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

Epstein-Barr virus (EBV) persistently infects more than 90% of the human population,causing infectious mononucleosis,susceptibility to autoimmune diseases and multiple malignancies of epithelial or B cell-origin. EBV infects epithelial cells and B cells through interaction between viral glycoproteins and different host receptors,but it has remained unknown whether a common receptor mediates infection of its two major host cell targets. Here,we establish R9AP as a crucial EBV receptor for entry into epithelial and B cells. R9AP silencing or knockout,R9AP-derived peptide and R9AP monoclonal antibody each significantly inhibit,whereas R9AP overexpression promotes,EBV uptake into both cell types. R9AP binds directly to the EBV glycoprotein gH/gL complex to initiate gH/gL-gB-mediated membrane fusion. Notably,the interaction of R9AP with gH/gL is inhibited by the highly competitive gH/gL-neutralizing antibody AMMO1,which blocks EBV epithelial and B cell entry. Moreover,R9AP mediates viral and cellular membrane fusion in cooperation with EBV gp42-human leukocyte antigen class II or gH/gL-EPHA2 complexes in B cells or epithelial cells,respectively. We propose R9AP as the crucial common receptor of B cells and epithelial cells and a potential prophylactic and vaccine target for EBV. View Publication

To search for new indicators of self-renewing hematopoietic stem cells (HSCs),highly purified populations were isolated from adult mouse marrow,micromanipulated into a specially designed microscopic array,and cultured for 4 days in 300 ng/ml Steel factor,20 ng/ml IL-11,and 1 ng/ml flt3-ligand. During this period,each cell and its progeny were imaged at 3-min intervals by using digital time-lapse photography. Individual clones were then harvested and assayed for HSCs in mice by using a 4-month multilineage repopulation endpoint (textgreater1% contribution to lymphoid and myeloid lineages). In a first experiment,6 of 14 initial cells (43%) and 17 of 61 clones (28%) had HSC activity,demonstrating that HSC self-renewal divisions had occurred in vitro. Characteristics associated with HSC activity included longer cell-cycle times and the absence of uropodia on a majority of cells within the clone during the final 12 h of culture. Combining these criteria maximized the distinction of clones with HSC activity from those without and identified a subset of 27 of the 61 clones. These 27 clones included all 17 clones that had HSC activity; a detection efficiency of 63% (2.26 times more frequently than in the original group). The utility of these characteristics for discriminating HSC-containing clones was confirmed in two independent experiments where all HSC-containing clones were identified at a similar 2- to 3-fold-greater efficiency. These studies illustrate the potential of this monitoring system to detect new features of proliferating HSCs that are predictive of self-renewal divisions. View Publication

BACKGROUND: Human cord blood (hCB) is the main source of hematopoietic stem and progenitor cells (HSCs/PCs) for transplantation. Efforts to overcome relative shortages of HSCs/PCs have led to technologies to expand HSCs/PCs ex vivo. However,methods suitable for clinical practice have yet to be fully established. METHODOLOGY/PRINCIPAL FINDINGS: In this study,we screened biologically active natural products for activity to promote expansion of hCB HSCs/PCs ex vivo,and identified Garcinol,a plant-derived histone acetyltransferase (HAT) inhibitor,as a novel stimulator of hCB HSC/PC expansion. During a 7-day culture of CD34(+)CD38(-) HSCs supplemented with stem cell factor and thrombopoietin,Garcinol increased numbers of CD34(+)CD38(-) HSCs/PCs more than 4.5-fold and Isogarcinol,a derivative of Garcinol,7.4-fold. Furthermore,during a 7-day culture of CD34(+) HSCs/PCs,Garcinol expanded the number of SCID-repopulating cells (SRCs) 2.5-fold. We also demonstrated that the capacity of Garcinol and its derivatives to expand HSCs/PCs was closely correlated with their inhibitory effect on HAT. The Garcinol derivatives which expanded HSCs/PCs inhibited the HAT activity and acetylation of histones,while inactive derivatives did not. CONCLUSIONS/SIGNIFICANCE: Our findings identify Garcinol as the first natural product acting on HSCs/PCs and suggest the inhibition of HAT to be an alternative approach for manipulating HSCs/PCs. View Publication

The neurosphere assay can detect and expand neural stem cells (NSCs) and progenitor cells,but it cannot discriminate between these two populations. Given two assays have purported to overcome this shortfall,we performed a comparative analysis of the distribution and frequency of NSCs and progenitor cells detected in 400 mum coronal segments along the ventricular neuraxis of the adult mouse brain using the neurosphere assay,the neural colony forming cell assay (N-CFCA),and label-retaining cell (LRC) approach. We observed a large variation in the number of progenitor/stem cells detected in serial sections along the neuraxis,with the number of neurosphere-forming cells detected in individual 400 mum sections varying from a minimum of eight to a maximum of 891 depending upon the rostral-caudal coordinate assayed. Moreover,the greatest variability occurred in the rostral portion of the lateral ventricles,thereby explaining the large variation in neurosphere frequency previously reported. Whereas the overall number of neurospheres (3730 +/- 276) or colonies (4275 +/- 124) we detected along the neuraxis did not differ significantly,LRC numbers were significantly reduced (1186 +/- 188,7 month chase) in comparison to both total colonies and neurospheres. Moreover,approximately two orders of magnitude fewer NSC-derived colonies (50 +/- 10) were detected using the N-CFCA as compared to LRCs. Given only 5% of the LRCs are cycling (BrdU+/Ki-67+) or competent to divide (BrdU+/Mcm-2+),and proliferate upon transfer to culture,it is unclear whether this technique selectively detects endogenous NSCs. Overall,caution should be taken with the interpretation and employment of all these techniques. View Publication

Diffuse alveolar hemorrhage (DAH),although rare,is a life-threatening complication of systemic lupus erythematosus (SLE). Little is known about the pathophysiology of DAH in humans,although increasingly neutrophils,NETosis and inflammatory monocytes have been shown to play an important role in the pristane-induced model of SLE which develops lung hemorrhage and recapitulates many of the pathologic features of human DAH. Using this experimental model,we asked whether endoplasmic reticulum (ER) stress played a role in driving the pathology of pulmonary hemorrhage and what role infiltrating neutrophils had in this process. Analysis of lung tissue from pristane-treated mice showed genes associated with ER stress and NETosis were increased in a time-dependent manner and reflected the timing of CD11b+Ly6G+ neutrophil accumulation in the lung. Using precision cut lung slices from untreated mice we observed that neutrophils isolated from the peritoneal cavity of pristane-treated mice could directly induce the expression of genes associated with ER stress,namely Chop and Bip. Mice which had myeloid-specific deletion of PAD4 were generated and treated with pristane to assess the involvement of PAD4 and PAD4-dependent NET formation in pristane-induced lung inflammation. Specific deletion of PAD4 in myeloid cells resulted in decreased expression of ER stress genes in the pristane model,with accompanying reduction in IFN-driven genes and pathology. Lastly,coculture experiments of human neutrophils and human lung epithelial cell line (BEAS-2b) showed neutrophils from SLE patients induced significantly more ER stress and interferon-stimulated genes in epithelial cells compared to healthy control neutrophils. These results support a pathogenic role of neutrophils and NETs in lung injury during pristane-induced DAH through the induction of ER stress response and suggest that overactivation of neutrophils in SLE and NETosis may underlie development of DAH. View Publication

Mesenchymal stem cells (MSCs) are attractive alternatives to conventional anti-asthmatic drugs for severe asthma. Mechanisms underlying the anti-asthmatic effects of MSCs have not yet been elucidated. This study evaluated the anti-asthmatic effects of intravenously administered MSCs,focusing on macrophages and monocytes. Seven-week-old transgenic (Tg) mice with lung-specific overexpression of IL-13 were used to simulate chronic asthma. MSCs were intravenously administered four days before sampling. We examined changes in immune cell subpopulations,gene expression,and histological phenotypes. IL-13 Tg mice exhibited diverse features of chronic asthma,including severe type 2 inflammation,airway fibrosis,and mucus metaplasia. Intravenous administration of MSCs attenuated these asthmatic features just four days after a single treatment. MSC treatment significantly reduced SiglecF-CD11c-CD11b+ monocyte-derived macrophages (MoMs) and inhibited the polarization of MoMs into M2 macrophages,especially M2a and M2c. Furthermore,MSCs downregulated the excessive accumulation of Ly6c- monocytes in the lungs. While an intravenous adoptive transfer of Ly6c- monocytes promoted the infiltration of MoM and Th2 inflammation,that of MSC-exposed Ly6c- monocytes did not. Ex vivo Ly6c- MoMs upregulated M2-related genes,which were reduced by MSC treatment. Molecules secreted by Ly6c- MoMs from IL-13 Tg mice lungs upregulated the expression of fibrosis-related genes in fibroblasts,which were also suppressed by MSC treatment. In conclusion,intravenously administered MSCs attenuate asthma phenotypes of chronic asthma by modulating macrophages. Identifying M2 macrophage subtypes revealed that exposure to MSCs transforms the phenotype and function of macrophages. We suggest that Ly6c- monocytes could be a therapeutic target for asthma management. View Publication

Aldehyde dehydrogenase (ALDH) is an enzyme that is expressed in the liver and is required for the conversion of retinol (vitamin A) to retinoic acids. ALDH is also highly enriched in hematopoietic stem cells (HSCs) and is considered a selectable marker of human HSCs,although its contribution to stem cell fate remains unknown. In this study,we demonstrate that ALDH is a key regulator of HSC differentiation. Inhibition of ALDH with diethylaminobenzaldehyde (DEAB) delayed the differentiation of human HSCs that otherwise occurred in response to cytokines. Moreover,short-term culture with DEAB caused a 3.4-fold expansion in the most primitive assayable human cells,the nonobese diabetic/severe combined immunodeficiency mouse repopulating cells,compared with day 0 CD34(+)CD38(-)lin(-) cells. The effects of DEAB on HSC differentiation could be reversed by the coadministration of the retinoic acid receptor agonist,all-trans-retinoic acid,suggesting that the ability of ALDH to generate retinoic acids is important in determining HSC fate. DEAB treatment also caused a decrease in retinoic acid receptor-mediated signaling within human HSCs,suggesting directly that inhibition of ALDH promotes HSC self-renewal via reduction of retinoic acid activity. Modulation of ALDH activity and retinoid signaling is a previously unrecognized and effective strategy to amplify human HSCs. View Publication

The maintenance of a basal immunoinflammatory signature in hematopoietic stem/progenitor cells (HSPCs) constitutes a fundamental regulatory axis governing hematopoietic competence and immune effector generation. While epigenetic repressors constrain this inflammatory phenotype,the molecular amplifiers that preserve this critical state remain undefined. Through integrated single-cell transcriptomic/epigenomic profiling and functional interrogation,we identify Setd2-mediated H3K36me3 as an indispensable epigenetic amplifier sustaining baseline inflammation in murine HSPCs. Setd2 ablation specifically eliminated interferon (IFN)-enriched HSPC subpopulations and attenuated inflammatory signaling cascades. Functionally,Setd2-deficient HSPCs exhibited impaired IFNγ responsiveness,compromised B-lymphopoiesis,and diminished reconstitution capacity due to Lin−c-Kit+Sca1high cell depletion. Paradoxically,Setd2 loss conferred resistance to IFNγ-induced HSPCs exhaustion,which may contribute to the maintenance of Setd2-deficient HSPCs in our myelodysplastic syndrome (MDS) model under the inflammatory milieu. Mechanistically,Setd2 sustained chromatin accessibility and enhancer (H3K27ac) activity at inflammatory gene loci. This work delineates a critical link between Setd2-mediated chromatin regulation,baseline inflammation,HSPC function,and immune competence,providing insights into inflammatory dysregulation in hematopoietic malignancies like MDS. View Publication

Cytotoxic chemotherapies could cause the dysregulation of hematopoiesis and even put patients at increased risk of hematopoietic malignancy. Therapy-related leukemia is mainly caused by cytotoxic chemotherapy-induced genetic mutations in hematopoietic stem/progenitor cells (HSPCs). In addition to the intrinsic mechanism,some extrinsic events occurring in the bone marrow (BM) microenvironment are also possible mechanisms involved in genetic alteration. In the present study,we investigated the damage to BM stromal cells induced by a chemotherapy drug,daunorubicin (DNR) and further identified the DNA damage in hematopoietic cells caused by drug-treated stromal cells. It was found that treatment with DNR in mice caused a temporary reduction in cell number in each BM stromal cell subpopulation and the impairment of clonal growth potential in BM stromal cells. DNR treatment led to a tendency of senescence,generation of intracellular reactive oxygen species,production of cytokines and chemokines,and dysfunction of mitochondrial in stromal cells. Transcriptome microarray data and gene ontology (GO) or gene set enrichment analysis (GSEA) showed that differentially expressed genes that were down-regulated in response to DNR treatment were significantly enriched in mitochondrion function,and negative regulators of reactive oxygen species. Surprisingly,it was found that DNR-treated stromal cells secreted high levels of H2O2 into the culture supernatant. Furthermore,coculture of hematopoietic cells with DNR-treated stromal cells led to the accumulation of DNA damage as determined by the levels of histone H2AX phosphorylation and 8-oxo-2'-deoxyguanosine in hematopoietic cells. Overall,our results suggest that DNR-induced BM stromal cell damage can lead to genomic instability in hematopoietic cells. View Publication

Human hematopoietic stem cells are defined by their ability to repopulate multiple hematopoietic lineages in the bone marrow of transplanted recipients and therefore are functionally distinct from hematopoietic progenitors detected in vitro. Although factors capable of regulating progenitors are well established,in vivo regulators of hematopoietic repopulating function are unknown. By using a member of the vertebrate Wnt family,Wnt-5A,the proliferation and differentiation of progenitors cocultured on stromal cells transduced with Wnt-5A or treated with Wnt-5A conditioned medium (CM) was unaffected. However,i.p. injection of Wnt-5A CM into mice engrafted with human repopulating cells increased multilineage reconstitution by textgreater3-fold compared with controls. Furthermore,in vivo treatment of human repopulating cells with Wnt-5A CM produced a greater proportion of phenotypically primitive hematopoietic progeny that could be isolated and shown to possess enhanced progenitor function independent of continued Wnt-5A treatment. Our study demonstrates that Wnt-5A augments primitive hematopoietic development in vivo and represents an in vivo regulator of hematopoietic stem cell function in the human. Based on these findings,we suggest a potential role for activation of Wnt signaling in managing patients exhibiting poor hematopoietic recovery shortly after stem cell transplantation. View Publication

Human hematopoietic stem cells (HSCs) are commonly purified by the expression of cell surface markers such as CD34. Because cell phenotype can be altered by cell cycle progression or ex vivo culture,purification on the basis of conserved stem cell function may represent a more reliable way to isolate various stem cell populations. We have purified primitive HSCs from human umbilical cord blood (UCB) by lineage depletion (Lin(-)) followed by selection of cells with high aldehyde dehydrogenase (ALDH) activity. ALDH(hi)Lin(-) cells contained 22.6% +/- 3.0% of the Lin(-) population and highly coexpressed primitive HSC phenotypes (CD34(+) CD38(-) and CD34(+)CD133(+)). In vitro hematopoietic progenitor function was enriched in the ALDH(hi)Lin(-) population,compared with ALDH(lo)Lin(-) cells. Multilineage human hematopoietic repopulation was observed exclusively after transplantation of ALDH(hi)Lin(-) cells. Direct comparison of repopulation with use of the nonobese diabetic/severe combined immunodeficient (NOD/SCID) and NOD/SCID beta2 microglobulin (beta2M) null models demonstrated that 10-fold greater numbers of ALDH(hi)-Lin(-) cells were needed to engraft the NOD/SCID mouse as compared with the more permissive NOD/SCID beta2M null mouse,suggesting that the ALDH(hi)Lin(-) population contained committed progenitors as well as primitive repopulating cells. Cell fractionation according to lineage depletion and ALDH activity provides a viable and prospective purification of HSCs on the basis of cell function rather than cell surface phenotype. View Publication