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

Respiratory organoids have emerged as a powerful in vitro model for studying respiratory diseases and drug discovery. However,the high-throughput analysis of organoid images remains a challenge due to the lack of automated and accurate segmentation tools. This study presents a semi-automatic algorithm for image analysis of respiratory organoids (nasal and lung organoids),employing the U-Net architecture and CellProfiler for organoids segmentation. The algorithm processes bright-field images acquired through z-stack fusion and stitching. The model demonstrated a high level of accuracy,as evidenced by an intersection-over-union metric (IoU) of 0.8856,F1-score = 0.937 and an accuracy of 0.9953. Applied to forskolin-induced swelling assays of lung organoids,the algorithm successfully quantified functional differences in Cystic Fibrosis Transmembrane conductance Regulator (CFTR)-channel activity between healthy donor and cystic fibrosis patient-derived organoids,without fluorescent dyes. Additionally,an open-source dataset of 827 annotated respiratory organoid images was provided to facilitate further research. Our results demonstrate the potential of deep learning to enhance the efficiency and accuracy of high-throughput respiratory organoid analysis for future therapeutic screening applications. Author summaryIn this study,we developed a semi-automated tool to analyze images of respiratory organoids—3D cell structures that mimic the human respiratory system. These organoids are vital for studying diseases like cystic fibrosis and testing potential drugs,but manually analyzing their images is time-consuming and prone to errors. Our tool uses artificial intelligence (AI) to quickly and accurately measure organoid size and shape from bright-field microscope images,eliminating the need for fluorescent dyes that can harm cells. We trained our AI model on a publicly shared dataset of 827 annotated organoid images,achieving high accuracy in detecting and quantifying organoids. When applied to cystic fibrosis research,the tool successfully measured differences in organoid swelling (forskolin-induced swelling - a key test for drug response) between healthy and patient-derived samples. By making our dataset and method openly available,we hope to support further research into respiratory diseases. Our work bridges the gap between complex lab techniques and practical applications,offering a faster,more reliable way to study human health and disease. View Publication

RNA-binding motif protein 15 (RBM15) is involved in the RBM15-megakaryoblastic leukemia 1 fusion in acute megakaryoblastic leukemia. Although Rbm15 has been reported to be required for B-cell differentiation and to inhibit myeloid and megakaryocytic expansion,it is not clear what the normal functions of Rbm15 are in the regulation of hematopoietic stem cell (HSC) and megakaryocyte development. In this study,we report that Rbm15 may function in part through regulation of expression of the proto-oncogene c-Myc. Similar to c-Myc knockout (c-Myc-KO) mice,long-term (LT) HSCs are significantly increased in Rbm15-KO mice due to an apparent LT-HSC to short-term HSC differentiation defect associated with abnormal HSC-niche interactions caused by increased N-cadherin and beta(1) integrin expression on mutant HSCs. Both serial transplantation and competitive reconstitution capabilities of Rbm15-KO LT-HSCs are greatly compromised. Rbm15-KO and c-Myc-KO mice also share related abnormalities in megakaryocyte development,with mutant progenitors producing increased,abnormally small low-ploidy megakaryocytes. Consistent with a possible functional interplay between Rbm15 and c-Myc,the megakaryocyte increase in Rbm15-KO mice could be partially reversed by ectopic c-Myc. Thus,Rbm15 appears to be required for normal HSC-niche interactions,for the ability of HSCs to contribute normally to adult hematopoiesis,and for normal megakaryocyte development; these effects of Rbm15 on hematopoiesis may be mediated at least in part by c-Myc. View Publication

MafB is a member of the large Maf family of transcription factors that share similar basic region/leucine zipper DNA binding motifs and N-terminal activation domains. Although it is well known that MafB is specifically expressed in glomerular epithelial cells (podocytes) and macrophages,characterization of the null mutant phenotype in these tissues has not been previously reported. To investigate suspected MafB functions in the kidney and in macrophages,we generated mafB/green fluorescent protein (GFP) knock-in null mutant mice. MafB homozygous mutants displayed renal dysgenesis with abnormal podocyte differentiation as well as tubular apoptosis. Interestingly,these kidney phenotypes were associated with diminished expression of several kidney disease-related genes. In hematopoietic cells,GFP fluorescence was observed in both Mac-1- and F4/80-expressing macrophages in the fetal liver. Interestingly,F4/80 expression in macrophages was suppressed in the homozygous mutant,although development of the Mac-1-positive macrophage population was unaffected. In primary cultures of fetal liver hematopoietic cells,MafB deficiency was found to dramatically suppress F4/80 expression in nonadherent macrophages,whereas the Mac-1-positive macrophage population developed normally. These results demonstrate that MafB is essential for podocyte differentiation,renal tubule survival,and F4/80 maturation in a distinct subpopulation of nonadherent mature macrophages. View Publication

Canine leukocyte adhesion deficiency (CLAD) represents the canine counter-part of the human disease leukocyte adhesion deficiency (LAD). Defects in the leukocyte integrin CD18 adhesion molecule in both CLAD and LAD lead to recurrent,life-threatening bacterial infections. We evaluated ex vivo retroviral-mediated gene therapy in CLAD using 2 nonmyeloablative conditioning regimens--200 cGy total body irradiation (TBI) or 10 mg/kg busulfan--with or without posttransplantation immunosuppression. In 6 of 11 treated CLAD dogs,therapeutic levels of CD18(+) leukocytes were achieved. Conditioning with either TBI or busulfan allowed long-term engraftment,and immunosuppression was not required for efficacy. The percentage of CD18(+) leukocytes in the peripheral blood progressively increased over 6 to 8 months after infusion to levels ranging from 1.26% to 8.37% at 1-year follow-up in the 6 dogs. These levels resulted in reversal or moderation of the severe CLAD phenotype. Linear amplification-mediated polymerase chain reaction assays indicated polyclonality of insertion sites. These results describe ex vivo hematopoietic stem cell gene transfer in a disease-specific,large animal model using 2 clinically applicable conditioning regimens,and they provide support for the use of nonmyeloablative conditioning regimens in preclinical protocols of retroviral-mediated gene transfer for nonmalignant hematopoietic diseases such as LAD. View Publication

Most polyreactive and antinuclear antibodies are removed from the human antibody repertoire during B cell development. To elucidate how B cell receptor (BCR) signaling may regulate human B cell tolerance,we tested the specificity of recombinant antibodies from single peripheral B cells isolated from patients suffering from X-linked agammaglobulinemia (XLA). These patients carry mutations in the Bruton's tyrosine kinase (BTK) gene that encode an essential BCR signaling component. We find that in the absence of Btk,peripheral B cells show a distinct antibody repertoire consistent with extensive secondary V(D)J recombination. Nevertheless,XLA B cells are enriched in autoreactive clones. Our results demonstrate that Btk is essential in regulating thresholds for human B cell tolerance. View Publication

NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5(eGFP/w) hESCs facilitate quantification of cardiac differentiation,purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP(+) cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages. View Publication

Peroxisomes are eukaryotic organelles that are essential for multiple metabolic pathways,including fatty acid oxidation,degradation of amino acids,and biosynthesis of ether lipids. Consequently,peroxisome dysfunction leads to pediatric-onset neurodegenerative conditions,including Peroxisome Biogenesis Disorders (PBD). Due to the dynamic,tissue-specific,and context-dependent nature of their biogenesis and function,live cell imaging of peroxisomes is essential for studying peroxisome regulation,as well as for the diagnosis of PBD-linked abnormalities. However,the peroxisomal imaging toolkit is lacking in many respects,with no reporters for substrate import,nor cell-permeable probes that could stain dysfunctional peroxisomes. Here we report that the BODIPY-C12 fluorescent fatty acid probe stains functional and dysfunctional peroxisomes in live mammalian cells. We then go on to improve BODIPY-C12,generating peroxisome-specific reagents,PeroxiSPY650 and PeroxiSPY555. These probes combine high peroxisome specificity,bright fluorescence in the red and far-red spectrum,and fast non-cytotoxic staining,making them ideal tools for live cell,whole organism,or tissue imaging of peroxisomes. Finally,we demonstrate that PeroxiSPY enables diagnosis of peroxisome abnormalities in the PBD CRISPR/Cas9 cell models and patient-derived cell lines. The array of tools to image peroxisome regulation is still limited. Here,the authors develop improved fatty acid-based probes with high peroxisome specificity and bright fluorescence in the red/far-red spectrum,which makes them ideal to study peroxisomes in live cells and whole organisms. View Publication

AbstractBackgroundWhile recent clinical trials of combination immunotherapies for hepatocellular carcinoma (HCC) have shown promising clinical efficacy and survival improvements breakthroughs,there is still much room for further improvement. A key limiting factor for HCC immunotherapy is the intrinsic immunosuppression within the liver microenvironment,resulting in suboptimal priming of tumor-specific CD8 cytotoxic T cells and thus immune evasion by the tumor. Hence,identifying new key molecular pathways suppressing T-cell responses within the liver is critical for the rational design of more effective combination immunotherapies for HCC.MethodsWe identified the 5-HT2A serotonin receptor as a potential target for HCC immunotherapy in a chemical screening approach and validated that targeting 5-HT2A signaling could be a viable approach for HCC immunotherapy via in vitro and in vivo studies.ResultsDisruption of 5-HT2A signaling using either a selective antagonist small molecule,ketanserin,or by knockout of its coding gene Htr2a augments the cytotoxic effector phenotype of mouse CD8 T cells activated in vitro with immunosuppressive liver non-parenchymal cells. Ketanserin treatment of in vitro activated human CD8 T cells also increased expression of the cytotoxic effector molecules granzyme B and perforin. Abrogation of 5-HT2A signaling was associated with increased expression of cytotoxicity-related genes such as granzyme B and reduced expression of transcription factors downstream of MAP kinase signaling. In vivo,systemic ketanserin treatment significantly prolonged survival of HCC tumor-bearing mice and was non-inferior to α-programmed death ligand 1 (PD-L1)+α-vascular endothelial growth factor A (VEGFA) combination antibody treatment. Combining ketanserin with αPD-L1+αVEGFA antibodies also significantly prolonged survival relative to control-treated mice while preserving the occurrence of complete tumor regression observed with αPD-L1+αVEGFA treatment alone.ConclusionsTogether,our data describe a role for 5-HT2A as a negative regulator of the cytotoxic effector phenotype in CD8 T cells and highlight the therapeutic potential of targeting 5-HT2A for HCC immunotherapy. View Publication

Oxidative stress is implicated in the pathogenesis of neuronal degenerative diseases. Oxidative stress has been shown to activate extracellular signal-regulated kinases (ERK)1/2. We investigated the role of these mitogen-activated protein kinases (MAPKs) in oxidative neuronal injury by using a mouse hippocampal cell line (HT22) and rat primary cortical cultures. Here,we show that a novel MAPK/ERK kinase (MEK) specific inhibitor U0126 profoundly protected HT22 cells against oxidative stress induced by glutamate,which was accompanied by an inhibition of phosphorylation of ERK1/2. U0126 also protected rat primary cultured cortical neurons against glutamate or hypoxia. However,U0126 was not protective against death caused by tumor necrosis factor alpha (TNFalpha),A23187,or staurosporine. These results indicate that MEK plays a central role in the neuronal death caused by oxidative stress. View Publication

Threats of bioterrorism have renewed efforts to better understand poxvirus pathogenesis and to develop a safer vaccine against smallpox. Individuals with atopic dermatitis are excluded from smallpox vaccination because of their propensity to develop eczema vaccinatum,a disseminated vaccinia virus (VACV) infection. To study the underlying mechanism of the vulnerability of atopic dermatitis patients to VACV infection,we developed a mouse model of eczema vaccinatum. Virus infection of eczematous skin induced severe primary erosive skin lesions,but not in the skin of healthy mice. Eczematous mice exhibited lower natural killer (NK) cell activity but similar cytotoxic T lymphocyte activity and humoral immune responses. The role of NK cells in controlling VACV-induced skin lesions was demonstrated by experiments depleting or transferring NK cells. The proinflammatory cytokine interleukin (IL)-17 reduced NK cell activity in mice with preexisting dermatitis. Given low NK cell activities and increased IL-17 expression in atopic dermatitis patients,these results can explain the increased susceptibility of atopic dermatitis patients to eczema vaccinatum. View Publication

A collaborative study of the second international Reference Preparation of Erythropoietin,Human,Urinary,for Bioassay was carried out in 10 laboratories. Combined potency estimates obtained by comparison with the International Reference Preparation,indicate that ampoules of the second Preparation contain 10.0 IU (weighted mean potency) or,taking the unweighted mean potency,9.8 IU,with fiducial limits (P=0.95) of 8.4-11.5 IU. The second Preparation could be used as a standard in estimating the potency of a preparation of sheep plasma erythropoietin (68/307) although,as with the International Reference Preparation,there was a tendency for the sheep plasma preparation to produce log-dose-log-response regression lines that were steeper than those produced by the second Preparation.In accelerated degradation studies of the second Preparation stored as the dry product in ampoules for up to 1 year,there was no consistent trend to indicate instability of the preparation.Following its establishment in 1971,the Second International Reference Preparation was allocated a potency of 10 IU/ampoule. View Publication

Human embryonic stem cells (hESCs) can be differentiated into multiple cell types with great therapeutic potential. However,optimizing the often multi-week cultures to obtain sufficient differentiated cell yields has been in part limited by the high variability of even parallel hESC differentiation cultures. We describe the isolation and features of a subline of CA1 hESCs (CA1S) that display a very high 25% cloning efficiency while retaining many properties of the parental hESCs,including being karyotypically normal and their ability to generate teratomas containing all three germ layers. Although more detailed analysis revealed that CA1S cells have a 3.8 Mb genomic duplication on chromosome 20,they remain highly useful. In particular,CA1S cells are readily expanded at high yields in culture and possess greatly reduced well-to-well variation even when seeded at 100 cells/well. Thus,108 CA1S cells can be generated within one week from 106 cells to seed 106 wells. We determined that CA1S cells have the capacity to follow established in vitro differentiation protocols to pancreatic progenitors and subsequent hormone-positive cell types and used CA1S cells to explore definitive endoderm induction in a high performance screen (Z-factor = 0.97). This system revealed that CA1S cells do not require WNT3A to efficiently form definitive endoderm,a finding that was confirmed with H1 hESCs,although H1 cells did show modest benefits of high WNT3A doses. Proliferative index measurements of CA1S cells were shown to rapidly reflect their differentiation status in a high throughput system. Though results obtained with CA1S cells will need to be confirmed using conventional hESC lines,these cells should ease the development of optimized hESC growth and differentiation protocols. In particular,they should limit the more arduous secondary screens using hESCs to a smaller number of variables and doses. Biotechnol. Bioeng. 2013;110: 2706–2716. textcopyright 2013 Wiley Periodicals,Inc. View Publication