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

The recruitment of monocytes and their differentiation into macrophages at sites of inflammation are key events in determining the outcome of the inflammatory response and initiating the return to tissue homeostasis. To study monocyte trafficking and macrophage differentiation in vivo,we have generated a novel transgenic reporter mouse expressing a green fluorescent protein (GFP) under the control of the human CD68 promoter. CD68-GFP mice express high levels of GFP in both monocyte and embryo-derived tissue resident macrophages in adult animals. The human CD68 promoter drives GFP expression in all CD115(+) monocytes of adult blood,spleen,and bone marrow; we took advantage of this to directly compare the trafficking of bone marrow-derived CD68-GFP monocytes to that of CX3CR1(GFP) monocytes in vivo using a sterile zymosan peritonitis model. Unlike CX3CR1(GFP) monocytes,which downregulate GFP expression on differentiation into macrophages in this model,CD68-GFP monocytes retain high-level GFP expression for 72 hours after differentiation into macrophages,allowing continued cell tracking during resolution of inflammation. In summary,this novel CD68-GFP transgenic reporter mouse line represents a powerful resource for analyzing monocyte mobilization and monocyte trafficking as well as studying the fate of recruited monocytes in models of acute and chronic inflammation. View Publication

In this study,we trace developmental stages using epigenome changes in human embryonic stem cells (hESCs) treated with drugs modulating either self-renewal or differentiation. Based on microscopy,qPCR and flow cytometry,we classified the treatment outcome as inducing pluripotency (hESC,flurbiprofen and gatifloxacin),mesendoderm (sinomenine),differentiation (cyamarin,digoxin,digitoxin,selegeline and theanine) and lineage-commitment (RA). When we analyzed histone PTMs that imprinted these gene and protein expressions,the above classification was reassorted. Hyperacetylation at H3K4,9,14,18,56 and 122 as well as H4K5,8,12 and 16 emerged as the pluripotency signature of hESCs. Methylations especially of H3 at K9,K20,K27 and K36 characterized differentiation initiation as seen in no-drug control and fluribiprofen. Sinomenine-treated cells clustered close to differentiation initiators" View Publication

Alveolar macrophages (AM) perform a primary defense mechanism in the lung through phagocytosis of inhaled particles and microorganisms. AM are known to be relatively immunosuppressive consistent with the aim to limit alveolar inflammation and maintain effective gas exchange in the face of these constant challenges. How AM respond to T cell derived cytokine signals,which are critical to the defense against inhaled pathogens,is less well understood. For example,successful containment of Mycobacterium tuberculosis (Mtb) in lung macrophages is highly dependent on IFN-$\gamma$ secreted by Th-1 lymphocytes,however,the proteomic IFN-$\gamma$ response profile in AM remains mostly unknown. In this study,we measured IFN-$\gamma$ induced protein abundance changes in human AM and autologous blood monocytes (MN). AM cells were activated by IFN-$\gamma$ stimulation resulting in STAT1 phosphorylation and production of MIG/CXCL9 chemokine. However,the global proteomic response to IFN-$\gamma$ in AM was dramatically limited in comparison to that of MN (9 AM vs 89 MN differentially abundant proteins). AM hypo-responsiveness was not explained by reduced JAK-STAT1 signaling nor increased SOCS1 expression. These findings suggest that AM have a tightly regulated response to IFN-$\gamma$ which may prevent excessive pulmonary inflammation but may also provide a niche for the initial survival and growth of Mtb and other intracellular pathogens in the lung. View Publication

SummaryCardiac fibrosis is a key characteristic of heart failure. CTPR390,an experimental anti-fibrotic inhibitor targeting Hsp90,has shown success in animal models,but remains unexplored in human cardiac models. This study evaluated an engineered cardiac connective tissue (ECCT) model,focusing on changes in the extracellular matrix and fibroblasts. Results showed that CTPR390 prevented architectural changes in TGF?1-activated ECCT,preserving tissue perimeter,collagen fibers alignment while reducing structured areas and degree of collagen structuration. CTPR390 treatment reduced cell area of fibroblasts under tension,without changes in the internal rounded cells devoid of tension. Fibroblast recruitment to tension areas was diminished,showing biomechanical behavior similar to control ECCT. This treatment also lowered the gene and protein expression of key pro-fibrotic markers. Here,advanced biotechnology was employed to detect the detailed structure of tissue fibrosis reduction after administering CTPR390,representing a significant advancement toward clinical application for cardiac fibrosis treatment. Graphical abstract Highlights•CTPR390 prevented architectural changes in TGF?1-activated ECCT•CTPR390 preserves tissue perimeter,collagen fibers alignment•CTPR390 reduces structured areas and degree of collagen structuration•CTPR390-trested ECCTs presented a biomechanical behavior similar to control ECCT Molecular biology; Cell biology View Publication

Background: Alzheimer's disease (AD) is characterized by progressive amyloid beta (Aβ) deposition in the brain,with eventual widespread neurodegeneration. While the cell-specific molecular signature of end-stage AD is reasonably well characterized through autopsy material,less is known about the molecular pathways in the human brain involved in the earliest exposure to Aβ. Human model systems that not only replicate the pathological features of AD but also the transcriptional landscape in neurons,astrocytes and microglia are crucial for understanding disease mechanisms and for identifying novel therapeutic targets. Methods: In this study,we used a human 3D iPSC-derived neurosphere model to explore how resident neurons,microglia and astrocytes and their interplay are modified by chronic amyloidosis induced over 3-5 weeks by supplementing media with synthetic Aβ1 - 42 oligomers. Neurospheres under chronic Aβ exposure were grown with or without microglia to investigate the functional roles of microglia. Neuronal activity and oxidative stress were monitored using genetically encoded indicators,including GCaMP6f and roGFP1,respectively. Single nuclei RNA sequencing (snRNA-seq) was performed to profile Aβ and microglia driven transcriptional changes in neurons and astrocytes,providing a comprehensive analysis of cellular responses. Results: Microglia efficiently phagocytosed Aβ inside neurospheres and significantly reduced neurotoxicity,mitigating amyloidosis-induced oxidative stress and neurodegeneration following different exposure times to Aβ. The neuroprotective effects conferred by the presence of microglia was associated with unique gene expression profiles in astrocytes and neurons,including several known AD-associated genes such as APOE. These findings reveal how microglia can directly alter the molecular landscape of AD. Conclusions: Our human 3D neurosphere culture system with chronic Aβ exposure reveals how microglia may be essential for the cellular and transcriptional responses in AD pathogenesis. Microglia are not only neuroprotective in neurospheres but also act as key drivers of Aβ-dependent APOE expression suggesting critical roles for microglia in regulating APOE in the AD brain. This novel,well characterized,functional in vitro platform offers unique opportunities to study the roles and responses of microglia to Aβ modelling key aspects of human AD. This tool will help identify new therapeutic targets,accelerating the transition from discovery to clinical applications. View Publication

Cardiac ventricular pressure overload affects patients with congenital heart defects and can cause cardiac insufficiency. Grafts of stem cell–derived cardiomyocytes are proposed as a complementary treatment to surgical repair of the cardiac defect,aiming to support ventricular function. Here,we report successful engraftment of human induced pluripotent stem cell–derived cardiac lineage cells into the heart of immunosuppressed rhesus macaques with a novel surgical model of right ventricular pressure overload. The human troponin+ grafts were detected in low-dose (2 × 106 cells/kg) and high-dose (10 × 106 cells/kg) treatment groups up to 12 weeks post-injection. Transplanted cells integrated and progressively matched the organization of the surrounding host myocardium. Ventricular tachycardia occurred in five out of 16 animals receiving cells,with episodes of incessant tachycardia observed in two animals; ventricular tachycardia events resolved within 19 days. Our results demonstrate that grafted cardiomyocytes mature and integrate into the myocardium of nonhuman primates modeling right ventricular pressure overload. View Publication

RAB7,encoded by RAB7A in humans and Rab7 in mice,is a small GTPase that catalyzes endosome maturation. It mediates NF-κB activation through the assembly of intracellular membrane signalosomes in stimulated normal B cells and plays a B cell-intrinsic role in the antibody response in mice. Here we show RAB7A transcripts are expressed in primary diffuse large B-cell lymphomas (DLBCLs),and that RAB7 protein expression is heightened in activated human tonsil B cells as well as in DLBCL and Burkitt lymphoma cell lines. Treating these cell lines with CID1067700,a selective small-molecule RAB7 inhibitor,results in a dose-dependent decrease in cell growth,associated with impaired proliferation and survival. CID1067700 also suppressed tumor development from Daudi cells,a Burkitt lymphoma cell line,in Foxn1nu/nu nude mice. The inhibitory effect of CID1067700 on Daudi cell growth in vitro is further enhanced by methyl-β-cyclodextrin,which disrupts plasma membrane lipid rafts,and by FX1,a BCL6 inhibitor. These findings,together with the unfavorable prognosis of DLBCL patients showing high RAB7A expression,suggest that targeting RAB7 is a promising therapeutic approach for mature B cell-derived lymphomas. View Publication

Chimeric antigen receptor (CAR) T cell therapies targeting B cell-restricted antigens CD19,CD20,or CD22 can produce potent clinical responses for some B cell malignancies,but relapse remains common. Camelid single-domain antibodies (sdAbs or nanobodies) are smaller,simpler,and easier to recombine than single-chain variable fragments (scFvs) used in most CARs,but fewer sdAb-CARs have been reported. Thus,we sought to identify a therapeutically active sdAb-CAR targeting human CD22. Immunization of an adult Llama glama with CD22 protein,sdAb-cDNA library construction,and phage panning yielded >20 sdAbs with diverse epitope and binding properties. Expressing CD22-sdAb-CAR in Jurkat cells drove varying CD22-specific reactivity not correlated with antibody affinity. Changing CD28- to CD8-transmembrane design increased CAR persistence and expression in vitro . CD22-sdAb-CAR candidates showed similar CD22-dependent CAR-T expansion in vitro,although only membrane-proximal epitope targeting CD22-sdAb-CARs activated direct cytolytic killing and extended survival in a lymphoma xenograft model. Based on enhanced survival in blinded xenograft studies,a lead CD22sdCAR-T was selected,achieving comparable complete responses to a benchmark short linker m971-scFv CAR-T in high-dose experiments. Finally,immunohistochemistry and flow cytometry confirm tissue and cellular-level specificity of the lead CD22-sdAb. This presents a complete report on preclinical development of a novel CD22sdCAR therapeutic. View Publication

In Alzheimer’s disease,amyloid beta (Aβ) and tau pathology are thought to drive synapse loss. However,there is limited information on how endogenous levels of tau,Aβ and other biomarkers relate to patient characteristics,or how manipulating physiological levels of Aβ impacts synapses in living adult human brain. Using live human brain slice cultures,we report that Aβ 1-40 and tau release levels vary with donor age and brain region,respectively. Release of other biomarkers such as KLK-6,NCAM-1,and Neurogranin vary between brain region,while TDP-43 and NCAM-1 release is impacted by sex. Pharmacological manipulation of Aβ in either direction results in a loss of synaptophysin puncta,with increased physiological Aβ triggering potentially compensatory synaptic transcript changes. In contrast,treatment with Aβ-containing Alzheimer’s disease brain extract results in post-synaptic Aβ uptake and pre-synaptic puncta loss without affecting synaptic transcripts. These data reveal distinct effects of physiological and pathological Aβ on synapses in human brain tissue. Subject terms: Alzheimer's disease,Alzheimer's disease View Publication

The upper airway epithelium,which is mainly composed of multiciliated,goblet,club and basal cells,ensures proper mucociliary function and can regenerate in response to assaults. In chronic airway diseases,defective repair leads to tissue remodeling. Delineating key drivers of differentiation dynamics can help understand how normal or pathological regeneration occurs. Using single-cell transcriptomics and lineage inference,we have unraveled trajectories from basal to luminal cells,providing novel markers for specific populations. We report that: (1) a precursor subgroup of multiciliated cells,which we have entitled deuterosomal cells,is defined by specific markers,such as DEUP1,FOXN4,YPEL1,HES6 and CDC20B; (2) goblet cells can be precursors of multiciliated cells,thus explaining the presence of hybrid cells that co-express markers of goblet and multiciliated cells; and (3) a repertoire of molecules involved in the regeneration process,such as keratins or components of the Notch,Wnt or BMP/TGF$\beta$ pathways,can be identified. Confirmation of our results on fresh human and pig airway samples,and on mouse tracheal cells,extend and confirm our conclusions regarding the molecular and cellular choreography at work during mucociliary epithelial differentiation. View Publication

A promising strategy to limit cholera severity involves blockers mimicking the canonical cholera toxin ligand (CT) ganglioside GM1. However,to date the efficacies of most of these blockers have been evaluated in noncellular systems that lack ligands other than GM1. Importantly,the CT B subunit (CTB) has a noncanonical site that binds fucosylated structures,which in contrast to GM1 are highly expressed in the human intestine. Here we evaluate the capacity of norbornene polymers displaying galactose and/or fucose to block CTB binding to immobilized protein-linked glycan structures and also to primary human and murine small intestine epithelial cells (SI ECs). We show that the binding of CTB to human SI ECs is largely dependent on the noncanonical binding site,and interference with the canonical site has a limited effect while the opposite is observed with murine SI ECs. The galactose-fucose polymer blocks binding to fucosylated glycans but not to GM1. However,the preincubation of CT with the galactose-fucose polymer only partially blocks toxic effects on cultured human enteroid cells,while preincubation with GM1 completely blocks CT-mediated secretion. Our results support a model whereby the binding of fucose to the noncanonical site places CT in close proximity to scarcely expressed galactose receptors such as GM1 to enable binding via the canonical site leading to CT internalization and intoxication. Our finding also highlights the importance of complementing CTB binding studies with functional intoxication studies when assessing the efficacy inhibitors of CT. View Publication

In humans,recent clinical and experimental data from hematopoietic stem cell transplantation revealed that donor-derived alloreactive NK cells exert a beneficial graft versus leukemia effect. The existence of donor-derived alloreactive NK cells can be predicted on the basis of donor killer cell Ig-like receptor (KIR) gene profile and HLA class I typing of both donor and recipient. Moreover,the size of the alloreactive NK cell population can be directly assessed by the combined use of anti-KIR-specific mAb. In this study,in an attempt to improve the definition of alloreactive NK cell subsets,we assessed the KIR genotype and phenotype in a cohort of 44 donors. This approach allowed the identification of two different KIR2DL3 alleles (KIR2DL3*005 and the novel allele KIR2DL3*015) that did not react with the anti-KIR2DL3-specific ECM41 mAb. In contrast,both alleles were recognized at the cell surface by several mAb reacting with KIR2DL2/L3/S2. Notably,KIR2DL3*005 was also stained by the anti-KIR2DL1/S1-specific EB6B and 11PB6 mAb. Functional analysis revealed that,despite its particular mAb reactivity,the specificity of KIR2DL3*005 for HLA-C molecules did not differ from that of other KIR2DL2/L3 alleles. Finally,site-directed mutagenesis demonstrated that glutamine at position 35 is required for ECM41 staining,whereas glutamic acid 35 and arginine 50 are relevant for staining with EB6B or 11PB6 mAb. Our present data represent a substantial progress in the characterization of the NK cell repertoire and an improved phenotypic/functional definition of given KIR(+) subsets. View Publication