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

Acute megakaryoblastic leukemia (AMKL) represents ∼10{\%} of pediatric acute myeloid leukemia cases and typically affects young children ({\textless}3 years of age). It remains plagued with extremely poor treatment outcomes ({\textless}40{\%} cure rates),mostly due to primary chemotherapy refractory disease and/or early relapse. Recurrent and mutually exclusive chimeric fusion oncogenes have been detected in 60{\%} to 70{\%} of cases and include nucleoporin 98 (NUP98) gene rearrangements,most commonly NUP98-KDM5A. Human models of NUP98-KDM5A-driven AMKL capable of faithfully recapitulating the disease have been lacking,and patient samples are rare,further limiting biomarkers and drug discovery. To overcome these impediments,we overexpressed NUP98-KDM5A in human cord blood hematopoietic stem and progenitor cells using a lentiviral-based approach to create physiopathologically relevant disease models. The NUP98-KDM5A fusion oncogene was a potent inducer of maturation arrest,sustaining long-term proliferative and progenitor capacities of engineered cells in optimized culture conditions. Adoptive transfer of NUP98-KDM5A-transformed cells into immunodeficient mice led to multiple subtypes of leukemia,including AMKL,that phenocopy human disease phenotypically and molecularly. The integrative molecular characterization of synthetic and patient NUP98-KDM5A AMKL samples revealed SELP,MPIG6B,and NEO1 as distinctive and novel disease biomarkers. Transcriptomic and proteomic analyses pointed to upregulation of the JAK-STAT signaling pathway in the model AMKL. Both synthetic models and patient-derived xenografts of NUP98-rearranged AMKL showed in vitro therapeutic vulnerability to ruxolitinib,a clinically approved JAK2 inhibitor. Overall,synthetic human AMKL models contribute to defining functional dependencies of rare genotypes of high-fatality pediatric leukemia,which lack effective and rationally designed treatments. View Publication

BackgroundSevere peripheral nerve damage always requires surgical treatment. Autologous nerve transplantation is a standard treatment,but it is not sufficient due to length limitations and extended surgical time. Even with the available artificial nerves,there is still large room for improvement in their therapeutic effects. Novel treatments for peripheral nerve injury are greatly expected.MethodsUsing a specialized microfluidic device,we generated artificial neurite bundles from human iPSC-derived motor and sensory nerve organoids. We developed a new technology to isolate cell-free neurite bundles from spheroids. Transplantation therapy was carried out for large nerve defects in rat sciatic nerve with novel artificial nerve conduit filled with lineally assembled sets of human neurite bundles. Quantitative comparisons were performed over time to search for the artificial nerve with the therapeutic effect,evaluating the recovery of motor and sensory functions and histological regeneration. In addition,a multidimensional unbiased gene expression profiling was carried out by using next-generation sequencing.ResultAfter transplantation,the neurite bundle-derived artificial nerves exerted significant therapeutic effects,both functionally and histologically. Remarkably,therapeutic efficacy was achieved without immunosuppression,even in xenotransplantation. Transplanted neurite bundles fully dissolved after several weeks,with no tumor formation or cell proliferation,confirming their biosafety. Posttransplant gene expression analysis highlighted the immune system’s role in recovery.ConclusionThe combination of newly developed microfluidic devices and iPSC technology enables the preparation of artificial nerves from organoid-derived neurite bundles in advance for future treatment of peripheral nerve injury patients. A promising,safe,and effective peripheral nerve treatment is now ready for clinical application.Supplementary InformationThe online version contains supplementary material available at 10.1186/s41232-024-00319-4. View Publication

Acute myeloid leukemia (AML) is fatal in the majority of adults. Identification of new therapeutic targets and their pharmacologic modulators are needed to improve outcomes. Previous studies had shown that immunization of rabbits with normal peripheral WBCs that had been incubated with fluorodinitrobenzene elicited high titer antibodies that bound to a spectrum of human leukemias. We report that proteomic analyses of immunoaffinity-purified lysates of primary AML cells showed enrichment of scaffolding protein IQGAP1. Immunohistochemistry and gene-expression analyses confirmed IQGAP1 mRNA overexpression in various cytogenetic subtypes of primary human AML compared to normal hematopoietic cells. shRNA knockdown of IQGAP1 blocked proliferation and clonogenicity of human leukemia cell-lines. To develop small molecules targeting IQGAP1 we performed in-silico screening of 212,966 compounds,selected 4 hits targeting the IQGAP1-GRD domain,and conducted SAR of the ‘fittest hit’ to identify UR778Br,a prototypical agent targeting IQGAP1. UR778Br inhibited proliferation,induced apoptosis,resulted in G2/M arrest,and inhibited colony formation by leukemia cell-lines and primary-AML while sparing normal marrow cells. UR778Br exhibited favorable ADME/T profiles and drug-likeness to treat AML. In summary, AML shows response to IQGAP1 inhibition,and UR778Br,identified through in-silico studies,selectively targeted AML cells while sparing normal marrow. Subject terms: Cancer,Cell biology,Drug discovery,Immunology,Oncology View Publication

Pseudomonas aeruginosa is a Gram-negative bacterium that is notorious for airway infections in cystic fibrosis (CF) subjects. Bacterial quorum sensing (QS) coordinates virulence factor expression and biofilm formation at population level. Better understanding of QS in the bacterium-host interaction is required. Here,we set up a new P. aeruginosa infection model,using 2D upper airway nasal organoids that were derived from 3D organoids. Using dual RNA-sequencing,we dissected the interaction between organoid epithelial cells and WT or QS-mutant P. aeruginosa strains. Since only a single healthy individual and a single CF subject were used as donors for the organoids,conclusions about CF-specific effects could not be deduced. However,P. aeruginosa induced epithelial inflammation,whereas QS signaling did not affect the epithelial airway cells. Conversely,the epithelium influenced infection-related processes of P. aeruginosa,including QS-mediated regulation. Comparison of our model with samples from the airways of CF subjects indicated that our model recapitulates important aspects of infection in vivo. Hence,the 2D airway organoid infection model is relevant and may help to reduce the future burden of P. aeruginosa infections in CF. The online version contains supplementary material available at 10.1038/s41598-024-82500-w. View Publication

Graphical Abstract Highlights d SMAD activity is active in suprabasal cells but is weaker in basal epithelial cells d SMAD signaling activity correlates with mucociliary differentiation in the airway d Dual TGFb/BMP inhibition prevents spontaneous differentiation in culture d Dual TGFb/BMP inhibition allows prolonged culture of diverse epithelial basal cells Correspondence jrajagopal@partners.org In Brief Mou et al. show that small-molecule-mediated SMAD signaling inhibition allows prolonged feeder-free culture of diverse functional epithelial basal stem cells in a 2D format. This methodology provides a facile patient-specific epithelial disease modeling platform,as shown by the expansion of airway epithelium from non-invasively obtained specimens from cystic fibrosis patients. View Publication

BACKGROUND: Cardiotoxicity is a leading cause for drug attrition during pharmaceutical development and has resulted in numerous preventable patient deaths. Incidents of adverse cardiac drug reactions are more common in patients with preexisting heart disease than the general population. Here we generated a library of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with various hereditary cardiac disorders to model differences in cardiac drug toxicity susceptibility for patients of different genetic backgrounds.backslashnbackslashnMETHODS AND RESULTS: Action potential duration and drug-induced arrhythmia were measured at the single cell level in hiPSC-CMs derived from healthy subjects and patients with hereditary long QT syndrome,familial hypertrophic cardiomyopathy,and familial dilated cardiomyopathy. Disease phenotypes were verified in long QT syndrome,hypertrophic cardiomyopathy,and dilated cardiomyopathy hiPSC-CMs by immunostaining and single cell patch clamp. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and the human ether-a-go-go-related gene expressing human embryonic kidney cells were used as controls. Single cell PCR confirmed expression of all cardiac ion channels in patient-specific hiPSC-CMs as well as hESC-CMs,but not in human embryonic kidney cells. Disease-specific hiPSC-CMs demonstrated increased susceptibility to known cardiotoxic drugs as measured by action potential duration and quantification of drug-induced arrhythmias such as early afterdepolarizations and delayed afterdepolarizations.backslashnbackslashnCONCLUSIONS: We have recapitulated drug-induced cardiotoxicity profiles for healthy subjects,long QT syndrome,hypertrophic cardiomyopathy,and dilated cardiomyopathy patients at the single cell level for the first time. Our data indicate that healthy and diseased individuals exhibit different susceptibilities to cardiotoxic drugs and that use of disease-specific hiPSC-CMs may predict adverse drug responses more accurately than the standard human ether-a-go-go-related gene test or healthy control hiPSC-CM/hESC-CM screening assays. View Publication

Cancer-targeting alkylphosphocholine (APC) analogs are being clinically developed for diagnostic imaging,intraoperative visualization,and therapeutic applications. These APC analogs derived from chemically-synthesized phospholipid ethers were identified and optimized for cancer-targeting specificity using extensive structure-activity studies. While they strongly label human brain cancers associated with disrupted blood-brain barriers (BBB),APC permeability across intact BBB remains unknown. Three of our APC analogs,CLR1404 (PET radiotracer),CLR1501 (green fluorescence),and CLR1502 (near infrared fluorescence),were tested for permeability across a BBB model composed of human induced pluripotent stem cell-derived brain microvascular endothelial cells (iPSC-derived BMECs). This in vitro BBB system has reproducibly consistent high barrier integrity marked by high transendothelial electrical resistance (TEERtextgreater1500 Ω-cm(2)) and functional expression of drug efflux transporters. Our radioiodinated and fluorescent APC analogs demonstrated fairly low permeability across the iPSC-BMEC (35±5.7 (CLR1404),54±3.2 (CLR1501),and 26±4.9 (CLR1502) x10(-5) cm/min) compared with BBB-impermeable sucrose (13±2.5) and BBB-permeable diazepam (170±29). Only our fluorescent APC analogs (CLR1501,CLR1502) underwent BCRP and MRP polarized drug efflux transport in the brain-to-blood direction of the BBB model and this efflux can be specifically blocked with pharmacological inhibition. None of our tested APC analogs appeared to undergo substantial P-gp transport. Limited permeability of our APC analogs across an intact BBB into normal brain likely contributes to the high tumor to background ratios observed in initial human trials. Moreover,addition of fluorescent moieties to APCs resulted in greater BMEC efflux via MRP and BCRP,and may affect fluorescence-guided applications. Overall,the characterization of APC analog permeability across human BBB is significant for advancing future brain tumor-targeted applications of these agents. View Publication

Spinocerebellar ataxia type3 (SCA3) is an autosomal dominant neurodegenerative disorder. Human embryonic stem cell line chHES-472 was derived from abnormal embryo donated by SCA3 patient after preimplantation genetic diagnosis (PGD) treatment. This cell line had a normal karyotype and retained the disease-causing mutant in ATXN3 gene. Characteristic tests proved that the embryonic stem cell line presented typical markers of pluripotency and had the capability to form the three germlayers in vivo. View Publication

Capsular polysaccharides are common virulence factors of extracellular,but not intracellular bacterial pathogens,due to the antiphagocytic properties of these surface structures. It is therefore paradoxical that Salmonella enterica subspecies enterica serovar Typhi,an intracellular pathogen,synthesizes a virulence-associated (Vi) capsule,which exhibits antiphagocytic properties. Here,we show that the Vi capsular polysaccharide has different functions when S. Typhi interacts with distinct subsets of host phagocytes. The Vi capsular polysaccharide allowed S. Typhi to selectively evade phagocytosis by human neutrophils while promoting human macrophage phagocytosis. A screen of C-type lectin receptors identified human DC-SIGN as the receptor involved in macrophage binding and phagocytosis of capsulated S. Typhi. Consistent with the anti-inflammatory activity of DC-SIGN,purified Vi capsular polysaccharide reduced inflammatory responses in macrophages. These data suggest that binding of the human C-type lectin receptor DC-SIGN by the Vi capsular polysaccharide contributes to the pathogenesis of typhoid fever. IMPORTANCE Salmonella enterica subspecies enterica serovar Typhi is the causative agent of typhoid fever. The recent emergence of S. Typhi strains which are resistant to antibiotic therapy highlights the importance of vaccination in managing typhoid fever. The virulence-associated (Vi) capsular polysaccharide is an effective vaccine against typhoid fever,but the role the capsule plays during pathogenesis remains incompletely understood. Here,we identify the human C-type lectin receptor DC-SIGN as the receptor for the Vi capsular polysaccharide. Binding of capsulated S. Typhi to DC-SIGN resulted in phagocytosis of the pathogen by macrophages and induction of an anti-inflammatory cytokine response. Thus,the interaction of the Vi capsular polysaccharide with human DC-SIGN contributes to the pathogenesis of typhoid fever and should be further investigated in the context of vaccine development. View Publication

Phosphoinositide 3-kinase (PI3K) p110$\delta$ signalling negatively regulates the production of mouse IgE. However,there are disparities between the mouse and human IgE biology,and the role of PI3K p110$\delta$ in the production of human IgE is yet to be determined. To investigate the effect of PI3K p110$\delta$ inhibition in the production of human IgE we isolated human B cells from tonsil tissue and stimulated them with IL-4 and anti-CD40 antibody to induce class switching to IgE and IgG1 in the presence or absence of IC87114,a small molecule inhibitor of PI3K p110$\delta$. Using FACS,RT-PCR and ELISA we examined the effect of PI3K p110$\delta$ inhibition on IgE production and determined the mechanisms involved. Unlike in mice,we observed that PI3K p110$\delta$ inhibition significantly reduces the number of IgE+ switched cells and the amounts of secreted IgE in IL4 and anti-CD40 cultures. However,the number of IgG1+ cells and secreted IgG1 were largely unaffected by PI3K p110$\delta$ inhibition. The expression levels of AID,$\epsilon$ and $\gamma$1 germinal transcripts or other factors involved in the regulation of CSR to IgE and IgG1 were also unaffected by IC87114. However,we found that IC87114 significantly decreases the proliferation of tonsil B cells stimulated with IL-4 and anti-CD40,specifically reducing the frequency of cells that had undergone 4 divisions or more. In addition,PI3K p110$\delta$ inhibition reduced the levels of IRF4 expression in IgE+ germinal centre-like B cells leading to a block in plasma cell differentiation. In conclusion,PI3K p110$\delta$ signalling is required for the production of human IgE,which makes it a pharmacological target for the treatment of allergic disease. View Publication

Antibodies are a principal determinant of immunity for most RNA viruses and have promise to reduce infection or disease during major epidemics. The novel coronavirus SARS-CoV-2 has caused a global pandemic with millions of infections and hundreds of thousands of deaths to date1,2. In response,we used a rapid antibody discovery platform to isolate hundreds of human monoclonal antibodies (mAbs) against the SARS-CoV-2 spike (S) protein. We stratify these mAbs into five major classes on the basis of their reactivity to subdomains of S protein as well as their cross-reactivity to SARS-CoV. Many of these mAbs inhibit infection of authentic SARS-CoV-2 virus,with most neutralizing mAbs recognizing the receptor-binding domain (RBD) of S. This work defines sites of vulnerability on SARS-CoV-2 S and demonstrates the speed and robustness of advanced antibody discovery platforms. View Publication