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

Evidence suggests the transcription factor GATA-2 is a critical regulator of murine hematopoietic stem cells. Here,we explore the relation between GATA-2 and cell proliferation and show that inducing GATA-2 increases quiescence (G(0) residency) of murine and human hematopoietic cells. In human cord blood,quiescent fractions (CD34(+)CD38(-)Hoechst(lo)Pyronin Y(lo)) express more GATA-2 than cycling counterparts. Enforcing GATA-2 expression increased quiescence of cord blood cells,reducing proliferation and performance in long-term culture-initiating cell and colony-forming cell (CFC) assays. Gene expression analysis places GATA-2 upstream of the quiescence regulator MEF,but enforcing MEF expression does not prevent GATA-2-conferred quiescence,suggesting additional regulators are involved. Although known quiescence regulators p21(CIP1) and p27(KIP1) do not appear to be responsible,enforcing GATA-2 reduced expression of regulators of cell cycle such as CCND3,CDK4,and CDK6. Enforcing GATA-2 inhibited human hematopoiesis in vivo: cells with highest exogenous expression (GATA-2(hi)) failed to contribute to hematopoiesis in nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice,whereas GATA-2(lo) cells contributed with delayed kinetics and low efficiency,with reduced expression of Ki-67. Thus,GATA-2 activity inhibits cell cycle in vitro and in vivo,highlighting GATA-2 as a molecular entry point into the transcriptional program regulating quiescence in human hematopoietic stem and progenitor cells. View Publication

The low frequency of hematopoietic stem and progenitor cells (HSPCs) in human BM has precluded analysis of the direct biochemical effects elicited by cytokines in these populations,and their functional consequences. Here,single-cell phospho-specific flow cytometry was used to define the signaling networks active in 5 previously defined human HSPC subsets. This analysis revealed that the currently defined HSC compartment is composed of biochemically distinct subsets with the ability to respond rapidly and directly in vitro to a broader array of cytokines than previously appreciated,including G-CSF. The G-CSF response was physiologically relevant-driving cell-cycle entry and increased proliferation in a subset of single cells within the HSC compartment. The heterogeneity in the single-cell signaling and proliferation responses prompted subfractionation of the adult BM HSC compartment by expression of CD114 (G-CSF receptor). Xenotransplantation assays revealed that HSC activity is significantly enriched in the CD114(neg/lo) compartment,and almost completely absent in the CD114(pos) subfraction. The single-cell analyses used here can be adapted for further refinement of HSPC surface immunophenotypes,and for examining the direct regulatory effects of other factors on the homeostasis of stem and progenitor populations in normal or diseased states. View Publication

OVERVIEW: All current antipsychotic medications work by binding to Gi-coupled dopamine (DA) D2 receptors. Such medications are thought to affect cellular function primarily by decreasing DA-mediated regulation of intracellular cyclic adenosine monophosphate (cAMP).However,several studies indicate that cAMP signal transduction abnormalities in schizophrenia may not be limited to D2-containing cells. The current study examines the potential of using non-receptor-based agents that modify intracellular signal transduction as potential antipsychotic medications. METHODS: The indirect DA agonist amphetamine has been used to model the auditory sensory processing deficits in schizophrenia. Such pharmacologically induced abnormalities are reversed by current antipsychotic treatments. This study examines the ability of the phosphodiesterase-4 inhibitor,rolipram,to reverse amphetamine-induced abnormalities in auditory-evoked potentials that are characteristic of schizophrenia. RESULTS: Rolipram reverses amphetamine-induced reductions in auditory-evoked potentials. CONCLUSION: This finding could lead to novel approaches to receptor-independent treatments for schizophrenia. View Publication

Small-molecule inhibitors of the serine dipeptidases DPP8 and DPP9 (DPP8/9) induce a lytic form of cell death called pyroptosis in mouse and human monocytes and macrophages1,2. In mouse myeloid cells,Dpp8/9 inhibition activates the inflammasome sensor Nlrp1b,which in turn activates pro-caspase-1 to mediate cell death3,but the mechanism of DPP8/9 inhibitor-induced pyroptosis in human myeloid cells is not yet known. Here we show that the CARD-containing protein CARD8 mediates DPP8/9 inhibitor-induced pro-caspase-1-dependent pyroptosis in human myeloid cells. We further show that DPP8/9 inhibitors induce pyroptosis in the majority of human acute myeloid leukemia (AML) cell lines and primary AML samples,but not in cells from many other lineages,and that these inhibitors inhibit human AML progression in mouse models. Overall,this work identifies an activator of CARD8 in human cells and indicates that its activation by small-molecule DPP8/9 inhibitors represents a new potential therapeutic strategy for AML. View Publication

The heart wall tissue,or the myocardium,is one of the main targets in cardiovascular disease prevention and treatment. Animal models have not been sufficient in mimicking the human myocardium as evident by the very low clinical translation rates of cardiovascular drugs. Additionally,current in vitro models of the human myocardium possess several shortcomings such as lack of physiologically relevant co-culture of myocardial cells,lack of a 3D biomimetic environment,and the use of non-human cells. In this study,we address these shortcomings through the design and manufacture of a myocardium-on-chip (MOC) using 3D cell-laden hydrogel constructs and human induced pluripotent stem cell (hiPSC) derived myocardial cells. The MOC utilizes 3D spatially controlled co-culture of hiPSC derived cardiomyocytes (iCMs) and hiPSC derived endothelial cells (iECs) integrated among iCMs as well as in capillary-like side channels,to better mimic the microvasculature seen in native myocardium. We first fully characterized iCMs using immunostaining,genetic,and electrochemical analysis and iECs through immunostaining and alignment analysis to ensure their functionality,and then seeded these cells sequentially into the MOC device. We showed that iECs could be cultured within the microfluidic device without losing their phenotypic lineage commitment,and align with the flow upon physiological level shear stresses. We were able to incorporate iCMs within the device in a spatially controlled manner with the help of photocrosslinkable polymers. The iCMs were shown to be viable and functional within the device up to 7 days,and were integrated with the iECs. The iCMs and iECs in this study were derived from the same hiPSC cell line,essentially mimicking the myocardium of an individual human patient. Such devices are essential for personalized medicine studies where the individual drug response of patients with different genetic backgrounds can be tested in a physiologically relevant manner. View Publication

New small molecules that regulate the step-wise differentiation of human pluripotent stem cells into dopaminergic neurons have been identified. The steroid,guggulsterone,was found to be the most effective inducer of neural stem cells into dopaminergic neurons. These neurons are extensively characterized and shown to be functional. We believe this new approach offers a practical route to creating neurons of sufficient quality to be used to treat Parkinson's disease patients. View Publication

The complete array of genes required for terminal erythroid differentiation remains unknown. To address this knowledge gap,we perform a genome-scale CRISPR knock-out screen in the human erythroid progenitor cell line HUDEP-2 and validate candidate regulators of erythroid differentiation in a custom secondary screen. Comparison of sgRNA abundance in the CRISPR library,proerythroblasts,and orthochromatic erythroblasts,resulted in the identification of genes that are essential for proerythroblast survival and genes that are required for terminal erythroid differentiation. Among the top genes identified are known regulators of erythropoiesis,underscoring the validity of this screen. Notably,using a Log2 fold change of <−1 and false discovery rate of <0.01,the screen identified 277 genes that are required for terminal erythroid differentiation,including multiple genes not previously nominated through GWAS. NHLRC2,which was previously implicated in hemolytic anemia,was a highly ranked gene. We suggest that anemia due to NHLRC2 mutation results at least in part from a defect in erythroid differentiation. Another highly ranked gene in the screen is VAC14,which we validated for its requirement in erythropoiesis in vitro and in vivo. Thus,data from this CRISPR screen may help classify the underlying mechanisms that contribute to erythroid disorders. Subject terms: Erythropoiesis,CRISPR-Cas9 genome editing,Haematopoietic stem cells View Publication

Stem cell differentiation involves critical changes in gene expression. Identification of these should provide endpoints useful for optimizing stem cell propagation as well as potential clues about mechanisms governing stem cell maintenance. Here we describe the results of a new meta-analysis methodology applied to multiple gene expression datasets from three mouse embryonic stem cell (ESC) lines obtained at specific time points during the course of their differentiation into various lineages. We developed methods to identify genes with expression changes that correlated with the altered frequency of functionally defined,undifferentiated ESC in culture. In each dataset,we computed a novel statistical confidence measure for every gene which captured the certainty that a particular gene exhibited an expression pattern of interest within that dataset. This permitted a joint analysis of the datasets,despite the different experimental designs. Using a ranking scheme that favored genes exhibiting patterns of interest,we focused on the top 88 genes whose expression was consistently changed when ESC were induced to differentiate. Seven of these (103728at,8430410A17Rik,Klf2,Nr0b1,Sox2,Tcl1,and Zfp42) showed a rapid decrease in expression concurrent with a decrease in frequency of undifferentiated cells and remained predictive when evaluated in additional maintenance and differentiating protocols. Through a novel meta-analysis,this study identifies a small set of genes whose expression is useful for identifying changes in stem cell frequencies in cultures of mouse ESC. The methods and findings have broader applicability to understanding the regulation of self-renewal of other stem cell types. View Publication