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

Human embryonic stem cell (hESC)-based assay systems and genetically modified hESCs are very useful tools for screening drugs that regulate stemness and differentiation and for studying the molecular mechanisms involved in hESC fate determination. For these types of studies,feeder cell-dependent cultures of hESCs are often problematic because the physiology of the feeder cells is perturbed by the drug treatments or genetic modifications,which potentially obscures research outcomes. In this study,we evaluated three commonly used feeder-free culture conditions to determine whether they supported the undifferentiated growth of hESCs and to determine whether the hESCs grown in these conditions displayed gene expression patterns that were similar to the expression patterns of feeder cell-dependent hESCs. Our results demonstrate that hESCs grown in the three feeder-free conditions expressed undifferentiation marker genes as strongly as hESCs that were grown in the feeder-dependent cultures. Furthermore,genome-wide gene expression profiles indicated that the gene expression patterns of hESCs that were grown under feeder-free or feeder-dependent culture conditions were highly similar. These results indicate that the feeder-free culture conditions support the undifferentiated growth of hESCs as effectively as the feeder-dependent culture conditions. Therefore,feeder-free culture conditions are potentially suitable for drug screening and for the genetic manipulation of hESCs in basic research. View Publication

Stem cells from bone marrow,skeletal muscle and possibly other tissues can be identified by the 'side-population' (SP) phenotype. Although it has been assumed that expression of ABC transporters is responsible for this phenotype,the specific molecules involved have not been defined. Here we show that expression of the Bcrp1 (also known as Abcg2 murine/ABCG2 human) gene is a conserved feature of stem cells from a wide variety of sources. Bcrp1 mRNA was expressed at high levels in primitive murine hematopoietic stem cells,and was sharply downregulated with differentiation. Enforced expression of the ABCG2 cDNA directly conferred the SP phenotype to bone-marrow cells and caused a reduction in maturing progeny both in vitro and in transplantation-based assays. These results show that expression of the Bcrp1/ABCG2 gene is an important determinant of the SP phenotype,and that it might serve as a marker for stem cells from various sources. View Publication

Systemic mastocytosis (SM) is a myeloid neoplasm involving mast cells (MCs) and their progenitors. In most cases,neoplastic cells display the D816V-mutated variant of KIT. KIT D816V exhibits constitutive tyrosine kinase (TK) activity and has been implicated in increased survival and growth of neoplastic MCs. Recent data suggest that the proapoptotic BH3-only death regulator Bim plays a role as a tumor suppressor in various myeloid neoplasms. We found that KIT D816V suppresses expression of Bim in Ba/F3 cells. The KIT D816-induced down-regulation of Bim was rescued by the KIT-targeting drug PKC412/midostaurin. Both PKC412 and the proteasome-inhibitor bortezomib were found to decrease growth and promote expression of Bim in MC leukemia cell lines HMC-1.1 (D816V negative) and HMC-1.2 (D816V positive). Both drugs were also found to counteract growth of primary neoplastic MCs. Furthermore,midostaurin was found to cooperate with bortezomib and with the BH3-mimetic obatoclax in producing growth inhibition in both HMC-1 subclones. Finally,a Bim-specific siRNA was found to rescue HMC-1 cells from PKC412-induced cell death. Our data show that KIT D816V suppresses expression of proapoptotic Bim in neoplastic MCs. Targeting of Bcl-2 family members by drugs promoting Bim (re)-expression,or by BH3-mimetics such as obatoclax,may be an attractive therapy concept in SM. View Publication

Integrating viruses represent robust tools for cellular reprogramming; however,the presence of viral transgenes in induced pluripotent stem cells (iPSCs) is deleterious because it holds the risk of insertional mutagenesis leading to malignant transformation. Here,we combine the robustness of lentiviral reprogramming with the efficacy of Cre recombinase protein transduction to derive iPSCs devoid of transgenes. By genome-wide analysis and targeted differentiation towards the cardiomyocyte lineage,we show that transgene-free iPSCs are superior to iPSCs before Cre transduction. Our study provides a simple,rapid and robust protocol for the generation of clinical-grade iPSCs suitable for disease modeling,tissue engineering and cell replacement therapies. View Publication

BACKGROUND Definitive endoderm (DE) is one of the three germ layers which during in vivo vertebrate development gives rise to a variety of organs including liver,lungs,thyroid and pancreas; consequently efficient in vitro initiation of stem cell differentiation to DE cells is a prerequisite for successful cellular specification to subsequent DE-derived cell types [1,2]. In this study we present a novel approach to rapidly and efficiently down regulate pluripotency genes during initiation of differentiation to DE cells by addition of dimethyl sulfoxide (DMSO) to Activin A-based culture medium and report its effects on the downstream differentiation to hepatocyte-like cells. MATERIALS AND METHODS Human embryonic stem cells (hESC) were differentiated to DE using standard methods in medium supplemented with 100ng/ml of Activin A and compared to cultures where DE specification was additionally enhanced with different concentrations of DMSO. DE cells were subsequently primed to generate hepatic-like cells to investigate whether the addition of DMSO during formation of DE improved subsequent expression of hepatic markers. A combination of flow cytometry,real-time quantitative reverse PCR and immunofluorescence was applied throughout the differentiation process to monitor expression of pluripotency (POUF5/OCT4 & NANOG),definitive endoderm (SOX17,CXCR4 & GATA4) and hepatic (AFP & ALB) genes to generate differentiation stage-specific signatures. RESULTS Addition of DMSO to the Activin A-based medium during DE specification resulted in rapid down regulation of the pluripotency genes OCT4 and NANOG,accompanied by an increase expression of the DE genes SOX17,CXCR4 and GATA4. Importantly,the expression level of ALB in DMSO-treated cells was also higher than in cells which were differentiated to the DE stage via standard Activin A treatment. View Publication

Recent evidence suggests that Notch signaling may play a role in regulation of cancer stem cell (CSC) self-renewal and differentiation hence presenting a promising target for development of novel therapies for aggressive cancers such as triple negative breast cancer (TNBC). We generated Notch1 monoclonal antibodies (mAbs) that specifically bind to the negative regulatory region of human Notch1. Notch1 inhibition in TNBC Sum149 and patient derived xenograft (PDX) 144580 models led to significant TGI particularly in combination with docetaxel. More interestingly,Notch1 mAbs caused a reduction in mammosphere formation and CD44+/CD24-/lo cell population. It also resulted in decreased tumor incidence upon re-implantation and delay in tumor recurrence. Our data demonstrated a potent antitumor efficacy of Notch1 mAbs,with a remarkable activity against CSCs. These findings suggest that anti-Notch1 mAbs may provide novel therapies to improve the efficacy of conventional therapies by directly targeting the CSC niche. They may also delay tumor recurrence and hence have a major impact on cancer patient survival. View Publication

Primary antiphospholipid syndrome (PAPS) is a life-threatening clotting disorder mediated by pathogenic autoantibodies. Here we dissect the origin of self-reactive B cells in human PAPS using peripheral blood and bone marrow of patients with triple-positive PAPS via combined single-cell RNA sequencing,B cell receptors (BCR) repertoire profiling,CITEseq analysis and single cell immortalization. We find that antiphospholipid (aPL)-specific B cells are present in the naive compartment,polyreactive,and derived from the natural repertoire. Furthermore,B cells with aPL specificities are not eliminated in patients with PAPS,persist until the memory and long-lived plasma cell stages,likely after defective germinal center selection,while becoming less polyreactive. Lastly,compared with the non-PAPS cells,PAPS B cells exhibit distinct IFN and APRIL signature as well as dysregulated mTORC1 and MYC pathways. Our findings may thus elucidate the survival mechanisms of these autoreactive B cells and suggest potential therapeutic targets for the treatment of PAPS. Primary antiphospholipid syndrome (PAPS) is a clotting disorder attributed to autoreactive antibodies produced by B cells. Here the authors show,using single cell omics and B cell repertoire data,that autoreactive B cells originate from the natural B cell repertoire and escape germinal center selection to persist in PAPS patient via potential dysregulation of mTORC1 and MYC pathways. View Publication

The human nasal epithelium is the primary site of exposure to influenza virus,the initiator of host responses to influenza and the resultant pathologies. Influenza virus may cause serious respiratory infection resulting in major complications,as well as severe impairment of the airways. Here,we elucidated the global transcriptomic changes during H3N2 infection of human nasal epithelial cells from multiple individuals. Using RNA sequencing,we characterized the differentially-expressed genes and pathways associated with changes occurring at the nasal epithelium following infection. We used in vitro differentiated human nasal epithelial cell culture model derived from seven different donors who had no concurrent history of viral infections. Statistical analysis highlighted strong transcriptomic signatures significantly associated with 24 and 48 h after infection,but not at the earlier 8-h time point. In particular,we found that the influenza infection induced in the nasal epithelium early and altered responses in interferon gamma signaling,B-cell signaling,apoptosis,necrosis,smooth muscle proliferation,and metabolic alterations. These molecular events initiated at the infected nasal epithelium may potentially adversely impact the airway,and thus the genes we identified could serve as potential diagnostic biomarkers or therapeutic targets for influenza infection and associated disease management. View Publication

The role of epigenetic regulators in the control of adult neurogenesis is largely undefined. We show that the histone demethylase enzyme Kdm5b (Jarid1b) negatively regulates neurogenesis from adult subventricular zone (SVZ) neural stem cells (NSCs) in culture. shRNA-mediated depletion of Kdm5b in proliferating adult NSCs decreased proliferation rates and reduced neurosphere formation in culture. When transferred to differentiation culture conditions,Kdm5b-depleted adult NSCs migrated from neurospheres with increased velocity. Whole-genome expression screening revealed widespread transcriptional changes with Kdm5b depletion,notably the up-regulation of reelin (Reln),the inhibition of steroid biosynthetic pathway component genes and the activation of genes with intracellular transport functions in cultured adult NSCs. Kdm5b depletion increased extracellular reelin concentration in the culture medium and increased phosphorylation of the downstream reelin signaling target Disabled-1 (Dab1). Sequestration of extracellular reelin with CR-50 reelin-blocking antibodies suppressed the increase in migratory velocity of Kdm5b-depleted adult NSCs. Chromatin immunoprecipitation revealed that Kdm5b is present at the proximal promoter of Reln,and H3K4me3 methylation was increased at this locus with Kdm5b depletion in differentiating adult NSCs. Combined the data suggest Kdm5b negatively regulates neurogenesis and represses Reln in neural stem cells from the adult SVZ. View Publication

Activation of the transcription factor,nuclear factor-kappaB (NF-kappaB),results in up-regulation of not only antiapoptotic genes but also proapoptotic genes,including death receptor 4 (DR4) and death receptor 5 (DR5). Therefore,NF-kappaB activation either suppresses or promotes apoptosis depending on the type of stimulus or cell context. We showed previously that the synthetic retinoid,6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437),effectively induces apoptosis particularly in androgen-independent prostate carcinoma cells. This effect was associated with the ability of CD437 to induce the expression of DR4 and DR5. In the present study,we examined the hypothesis that NF-kappaB activation plays a role in CD437-induced death receptor expression and apoptosis. Treatment of DU145 cells with CD437 resulted in a rapid decrease (textgreater or = 3 hours) of IkappaBalpha,which was accompanied by increased translocation of the NF-kappaB subunit p65 from the cytoplasm to the nucleus and increased NF-kappaB DNA-binding activity (textgreater or = 4 hours). The NF-kappaB inhibitor,helenalin,inhibited CD437-induced IkappaBalpha reduction and p65 nuclear translocation. Accordingly,it also abrogated CD437-induced up-regulation of DR4,activation of caspase-8 and caspase-3,and increased DNA fragmentation. Overexpression of an IkappaBalpha dominant-negative mutant blocked not only CD437-induced p65 nuclear translocation but also DR4 up-regulation,caspase activation,and DNA fragmentation. CD437 was unable to decrease IkappaBalpha protein levels and up-regulate DR4 expression in CD437-resistant DU145 cells. Moreover,knockdown of Fas-associated death domain,caspase-8,and DR4,respectively,suppressed CD437-induced apoptosis. Collectively,these results indicate that CD437 activates NF-kappaB via decreasing IkappaBalpha protein and thereby induces DR4 expression and subsequent apoptosis in DU145 cells. View Publication

BACKGROUND: Age and cardiovascular disease status appear to alter numbers and function of circulating endothelial progenitor cells (EPCs). Despite no universal phenotypic definition,numerous studies have implicated progenitors with apparent endothelial potential in local responses to vascular injury and with cardiovascular disease in general. To further define the role of this lineage in peripheral artery disease (PAD),we developed a multiparameter flow cytometry assay to analyze multiple phenotypic definitions of progenitor cells (PCs),EPCs,and mature endothelial cells (ECs) and evaluate effects of age and PAD on baseline levels of each subset. METHODS: Blood was collected from young healthy subjects (N = 9,mean age 33 +/- 8 years),older healthy subjects (N = 13,mean age 66 +/- 8 years),and older subjects with PAD (N = 15,mean age 69 +/- 8 years). After ammonium chloride lysis,cells were stained and analyzed on a Becton-Dickinson LSR II with a 5-color antibody panel: FITC-anti-CD31,PE-anti-CD146,PE-anti-CD133,PerCP-Cy5.5-anti-CD3,-CD19,-CD33 (lineage panel),PE-Cy7-anti-CD34,and APC-anti-VEGF-R2. Viability was assessed by propidium iodide exclusion,and only viable,low to medium side scatter lineage-negative singlets were analyzed. In some studies,cells were sorted for morphological studies. Subsets were defined as indicated later. RESULTS: Our results,using a comprehensive flow cytometric panel,indicate that CD133+,CD34+,and CD133+/CD34+ PCs are elevated in younger healthy individuals compared to older individuals,both healthy and with PAD. However,the number of EPCs and mature ECs did not significantly differ among the three groups. Assessment of endothelial colony forming units and dual acLDL-lectin staining supported the flow cytometric findings. CONCLUSIONS: We describe a comprehensive flow cytometric method to detect circulating mature and progenitor endothelial populations confirmed by conventional morphological and functional assays. Our findings suggest that aging may influence circulating levels of PCs,but not EPCs or ECs; PAD had no effect on baseline levels of any populations investigated. This study provides the basis for evaluating the potential effects of acute stress and therapeutic intervention on circulating progenitor and endothelial populations as a biomarker for cardiovascular status. View Publication

Increasing occurrence of moderate to severe intrauterine adhesion (IUA) is seriously affecting the quality of human life. The aim of the study was to establish IUA models in nonhuman primates and to explore the dual repair effects of human umbilical cord-derived mesenchymal stem cells (huMSCs) loaded on autocrosslinked hyaluronic acid gel (HA-GEL) on endometrial damage and adhesion. Here,we recorded the menstrual cycle data in detail with uterine cavities observed and endometrial tissues detected after intervention,and the thicker endometria,decreased amount of fibrotic formation,increased number of endometrium glands,etc.,suggested that both HA-GEL and huMSC/HA-GEL complexes could partially repair IUA caused by mechanical injury,but huMSC/HA-GEL complex transplantation had notable dual repair effects: a reliable antiadhesion property and the promotion of endometrial regeneration. View Publication