stemness in the suboptimal environment outside their niches during invasion. Postnatal co-deletion of Pten and Trp53 in mouse neural stem cells (NSCs) leads to the expansion of these cells in their subventricular zone (SVZ) niches but fails to maintain stemness outside the SVZ. We discovered that Qki is a major regulator of NSC stemness. Qk deletion on a Pten-/-; Trp53-/- background helps NSCs maintain their stemness outside the SVZ in Nes-CreERT2; QkL/L; PtenL/L; Trp53L/L mice,which develop glioblastoma with a penetrance of 92% and a median survival time of 105 d. Mechanistically,Qk deletion decreases endolysosome-mediated degradation and enriches receptors essential for maintaining self-renewal on the cytoplasmic membrane to cope with low ligand levels outside niches. Thus,downregulation of endolysosome levels by Qki loss helps glioma stem cells (GSCs) maintain their stemness in suboptimal environments outside their niches. View Publication

Lgr5-expressing intestinal stem cells (ISCs) maintain continuous and rapid generation of the intestinal epithelium. Here we present evidence that dedifferentiation of committed enteroendocrine cells (EECs) contributes to maintenance of the epithelium under both basal conditions and in response to injury. Lineage tracing studies identified a subset of EECs that reside at +4 position for more than 2 weeks,most of which were BrdU-label-retaining cells. Under basal conditions,cells derived from these EECs grow from the bottom of the crypt to generate intestinal epithelium according to neutral drift kinetics that is consistent with dedifferentiation of mature EECs to ISCs. The lineage tracing of EECs demonstrated reserve stem cell properties in response to radiation-induced injury with the generation of reparative EEC-derived epithelial patches. Finally,the enterochromaffin (EC) cell was the predominant EEC type participating in these stem cell dynamics. These results provide novel insights into the +4 reserve ISC hypothesis,stem cell dynamics of the intestinal epithelium and novel insight in the development of EC-derived small intestinal tumors. View Publication

Many natural prion diseases of humans and animals are considered to be acquired through oral consumption of contaminated food or pasture. Determining the route by which prions establish host infection will identify the important factors that influence oral prion disease susceptibility and to which intervention strategies can be developed. After exposure,the early accumulation and replication of prions within small intestinal Peyer's patches is essential for the efficient spread of disease to the brain. To replicate within Peyer's patches,the prions must first cross the gut epithelium. M cells are specialised epithelial cells within the epithelia covering Peyer's patches that transcytose particulate antigens and microorganisms. M cell-development is dependent upon RANKL-RANK-signalling,and mice in which RANK is deleted only in the gut epithelium completely lack M cells. In the specific absence of M cells in these mice,the accumulation of prions within Peyer's patches and the spread of disease to the brain was blocked,demonstrating a critical role for M cells in the initial transfer of prions across the gut epithelium in order to establish host infection. Since pathogens,inflammatory stimuli and aging can modify M cell-density in the gut,these factors may also influence oral prion disease susceptibility. Mice were therefore treated with RANKL to enhance M cell density in the gut. We show that prion uptake from the gut lumen was enhanced in RANKL-treated mice,resulting in shortened survival times and increased disease susceptibility,equivalent to a 10-fold higher infectious titre of prions. Together these data demonstrate that M cells are the critical gatekeepers of oral prion infection,whose density in the gut epithelium directly limits or enhances disease susceptibility. Our data suggest that factors which alter M cell-density in the gut epithelium may be important risk factors which influence host susceptibility to orally acquired prion diseases. View Publication

AIMS: Mesenchymal stem cells (MSCs) are widely used for cell therapy,particularly for the treatment of ischaemic heart disease. Mechanisms underlying control of their metabolism and proliferation capacity,critical elements for their survival and differentiation,have not been fully characterized. AMP-activated protein kinase (AMPK) is a key regulator known to metabolically protect cardiomyocytes against ischaemic injuries and,more generally,to inhibit cell proliferation. We hypothesized that AMPK plays a role in control of MSC metabolism and proliferation. METHODS AND RESULTS: MSCs isolated from murine bone marrow exclusively expressed the AMPKα1 catalytic subunit. In contrast to cardiomyocytes,a chronic exposure of MSCs to hypoxia failed to induce cell death despite the absence of AMPK activation. This hypoxic tolerance was the consequence of a preference of MSC towards glycolytic metabolism independently of oxygen availability and AMPK signalling. On the other hand,A-769662,a well-characterized AMPK activator,was able to induce a robust and sustained AMPK activation. We showed that A-769662-induced AMPK activation inhibited MSC proliferation. Proliferation was not arrested in MSCs derived from AMPKα1-knockout mice,providing genetic evidence that AMPK is essential for this process. Among AMPK downstream targets proposed to regulate cell proliferation,we showed that neither the p70 ribosomal S6 protein kinase/eukaryotic elongation factor 2-dependent protein synthesis pathway nor p21 was involved,whereas p27 expression was increased by A-769662. Silencing p27 expression partially prevented the A-769662-dependent inhibition of MSC proliferation. CONCLUSION: MSCs resist hypoxia independently of AMPK whereas chronic AMPK activation inhibits MSC proliferation,p27 being involved in this regulation. View Publication

HIV-1 infection leads to numerous B cell abnormalities,including hypergammaglobulinemia,nonspecific B cell activation,nonspecific class switching,increased cell turnover,breakage of tolerance,increased immature/transitional B cells,B cell malignancies,as well as a loss of capacity to generate and maintain memory,all of which contribute to a global impairment of the immune humoral compartment. Several cytokines and soluble factors,which are increased in sera of HIV-1-infected individuals,have been suggested to directly or indirectly contribute to these B cell dysfunctions,and one of these is the B cell-activating factor (BAFF). We report in this study that HIV-1 (X4- and R5-tropic) upregulates BAFF expression and secretion by human monocytes. Moreover,we show that the virus-mediated production of BAFF by monocytes relies on a type I IFN response by a small percentage of plasmacytoid dendritic cells (pDCs) present in the monocyte cultures. HIV-1-induced type I IFN by pDCs triggers BAFF production in both classical and intermediate monocytes,but not in nonclassical monocytes,which nonetheless display a very strong basal BAFF production. We report also that basal BAFF secretion was higher in monocytes obtained from females compared with those from male donors. This study provides a novel mechanistic explanation for the increased BAFF levels observed during HIV-1 infection and highlights the importance of pDC/monocyte crosstalk to drive BAFF secretion. View Publication

The aggressiveness of glioblastoma multiforme (GBM) is defined by local invasion and resistance to therapy. Within established GBM,a subpopulation of tumor-initiating cells with stem-like properties (GBM stem cells,GSCs) is believed to underlie resistance to therapy. The metabolic pathway autophagy has been implicated in the regulation of survival in GBM. However,the status of autophagy in GBM and its role in the cancer stem cell fraction is currently unclear. We found that a number of autophagy regulators are highly expressed in GBM tumors carrying a mesenchymal signature,which defines aggressiveness and invasion,and are associated with components of the MAPK pathway. This autophagy signature included the autophagy-associated genes DRAM1 and SQSTM1,which encode a key regulator of selective autophagy,p62. High levels of DRAM1 were associated with shorter overall survival in GBM patients. In GSCs,DRAM1 and SQSTM1 expression correlated with activation of MAPK and expression of the mesenchymal marker c-MET. DRAM1 knockdown decreased p62 localization to autophagosomes and its autophagy-mediated degradation,thus suggesting a role for DRAM1 in p62-mediated autophagy. In contrast,autophagy induced by starvation or inhibition of mTOR/PI-3K was not affected by either DRAM1 or p62 downregulation. Functionally,DRAM1 and p62 regulate cell motility and invasion in GSCs. This was associated with alterations of energy metabolism,in particular reduced ATP and lactate levels. Taken together,these findings shed new light on the role of autophagy in GBM and reveal a novel function of the autophagy regulators DRAM1 and p62 in control of migration/invasion in cancer stem cells. View Publication