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

Idorn et al. characterized a mouse strain harboring a mutation identified in an HSE patient. Defective IFN-driven antiviral responses led to hyperactivation of inflammatory responses,which contributed to disease development. The study identifies immunopathology as an important contributor to HSE pathogenesis. View Publication

Robust in vitro lung models are required for risk assessment to measure key events leading to respiratory diseases. Primary normal human bronchial epithelial cells (NHBE) represent a good lung model but obtaining well-differentiated 3D cultures can be challenging. Here,we evaluated the ability to expand primary NHBE cells in different culture conditions while maintaining their 3D culture characteristics such as ciliated and goblet cells,and ion channel function. Differentiated cultures were optimally obtained with PneumaCult-Ex Plus (expansion medium)/PneumaCult-ALI (differentiation medium). Primary cells passaged up to four times maintained airway epithelial characteristics as evidenced by ciliated pseudostratified columnar epithelium with goblet cells,trans-epithelial electrical resistance (TEER) ({\textgreater}400 Ohms.cm2),and cystic fibrosis transmembrane conductance regulator-mediated short-circuit currents ({\textgreater}3 µA/cm2). No change in ciliary beat frequency (CBF) or airway surface liquid (ASL) meniscus length was observed up to passage six. For the first time,this study demonstrates that CFTR ion channel function and normal epithelial phenotypic characteristics are maintained in passaged primary NHBE cells. Furthermore,this study highlights the criticality of evaluating expansion and differentiation conditions for achieving optimal phenotypic and functional endpoints (CBF,ASL,ion channel function,presence of differentiated cells,TEER) when developing in vitro lung models. View Publication

In dynamic light microscopy applications,imaging specimens from multiple angles while maintaining controlled temperature conditions is crucial for comprehensive and accurate analysis. To address these challenges,we present iCAT,an open-source multifunctional accessory designed to enhance light microscopy. It enables specimen rotation along the axial plane,incorporates built-in modules for precise temperature control,features an integrated LED,and includes a camera for real-time specimen monitoring. It can be easily 3D-printed and assembled using readily available electrical components. Combined with any up-right microscope,this versatile device allows researchers to capture detailed images and videos of organoid cultures and live or fixed specimens,such as C. elegans,zebrafish,drosophila,or mouse embryos. The potential applications of iCAT in investigating dynamic cellular processes and complex developmental phenomena are vast,inspiring researchers to explore its possibilities and push the boundaries of biological research. iCAT,an open-source,3D-printable microscopy accessory,enables axial specimen rotation,temperature control,and live monitoring for high-resolution imaging of organoids and diverse model organisms. View Publication

Lenalidomide is the first karyotype-selective therapeutic approved for the treatment of myelodysplastic syndromes (MDS) owing to high rates of erythroid and cytogenetic response in patients with chromosome 5q deletion [del(5q)]. Although haploinsufficiency for the RPS14 gene and others encoded within the common deleted region (CDR) have been implicated in the pathogenesis of the del(5q) phenotype,the molecular basis of the karyotype specificity of lenalidomide remains unexplained. We focused our analysis on possible haplodeficient enzymatic targets encoded within the CDR that play key roles in cell-cycle regulation. We show that the dual specificity phosphatases,Cdc25C and PP2Acalpha,which are coregulators of the G(2)-M checkpoint,are inhibited by lenalidomide. Gene expression was lower in MDS and acute myeloid leukemia (AML) specimens with del(5q) compared with those with alternate karyotypes. Lenalidomide inhibited phosphatase activity either directly (Cdc25C) or indirectly (PP2A) with corresponding retention of inhibitory phospho-tyrosine residues. Treatment of del(5q) AML cells with lenalidomide induced G(2) arrest and apoptosis,whereas there was no effect in nondel(5q) AML cells. Small interfering RNA (shRNA) suppression of Cdc25C and PP2Acalpha gene expression recapitulated del(5q) susceptibility to lenalidomide with induction of G(2) arrest and apoptosis in both U937 and primary nondel(5q) MDS cells. These data establish a role for allelic haplodeficiency of the lenalidomide inhibitable Cdc25C and PP2Acalpha phosphatases in the selective drug sensitivity of del(5q) MDS. View Publication

HIV-infected slow progressors (SP) represent a heterogeneous group of subjects who spontaneously control HIV infection without treatment for several years while showing moderate signs of disease progression. Under conditions that remain poorly understood,a subgroup of these subjects experience failure of spontaneous immunological and virological control. Here we determined the frequency of SP subjects who showed loss of HIV control within our Canadian Cohort of HIV(+) Slow Progressors and identified the proinflammatory cytokine IL-32 as a robust biomarker for control failure. Plasmatic levels of the proinflammatory isoforms of IL-32 (mainly β and γ) at earlier clinic visits positively correlated with the decline of CD4 T-cell counts,increased viral load,lower CD4/CD8 ratio and levels of inflammatory markers (sCD14 and IL-6) at later clinic visits. We present here a proof-of-concept for the use of IL-32 as a predictive biomarker for disease progression in SP subjects and identify IL-32 as a potential therapeutic target. View Publication

Intestinal tuft cells are one of 4 secretory cell linages in the small intestine and the source of IL-25,a critical initiator of the type 2 immune response to parasite infection. When Raptor,a critical scaffold protein for mammalian target of rapamycin complex 1 (mTORC1),was acutely deleted in intestinal epithelium via Tamoxifen injection in Tritrichomonas muris (Tm) infected mice,tuft cells,IL-25 in epithelium and IL-13 in the mesenchyme were significantly reduced,but Tm burden was not affected. When Tm infected mice were treated with rapamycin,DCLK1 and IL-25 expression in enterocytes and IL-13 expression in mesenchyme were diminished. After massive small bowel resection,tuft cells and Tm were diminished due to the diet used postoperatively. The elimination of Tm and subsequent re-infection of mice with Tm led to type 2 immune response only in WT,but Tm colonization in both WT and Raptor deficient mice. When intestinal organoids were stimulated with IL-4,tuft cells and IL-25 were induced in both WT and Raptor deficient organoids. In summary,our study reveals that enterocyte specific Raptor is required for initiating a type 2 immune response which appears to function through the regulation of mTORC1 activity. View Publication

Multiple signalling pathways maintain human embryonic stem cells (hESC) in an undifferentiated state. Here we sought to define the significance of G protein signal transduction in the preservation of this state distinct from other cellular processes. Continuous treatment with drugs targeting G(αs)-,G(α-i/o)- and G(α-q/11)-subunit signalling mediators were assessed in independent hESC lines after 7days to discern effects on normalised alkaline phosphatase positive colony frequency vs total cell content. This identified PLCβ,intracellular free calcium and CAMKII kinase activity downstream of G(α-q/11) as of particular importance to the former. To confirm the significance of this finding we generated an agonist-responsive hESC line transgenic for a G(α-q/11) subunit-coupled receptor and demonstrated that an undifferentiated state could be promoted in the presence of an agonist without exogenously supplied bFGF and that this correlated with elevated intracellular calcium. Similarly,treatment of unmodified hESCs with a range of intracellular free calcium-modulating drugs in biologically defined mTESR culture system lacking exogenous bFGF promoted an hESC phenotype after 1week of continuous culture as defined by co-expression of OCT4 and NANOG. At least one of these drugs,lysophosphatidic acid significantly elevates phosphorylation of calmodulin and STAT3 in this culture system (ptextless0.05). These findings substantiate a role for G-protein and calcium signalling in undifferentiated hESC culture. View Publication

Fibroblast growth factor (FGF) signaling plays an important role in embryonic stem cells and adult tissue homeostasis,but the function of FGFs in mammary gland stem cells is less well defined. Both FGFR1 and FGFR2 are expressed in basal and luminal mammary epithelial cells (MECs),suggesting that together they might play a role in mammary gland development and stem cell dynamics. Previous studies have demonstrated that the deletion of FGFR2 resulted only in transient developmental defects in branching morphogenesis. Using a conditional deletion strategy,we investigated the consequences of FGFR1 deletion alone and then the simultaneous deletion of both FGFR1 and FGFR2 in the mammary epithelium. FGFR1 deletion using a keratin 14 promoter-driven Cre-recombinase resulted in an early,yet transient delay in development. However,no reduction in functional outgrowth potential was observed following limiting dilution transplantation analysis. In contrast,a significant reduction in outgrowth potential was observed upon the deletion of both FGFR1 and FGFR2 in MECs using adenovirus-Cre. Additionally,using a fluorescent reporter mouse model to monitor Cre-mediated recombination,we observed a competitive disadvantage following transplantation of both FGFR1/R2-null MECs,most prominently in the basal epithelial cells. This correlated with the complete loss of the mammary stem cell repopulating population in the FGFR1/R2-attenuated epithelium. FGFR1/R2-null MECs were partially rescued in chimeric outgrowths containing wild-type MECs,suggesting the potential importance of paracrine mechanisms involved in the maintenance of the basal epithelial stem cells. These studies document the requirement for functional FGFR signaling in mammary stem cells during development. View Publication

The impact of shipping temperatures and preservation media used during transport of either peripheral blood mononuclear cells (PBMCs) or Jurkat cells was assessed,in view of implementing of a proficiency testing scheme on mononuclear cell viability. Samples were analyzed before and after shipment at different temperatures (ambient temperature,dry ice,and liquid nitrogen) and in different preservation media (serum with cryoprotectant,commercial cryopreservation solution,and room temperature transport medium). Sample quality was assessed by viability assays (Trypan Blue dye exclusion,flow cytometry,Cell Analysis System cell counting (CASY)),and by ELISpot functional assay. The liquid nitrogen storage and shipment were found to be the most stable conditions to preserve cell viability and functionality. However,we show that alternative high quality shipment conditions for viable cells are dry ice shipment and commercial cryopreservation solution. These were also cost-efficient shipment conditions,satisfying the requirements of a proficiency testing scheme for viable mononuclear cells. Room temperature transport medium dramatically and adversely affected the integrity of mononuclear cells. View Publication

Creatine transporter deficiency (CTD) is an X-linked disease caused by mutations in the Slc6a8 gene. The impaired creatine uptake in the brain leads to developmental delays with intellectual disability. We hypothesized that deficient creatine uptake in CTD cerebral cells impact methylation balance leading to alterations of genes and proteins expression by epigenetic mechanism. In this study,we determined the status of nucleic acid methylation in both Slc6a8 knockout mouse model and brain organoids derived from CTD patients’ cells. We also investigated the effect of dodecyl creatine ester (DCE),a promising prodrug that increases brain creatine content in the mouse model of CTD. The level of nucleic acid methylation was significantly reduced compared to healthy controls in both in vivo and in vitro CTD models. This hypo-methylation tended to be regulated by DCE treatment in vivo. These results suggest that increased brain creatine after DCE treatment restores normal levels of DNA methylation,unveiling the potential of using DNA methylation as a marker to monitor the drug efficacy. View Publication