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The neurodegenerative disease spinocerebellar ataxia type 3 (SCA3) is caused by a CAG-repeat expansion in the ATXN3 gene. In this study,induced pluripotent stem cell (iPSC) lines were established from two SCA3 patients. Dermal fibroblasts were reprogrammed using an integration-free method and the resulting SCA3 iPSCs were differentiated into neurons. These neuronal lines harbored the disease causing mutation,expressed comparable levels of several neuronal markers and responded to the neurotransmitters,glutamate/glycine,GABA and acetylcholine. Additionally,all neuronal cultures formed networks displaying synchronized spontaneous calcium oscillations within 28days of maturation,and expressed the mature neuronal markers NeuN and Synapsin 1 implying a relatively advanced state of maturity,although not comparable to that of the adult human brain. Interestingly,we were not able to recapitulate the glutamate-induced ataxin-3 aggregation shown in a previously published iPSC-derived SCA3 model. In conclusion,we have generated a panel of SCA3 patient iPSCs and a robust protocol to derive neurons of relatively advanced maturity,which could potentially be valuable for the study of SCA3 disease mechanisms. View Publication

Thiazolidinedione derivatives are antidiabetic agents that increase the insulin sensitivity of target tissues in animal models of non-insulin-dependent diabetes mellitus. In vitro,thiazolidinediones promote adipocyte differentiation of preadipocyte and mesenchymal stem cell lines; however,the molecular basis for this adipogenic effect has remained unclear. Here,we report that thiazolidinediones are potent and selective activators of peroxisome proliferator-activated receptor gamma (PPAR gamma),a member of the nuclear receptor superfamily recently shown to function in adipogenesis. The most potent of these agents,BRL49653,binds to PPAR gamma with a Kd of approximately 40 nM. Treatment of pluripotent C3H10T1/2 stem cells with BRL49653 results in efficient differentiation to adipocytes. These data are the first demonstration of a high affinity PPAR ligand and provide strong evidence that PPAR gamma is a molecular target for the adipogenic effects of thiazolidinediones. Furthermore,these data raise the intriguing possibility that PPAR gamma is a target for the therapeutic actions of this class of compounds. View Publication

Mitomycin C (MMC) is the prototype bioreductive DNA alkylating agent. To exploit its unique properties and maximize patient responses,different therapeutic approaches have been investigated. Recently,the focus has concentrated on monitoring the levels of the proteins metabolizing the drug and relating these to activity in a regimen referred to as enzyme-directed bioreductive drug development. To be successful,it is important to understand the enzymology of metabolic activation not only in cell lines but also in solid tumour models. A general mechanism of action for MMC has now emerged that is activated regardless of the source of reducing equivalents,comprising three competing pathways that give rise to unique reactive intermediates and different DNA adducts. Partitioning into the pathways is dictated by chemical considerations such as pH and drug concentration. DT-diaphorase stands out in this mechanism,since it is much less effective at metabolizing MMC at neutral pH. At least five different enzymes can catalyse MMC bioreduction in vitro,and as many activities may be present in solid tumours,including a series of novel mitochondrial reductases such as a cytochrome P450 reductase. Competition between reductases for MMC appears to be based solely on protein levels rather than enzyme kinetics. Consequentially,DT-diaphorase can occupy a central role in MMC metabolic activation since it is often highly overexpressed in cancer cells. Although a good correlation has been observed in cell lines between DT-diaphorase expression and aerobic cytotoxicity,this does not hold consistently in vivo for any single bioreductive enzyme,suggesting revision of the enzyme-directed hypothesis as originally formulated. View Publication

CD4+ T cells recognize antigens through their T cell receptors (TCRs); however,additional signals involving costimulatory receptors,for example,CD28,are required for proper T cell activation. Alternative costimulatory receptors have been proposed,including members of the Toll-like receptor (TLR) family,such as TLR5 and TLR2. To understand the molecular mechanism underlying a potential costimulatory role for TLR5,we generated detailed molecular maps and logical models for the TCR and TLR5 signaling pathways and a merged model for cross-interactions between the two pathways. Furthermore,we validated the resulting model by analyzing how T cells responded to the activation of these pathways alone or in combination,in terms of the activation of the transcriptional regulators CREB,AP-1 (c-Jun),and NF-kappaB (p65). Our merged model accurately predicted the experimental results,showing that the activation of TLR5 can play a similar role to that of CD28 activation with respect to AP-1,CREB,and NF-kappaB activation,thereby providing insights regarding the cross-regulation of these pathways in CD4+ T cells. View Publication

Background: Effective molecular diagnosis of congenital diseases hinges on comprehensive genomic analysis,traditionally reliant on various methodologies specific to each variant type-whole exome or genome sequencing for single nucleotide variants (SNVs),array CGH for copy-number variants (CNVs),and microscopy for structural variants (SVs). Methods: We introduce a novel,integrative approach combining exome sequencing with chromosome conformation capture,termed Exo-C. This method enables the concurrent identification of SNVs in clinically relevant genes and SVs across the genome and allows analysis of heterozygous and mosaic carriers. Enhanced with targeted long-read sequencing,Exo-C evolves into a cost-efficient solution capable of resolving complex SVs at base-pair accuracy. Results: Applied to 66 human samples Exo-C achieved 100% recall and 73% precision in detecting chromosomal translocations and SNVs. We further benchmarked its performance for inversions and CNVs and demonstrated its utility in detecting mosaic SVs and resolving diagnostically challenging cases. Conclusions: Through several case studies,we demonstrate how Exo-C's multifaceted application can effectively uncover diverse causative variants and elucidate disease mechanisms in patients with rare disorders. View Publication

SummaryCryosectioning remains the gold standard for antibody and transcriptomic/in situ hybridization tissue analysis. However,tissue processing is time-consuming and costly,limiting routine and diagnostic use. Currently,no commercially available protocols or products exist for multiplexing this process. Here,we introduce multiplexed tissue molds (MTMs) that enable high-throughput cryoprocessing—cutting costs and workload by up to 96% while permitting the processing of tissues of various sizes and origins. We demonstrate compatibility with heterogeneous tissues by processing 19 different adult mouse tissues in parallel. Furthermore,we process up to ?110 neural organoids of different ages and sizes simultaneously and assess their neural differentiation marker expression. MTMs allow sectioning-based tissue analysis when labor,time,and cost are limiting factors. MTMs could be used to compare high specimen numbers in histopathological settings,organism-wide antigen and antibody targeting studies,high-throughput tissue screens,and defined tissue section positioning for,e.g.,spatial transcriptomics experiments. Graphical abstract Highlights•Multiplexed tissue molds (MTMs) drastically upscale cryosectioning procedures•MTMs can simultaneously accommodate up to 19 mouse organs and ?110 cerebral organoids•MTMs reduce analysis costs and processing times of tissues by up to 96%•MTMs could be used to reduce diagnostic costs and for spatial transcriptomics MotivationEfficient cryosectioning remains a critical yet labor- and cost-intensive step for immunohistochemistry and in situ hybridization,limiting routine diagnostic and research applications. The increasing demand for high-throughput tissue analysis—driven by advances in organoid and three-dimensional (3D) culture systems and tissue analysis for diagnostics—necessitates methods capable of processing numerous heterogeneous samples simultaneously. Current protocols lack multiplexing capabilities,leading to variability and extended processing times. Our work introduces multiplexed tissue molds (MTMs),a scalable solution that drastically reduces costs and labor by up to 96% while maintaining tissue integrity and consistency,thereby enabling large-scale (>100 tissues) comparative analyses and enhanced experimental reproducibility as well as access to tissue analysis,where cost is a restrictive factor. Reumann et al. develop multiplexed tissue molds (MTMs),which allow upscaling of tissue processing (up to 19 mouse organs or ?110 cerebral organoids simultaneously) while reducing workload and associated analysis costs by up to 96%. MTMs allow cryosection-based tissue analysis when labor,time,and cost are limiting factors and could be used for patient sample analysis as well as spatial transcriptomics approaches. View Publication

SummaryA strong interest in drugs targeting the tumor microenvironment (TME) necessitates new experimental systems that incorporate key TME components. Compared to traditional 2D cell lines,3D ex vivo spheroids from patient-derived xenograft (PDX) materials may better capture patient tumor characteristics. We developed and validated a 3D tumor spheroid model from non-small cell lung cancer (NSCLC) PDXs to enable T cell infiltration. Histologic and transcriptomic analysis suggested that tumor spheroids closely recapitulate the source PDX tumor tissues. Consistent T cell infiltration into tumor spheroids was achieved using a well-established magnetic nanoparticle technology,which maintained T cell function and tumor-killing activity. Drug treatment studies with immunotherapy agents also demonstrated the potential scalability of 3D tumor-T cell spheroids in assessing drug activity,including tumor viability and cytokine secretion. This platform provides a useful tool for evaluating drug candidates that can be translated to patient tumor responses related to both tumor intrinsic and TME factors. Graphical abstract Highlights•We developed a 3D tumor spheroid model from lung cancer patient-derived xenografts•The model enabled robust T cell infiltration and preserved T cell cytotoxic functions•Histology and RNA-seq showed that tumor spheroids closely resembled source tumors•Proof-of-concept experiments showed this platform’s utility in preclinical drug testing Biological sciences; Biotechnology; Natural sciences; Tissue Engineering View Publication

Hematologic adverse events are common dose-limiting toxicities in drug development. Classical animal models for preclinical safety assessment of immunotherapies are often limited due to insufficient cross-reactivity with non-human homologous proteins,immune system differences,and ethical considerations. Therefore,we evaluate a human bone marrow (BM) microphysiological system (MPS) for its ability to predict expected hematopoietic liabilities of immunotherapeutics. The BM-MPS consists of a closed microfluidic circuit containing a ceramic scaffold covered with human mesenchymal stromal cells and populated with human BM-derived CD34+ cells in chemically defined growth factor-enriched media. The model supports on-chip differentiation of erythroid,myeloid and NK cells from CD34+ cells over 31 days. The hematopoietic lineage balance and output is responsive to pro-inflammatory factors and cytokines. Treatment with a transferrin receptor-targeting IgG1 antibody results in inhibition of on-chip erythropoiesis. The immunocompetence of the chip is established by the addition of peripheral blood T cells in a fully autologous setup. Treatment with T cell bispecific antibodies induces T cell activation and target cell killing consistent with expected on-target off-tumor toxicities. In conclusion,this study provides a proof-of-concept that this BM-MPS is applicable for in vitro hematopoietic safety profiling of immunotherapeutics. Subject terms: Biologics,Haematopoiesis,Lab-on-a-chip,Drug safety View Publication

The human pre-T-cell receptor alpha (TCRalpha; pTalpha) gene encodes a polypeptide which associates with the TCRbeta chain and CD3 molecules to form the pre-TCR complex. The surface expression of the pre-TCR is pTalpha dependent,and signaling through this complex triggers an early alphabeta T-cell developmental checkpoint inside the thymus,known as beta-selection. E2A transcription factors,which are involved at multiple stages of T-cell development,regulate the transcription of the pTalpha gene. Here we show that the regulatory protein Tax of the human T-cell leukemia virus type 1 (HTLV-1) efficiently suppresses the E47-mediated activation of the pTalpha promoter. Furthermore,we report that in Tax lentivirally transduced human MOLT-4 T cells,which constitutively express the pTalpha gene,the amount of pTalpha transcripts decreases. Such a decrease is not observed in MOLT-4 cells transduced by a vector encoding the Tax mutant K88A,which is unable to interact with p300. These data underline that Tax inhibits pTalpha transcription by recruiting this coactivator. Finally,we show that the expression of Tax in human immature thymocytes results in a decrease of pTalpha gene transcription but does not modify the level of E47 transcripts. These observations indicate that Tax,by silencing E proteins,down-regulates pTalpha gene transcription during early thymocyte development. They further provide evidence that Tax can interfere with an important checkpoint during T-cell differentiation in the thymus. View Publication

A human invitro cardiac tissue model would be a significant advancement for understanding,studying,and developing new strategies for treating cardiac arrhythmias and related cardiovascular diseases. We developed an invitro model of three-dimensional (3D) human cardiac tissue by populating synthetic filamentous matrices with cardiomyocytes derived from healthy wild-type volunteer (WT) and patient-specific long QT syndrome type 3 (LQT3) induced pluripotent stem cells (iPS-CMs) to mimic the condensed and aligned human ventricular myocardium. Using such a highly controllable cardiac model,we studied the contractility malfunctions associated with the electrophysiological consequences of LQT3 and their response to a panel of drugs. By varying the stiffness of filamentous matrices,LQT3 iPS-CMs exhibited different level of contractility abnormality and susceptibility to drug-induced cardiotoxicity. textcopyright 2013 Elsevier Ltd. View Publication