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

BACKGROUND: Many retinal degenerative diseases are caused by the loss of retinal ganglion cells (RGCs). Autosomal dominant optic atrophy is the most common hereditary optic atrophy disease and is characterized by central vision loss and degeneration of RGCs. Currently,there is no effective treatment for this group of diseases. However,stem cell therapy holds great potential for replacing lost RGCs of patients. Compared with embryonic stem cells,induced pluripotent stem cells (iPSCs) can be derived from adult somatic cells,and they are associated with fewer ethical concerns and are less prone to immune rejection. In addition,patient-derived iPSCs may provide us with a cellular model for studying the pathogenesis and potential therapeutic agents for optic atrophy.backslashnbackslashnMETHODS: In this study,iPSCs were obtained from patients carrying an OPA1 mutation (OPA1 (+/-) -iPSC) that were diagnosed with optic atrophy. These iPSCs were differentiated into putative RGCs,which were subsequently characterized by using RGC-specific expression markers BRN3a and ISLET-1.backslashnbackslashnRESULTS: Mutant OPA1 (+/-) -iPSCs exhibited significantly more apoptosis and were unable to efficiently differentiate into RGCs. However,with the addition of neural induction medium,Noggin,or estrogen,OPA1 (+/-) -iPSC differentiation into RGCs was promoted.backslashnbackslashnCONCLUSIONS: Our results suggest that apoptosis mediated by OPA1 mutations plays an important role in the pathogenesis of optic atrophy,and both noggin and β-estrogen may represent potential therapeutic agents for OPA1-related optic atrophy. View Publication

With increasing worldwide demand for safe blood,there is much interest in generating red blood cells in vitro as an alternative clinical product. However,available methods for in vitro generation of red cells from adult and cord blood progenitors do not yet provide a sustainable supply,and current systems using pluripotent stem cells as progenitors do not generate viable red cells. We have taken an alternative approach,immortalizing early adult erythroblasts generating a stable line,which provides a continuous supply of red cells. The immortalized cells differentiate efficiently into mature,functional reticulocytes that can be isolated by filtration. Extensive characterization has not revealed any differences between these reticulocytes and in vitro-cultured adult reticulocytes functionally or at the molecular level,and importantly no aberrant protein expression. We demonstrate a feasible approach to the manufacture of red cells for clinical use from in vitro culture. View Publication

Inflammation is a risk factor for cancer development. Individuals with preleukemic TET2 mutations manifest clonal hematopoiesis and are at a higher risk of developing leukemia. How inflammatory signals influence the survival of preleukemic hematopoietic stem and progenitor cells (HSPCs) is unclear. We show a rapid increase in the frequency and absolute number of Tet2-KO mature myeloid cells and HSPCs in response to inflammatory stress,which results in enhanced production of inflammatory cytokines,including interleukin-6 (IL-6),and resistance to apoptosis. IL-6 induces hyperactivation of the Shp2-Stat3 signaling axis,resulting in increased expression of a novel anti-apoptotic long non-coding RNA (lncRNAs),Morrbid,in Tet2-KO myeloid cells and HSPCs. Expression of activated Shp2 in HSPCs phenocopies Tet2 loss with regard to hyperactivation of Stat3 and Morrbid. In vivo,pharmacologic inhibition of Shp2 or Stat3 or genetic loss of Morrbid in Tet2 mutant mice rescues inflammatory-stress-induced abnormalities in HSPCs and mature myeloid cells,including clonal hematopoiesis. View Publication

HIV persists in latently infected CD4+ T cells during antiretroviral therapy (ART). Immune checkpoint molecules,including PD-1,are preferentially expressed at the surface of persistently infected cells. However,whether PD-1 plays a functional role in HIV latency and reservoir persistence remains unknown. Using CD4+ T cells from HIV-infected individuals,we show that the engagement of PD-1 inhibits viral production at the transcriptional level and abrogates T-cell receptor (TCR)-induced HIV reactivation in latently infected cells. Conversely,PD-1 blockade with the monoclonal antibody pembrolizumab enhances HIV production in combination with the latency reversing agent bryostatin without increasing T cell activation. Our results suggest that the administration of immune checkpoint blockers to HIV-infected individuals on ART may facilitate latency disruption. View Publication

Sialic acids are negatively charged carbohydrates that cap the glycans of glycoproteins and glycolipids. Sialic acids are involved in various biological processes including cell-cell adhesion and immune recognition. In dendritic cells (DCs),the major antigen-presenting cells of the immune system,sialic acids emerge as important regulators of maturation and interaction with other lymphocytes including T cells. Many aspects of how sialic acids regulate DC functions are not well understood and tools and model systems to address these are limited. Here,we have established cultures of murine bone marrow-derived DCs (BMDCs) that lack sialic acid expression using a sialic acid-blocking mimetic Ac53FaxNeu5Ac. Ac53FaxNeu5Ac treatment potentiated BMDC activation via toll-like receptor (TLR) stimulation without affecting differentiation and viability. Sialic acid blockade further increased the capacity of BMDCs to induce antigen-specific CD8+ T cell proliferation. Transcriptome-wide gene expression analysis revealed that sialic acid mimetic treatment of BMDCs induces differential expression of genes involved in T cell activation,cell-adhesion,and cell-cell interactions. Subsequent cell clustering assays and single cell avidity measurements demonstrated that BMDCs with reduced sialylation form higher avidity interactions with CD8+ T cells. This increased avidity was detectable in the absence of antigens,but was especially pronounced in antigen-dependent interactions. Together,our data show that sialic acid blockade in BMDCs ameliorates maturation and enhances both cognate T cell receptor-MHC-dependent and independent T cell interactions that allow for more robust CD8+ T cell responses. View Publication

Extracellular acidification occurs in inflamed tissue and the tumor microenvironment; however,a systematic study on how pH sensing contributes to tissue homeostasis is lacking. In the present study,we examine cell type-specific roles of the pH sensor G protein-coupled receptor 65 (GPR65) and its inflammatory disease-associated Ile231Leu-coding variant in inflammation control. GPR65 Ile231Leu knock-in mice are highly susceptible to both bacterial infection-induced and T cell-driven colitis. Mechanistically,GPR65 Ile231Leu elicits a cytokine imbalance through impaired helper type 17 T cell (TH17 cell) and TH22 cell differentiation and interleukin (IL)-22 production in association with altered cellular metabolism controlled through the cAMP-CREB-DGAT1 axis. In dendritic cells,GPR65 Ile231Leu elevates IL-12 and IL-23 release at acidic pH and alters endo-lysosomal fusion and degradation capacity,resulting in enhanced antigen presentation. The present study highlights GPR65 Ile231Leu as a multistep risk factor in intestinal inflammation and illuminates a mechanism by which pH sensing controls inflammatory circuits and tissue homeostasis. View Publication

Plasmacytoid dendritic cells (pDCs) have been implicated as having a role in antifungal immunity,but mechanisms of their interaction with fungi and the resulting cellular responses are not well understood. In this study,we identify the direct and indirect biological response of human pDCs to the fungal pathogen Aspergillus fumigatus and characterize the expression and regulation of antifungal receptors on the pDC surface. Results indicate pDCs do not phagocytose Aspergillus conidia,but instead bind hyphal surfaces and undergo activation and maturation via the upregulation of costimulatory and maturation markers. Measuring the expression of C-type lectin receptors dectin-1,dectin-2,dectin-3,and mannose receptor on human pDCs revealed intermediate expression of each receptor compared with monocytes. The specific dectin-1 agonist curdlan induced pDC activation and maturation in a cell-intrinsic and cell-extrinsic manner. The indirect activation of pDCs by curdlan was much stronger than direct stimulation and was mediated through cytokine production by other PBMCs. Overall,our data indicate pDCs express various C-type lectin receptors,recognize and respond to Aspergillus hyphal Ag,and serve as immune enhancers or modulators in the overarching fungal immune response. View Publication

Mesenchymal stem cells (MSCs) in bone marrow and adipose tissues are expected to be effective tools for regenerative medicine to treat various diseases. To obtain MSCs that possess both high differentiation and tissue regenerative potential,it is necessary to establish an isolation system that does not require long-term culture. It has previously been reported that the cytoskeletal protein vimentin,expressed on the surfaces of multiple cell types,possesses N-acetylglucosamine- (GlcNAc-) binding activity. Therefore,we tried to exploit this interaction to efficiently isolate MSCs from rat bone marrow cells using GlcNAc-bearing polymer-coated dishes. Cells isolated by this method were identified as MSCs because they were CD34-,CD45-,and CD11b/c-negative and CD90-,CD29-,CD44-,CD54-,CD73-,and CD105-positive. Osteoblast,adipocyte,and chondrocyte differentiation was observed in these cells. In total,yields of rat MSCs were threefold to fourfold higher using GlcNAc-bearing polymer-coated dishes than yields using conventional tissue-culture dishes. Interestingly,MSCs isolated with GlcNAc-bearing polymer-coated dishes strongly expressed CD106,whereas those isolated with conventional tissue-culture dishes had low CD106 expression. Moreover,senescence-associated $\beta$-galactosidase activity in MSCs from GlcNAc-bearing polymer-coated dishes was lower than that in MSCs from tissue-culture dishes. These results establish an improved isolation method for high-quality MSCs. View Publication

HNF4A and HNF1A encode transcription factors that are important for the development and function of the pancreas and liver. Mutations in both genes have been directly linked to Maturity Onset Diabetes of the Young (MODY) and type 2 diabetes (T2D) risk. To better define the pleiotropic gene regulatory roles of HNF4A and HNF1A,we generated a comprehensive genome-wide map of their binding targets in pancreatic and hepatic cells using ChIP-Seq. HNF4A was found to bind and regulate known (ACY3,HAAO,HNF1A,MAP3K11) and previously unidentified (ABCD3,CDKN2AIP,USH1C,VIL1) loci in a tissue-dependent manner. Functional follow-up highlighted a potential role for HAAO and USH1C as regulators of beta cell function. Unlike the loss-of-function HNF4A/MODY1 variant I271fs,the T2D-associated HNF4A variant (rs1800961) was found to activate AKAP1,GAD2 and HOPX gene expression,potentially due to changes in DNA-binding affinity. We also found HNF1A to bind to and regulate GPR39 expression in beta cells. Overall,our studies provide a rich resource for uncovering downstream molecular targets of HNF4A and HNF1A that may contribute to beta cell or hepatic cell (dys)function,and set up a framework for gene discovery and functional validation. Here,the authors generated a genome-wide map of the global targets bound by HNF4A and HNF1A in beta cells and hepatic cells,and highlighted notable downstream pathways and target genes that may influence beta cell function. This approach also shed light on a potentially activating effect of a HNF4A type 2 diabetes risk variant. View Publication

SummaryFollicular lymphoma (FL) is an indolent non-Hodgkin lymphoma of germinal center origin,which presents with significant biologic and clinical heterogeneity. Using RNA-seq on B cells sorted from 87 FL biopsies,combined with machine-learning approaches,we identify 3 transcriptional states that divide the biological ontology of FL B cells into inflamed,proliferative,and chromatin-modifying states,with relationship to prior GC B cell phenotypes. When integrated with whole-exome sequencing and immune profiling,we find that each state was associated with a combination of mutations in chromatin modifiers,copy-number alterations to TNFAIP3,and T follicular helper cells (Tfh) cell interactions,or primarily by a microenvironment rich in activated T cells. Altogether,these data define FL B cell transcriptional states across a large cohort of patients,contribute to our understanding of FL heterogeneity at the tumor cell level,and provide a foundation for guiding therapeutic intervention. Graphical abstract Highlights•B cells from follicular lymphoma exhibit 3 distinct transcriptional states•FL B cells differ by enhanced inflammation,proliferation,or chromatin remodeling•Tumor cell states correlate with unique immune-microenvironment features•Unique mutation and CNV profiles highlight potential genetic causes of heterogeneity Krull et al. analyzed bulk transcriptional,genomic,and immune profiles of B cells from follicular lymphoma and reveal 3 distinct transcriptional states. These cell states underscore the inherent variability of FL tumors,independent of stroma,and implicate intrinsic differences as an underpinning to FL heterogeneity. View Publication

The antitumor efficacy of adoptively transferred T cells is limited by their poor persistence,in part due to exhaustion,but the underlying mechanisms and potential interventions remain underexplored. Here,we show that targeting histone demethylase LSD1 by chemical inhibitors reshapes the epigenome of in vitro activated and expanded CD8+ T cells,and potentiates their antitumor efficacy. Upon T cell receptor activation and IL-2 signaling,a timely and transient inhibition of LSD1 suffices to improve the memory phenotype of mouse CD8+ T cells,associated with a better ability to produce multiple cytokines,resist exhaustion,and persist in both antigen-dependent and -independent manners after adoptive transfer. Consequently,OT1 cells primed with LSD1 inhibitors demonstrate an enhanced antitumor effect in OVA-expressing solid tumor models implanted in female mice,both as a standalone treatment and in combination with PD-1 blockade. Moreover,priming with LSD1 inhibitors promotes polyfunctionality of human CD8+ T cells,and increases the persistence and antitumor efficacy of human CD19-CAR T cells in both leukemia and solid tumor models. Thus,pharmacological inhibition of LSD1 could be exploited to improve adoptive T cell therapy. Phenotypic changes in exhausted T cells are linked to chromatin remodeling. Here the authors show that pharmacological inhibition of the H3K4me1/2 demethylase LSD1 promotes the persistence and enhances the therapeutic activity of adoptively transferred T cells for cancer therapy. View Publication