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

New approach methodologies (NAMs) for predicting embryotoxicity and developmental toxicity are urgently needed for generating human relevant data,while reducing turnover time and costs,and alleviating ethical concerns related to the use of animal models. We have previously developed the PluriLum assay,a NKX2.5-reporter gene 3D model using human-induced pluripotent stem cells (hiPSCs) that are genetically modified to enable the assessment of adverse effects of chemicals on the early-stage embryo. Aiming at improving the predictive value of the PluriLum assay for future screening purposes,we sought to introduce standardization steps to the protocol,improving the overall robustness of the PluriLum assay,as well as a shortening of the assay protocol. First,we showed that the initial size of embryoid bodies (EBs) is crucial for a proper differentiation into cardiomyocytes and overall reproducibility of the assay. When the starting diameter of the EBs exceeds 500 µm,robust differentiation can be anticipated. In terms of reproducibility,exposure to the fungicide epoxiconazole at smaller initial diameters resulted in a larger variation of the derived data,compared to more reliable concentration–response curves obtained using spheroids with larger initial diameters. We further investigated the ideal length of the differentiation protocol,resulting in a shortening of the PluriLum assay by 24 h to 7 days. Following exposure to the teratogens all-trans and 13-cis retinoic acid,both cardiomyocyte contraction and measurement of NKX2.5-derived luminescence were recorded with a similar or increased sensitivity after 6 days of differentiation when compared to the original 7 days. Finally,we have introduced an efficient step for enzymatic dissociation of the EBs at assay termination. This allows for an even splitting of the individual EBs and testing of additional endpoints other than the NKX2.5-luciferase reporter,which was demonstrated in this work by the simultaneous assessment of ATP levels. In conclusion,we have introduced standardizations and streamlined the PluriLum assay protocol to improve its suitability as a NAM for screening of a large number of chemicals for developmental toxicity testing. 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

Small non‐coding microRNAs (miRNAs) play essential roles in Alzheimer's disease (AD) pathogenesis. Repressor element 1‐silencing transcription factor (REST) is involved in AD,though its regulation remains unclear. We performed real‐time quantitative polymerase chain reaction (qPCR) in autopsied brain tissues to determine miR‐153‐3p and AD associations. A reporter‐based assay measured the activity of REST mRNA 3′‐untranslated region (3′‐UTR). Induced pluripotent stem cells (iPSC)‐derived neurons and human cell lines were applied to determine miR‐153‐3p regulation of endogenous proteins. Elevation of miR‐153‐3p is associated with a reduced probability of AD,while elevated REST is associated with a greater probability of AD. The 3′‐UTR functional assay pinpointed the miR‐153‐3p binding sites. miR‐153‐3p treatment reduced REST,amyloid precursor protein (APP),and α‐synuclein (SNCA) 3′‐UTR activities and protein levels. miR‐153‐3p treatment altered REST and neuronal differentiation in iPSC‐derived neuronal stem cells. RNA‐sequencing and proteomics revealed miR‐153‐3p‐associated networks. miR‐153‐3p reduces REST,APP,and SNCA expression,pointing toward its therapeutic and biomarker potential in neurodegenerative diseases. With the increased emphasis on comorbidities of Alzheimer's disease (AD) and other neurodegenerative diseases,we identified that miR‐153‐3p,as a master regulator,reduced a group of neurodegeneration related proteins: REST,amyloid precursor protein (APP) and α‐synuclein (SNCA) levels. The elevation of miR‐153‐3p levels is associated with reduced probability of AD in posterior cingulate cortex (PCC),while REST,by contrast,is associated with a greater probability of AD. miR‐153‐3p treatment alters REST protein levels and neuronal differentiation in induced pluripotent stem cells (iPSC) derived neuronal cells. RNA sequencing proteomics and interactome analysis revealed the role of miR‐153‐3p in axonal guidance. View Publication

Delivering macromolecules across biological barriers such as the blood–brain barrier limits their application in vivo. Previous work has demonstrated that Toxoplasma gondii,a parasite that naturally travels from the human gut to the central nervous system (CNS),can deliver proteins to host cells. Here we engineered T. gondii ’s endogenous secretion systems,the rhoptries and dense granules,to deliver multiple large (>100 kDa) therapeutic proteins into neurons via translational fusions to toxofilin and GRA16. We demonstrate delivery in cultured cells,brain organoids and in vivo,and probe protein activity using imaging,pull-down assays,scRNA-seq and fluorescent reporters. We demonstrate robust delivery after intraperitoneal administration in mice and characterize 3D distribution throughout the brain. As proof of concept,we demonstrate GRA16-mediated brain delivery of the MeCP2 protein,a putative therapeutic target for Rett syndrome. By characterizing the potential and current limitations of the system,we aim to guide future improvements that will be required for broader application. Subject terms: Parasitology,Biologics,Synthetic biology View Publication

Ovarian cancer (OV) has the highest mortality rate among gynecological cancers. As OV progresses,tumor cells spread outside the ovaries to the peritoneal and abdominal cavities,forming cell clusters that float in the ascitic fluid caused by peritonitis carcinomatosa,leading to further dissemination and metastasis. These cell clusters are enriched with cancer stem cells (CSCs) which are responsible for treatment resistance,recurrence,and metastasis. Therefore,targeting CSCs is a potentially effective approach for treating OV. However,understanding how CSCs acquire treatment resistance and identifying targets against CSCs remains challenging. In this study,we demonstrate that 3D-spheroids of OV cell lines exhibit higher stemness than conventional adherent cells. Metabolomics profiling studies have revealed that 3D-spheroids maintain a high-energy state through increased glucose utilization in the citric acid cycle (TCA),efficient nucleotide phosphorylation,and elevated phosphocreatine as an energy buffer. We also found that nicotinamide phosphoribosyltransferase (NAMPT),the rate-limiting enzyme for NAD + production,is highly expressed in OV. Furthermore,the approach based on NAMPT dependence rather than histology found NAMPT to be a potential therapeutic target against CSCs,while also serving as a prognostic indicator in OV. Moreover,we identified a previously unrecognized anti-tumor mechanism whereby disulfiram,an aldehyde dehydrogenase (ALDH) inhibitor,synergistically inhibited mitochondrial function when combined with NAMPT inhibitors - leading to cell cycle arrest in G2/M. Finally,the combination of a NAMPT inhibitor and disulfiram showed significant anti-tumor effects and extended survival in an animal model. Our findings demonstrate the potential of spheroids as a preclinical model for targeting OV CSCs and also indicate that the combination of NAMPT inhibitors and disulfiram is a promising therapeutic strategy to overcome recurrent OV. Subject terms: Ovarian cancer,Metabolomics,Apoptosis,Cancer stem cells View Publication

Metastasis is one of the leading causes of cancer-related death worldwide. Fascin,a protein that bundles actin filaments to produce protrusions in cancer cells,plays a significant role in the enhancement of cell migration. This protein has been shown that the overexpression of this protein is related to the appearance of different types of cancer,such as colorectal cancer. In this study,we conducted in silico screening of the Enamine library,a compound library with a broad chemical space. Using a ligand-based virtual screening approach based on the pharmacophore model of G2,we identified the predicted inhibitors. First,these compounds were validated by physicochemical analysis. Differential scanning calorimetry (DSF) was used to study the binding between the predicted compounds and fascin protein,followed by an F-actin bundling assay to determine which compounds inhibited the bundling function of fascin. Z1362873773,which exhibited binding to fascin and inhibited F-actin bundling,was further tested in cell cultures to assess its effects on cancer cell viability and migration as well as in organoid models to evaluate potential cytotoxicity. Finally,we established a protocol that can be applied to discover anti-fascin agents from diverse compound libraries. A new molecule has been identified with considerable fascin inhibitory and migration-arresting capacity,which may lead to the development of new therapies to treat cancer. The online version contains supplementary material available at 10.1038/s41598-025-96457-x. Subject terms: Biochemistry,Biophysics,Cancer,Drug discovery,Molecular biology,Virtual drug screening View Publication

NK cells protect hosts against viral pathogens and transformed cells,and dendritic cells (DCs) play important roles in activating NK cells. We now find that murine IL-15Ralpha-deficient DCs fail to support NK cell cytolytic activity and elaboration of IFN-gamma,despite the fact that these DCs express normal levels of costimulatory molecules and IL-12. By contrast,IL-15Ralpha expression on NK cells is entirely dispensable for their activation by DCs. In addition,blockade with anti-IL-15Ralpha and anti-IL-2Rbeta but not anti-IL-2Ralpha-specific Abs prevents NK cell activation by wild-type DCs. Finally,presentation of IL-15 by purified IL-15Ralpha/Fc in trans synergizes with IL-12 to support NK cell priming. These findings suggest that murine DCs require IL-15Ralpha to present IL-15 in trans to NK cells during NK cell priming. View Publication

Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and nontumorigenic cancer cells. Three clusters,miR-200c-141,miR-200b-200a-429,and miR-183-96-182 were downregulated in human BCSCs,normal human and murine mammary stem/progenitor cells,and embryonal carcinoma cells. Expression of BMI1,a known regulator of stem cell self-renewal,was modulated by miR-200c. miR-200c inhibited the clonal expansion of breast cancer cells and suppressed the growth of embryonal carcinoma cells in vitro. Most importantly,miR-200c strongly suppressed the ability of normal mammary stem cells to form mammary ducts and tumor formation driven by human BCSCs in vivo. The coordinated downregulation of three microRNA clusters and the similar functional regulation of clonal expansion by miR-200c provide a molecular link that connects BCSCs with normal stem cells. View Publication

Assessing relevant molecular differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) is important,given that such differences may impact their potential therapeutic use. Controversy surrounds recent gene expression studies comparing hiPSCs and hESCs. Here,we present an in-depth quantitative mass spectrometry-based analysis of hESCs,two different hiPSCs and their precursor fibroblast cell lines. Our comparisons confirmed the high similarity of hESCs and hiPSCS at the proteome level as 97.8% of the proteins were found unchanged. Nevertheless,a small group of 58 proteins,mainly related to metabolism,antigen processing and cell adhesion,was found significantly differentially expressed between hiPSCs and hESCs. A comparison of the regulated proteins with previously published transcriptomic studies showed a low overlap,highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature. View Publication

Corneal transparency depends on a unique extracellular matrix secreted by stromal keratocytes,mesenchymal cells of neural crest lineage. Derivation of keratocytes from human embryonic stem (hES) cells could elucidate the keratocyte developmental pathway and open a potential for cell-based therapy for corneal blindness. This study seeks to identify conditions inducing differentiation of pluripotent hES cells to the keratocyte lineage. Neural differentiation of hES cell line WA01(H1) was induced by co-culture with mouse PA6 fibroblasts. After 6 days of co-culture,hES cells expressing cell-surface NGFR protein (CD271,p75NTR) were isolated by immunoaffinity adsorption,and cultured as a monolayer for one week. Keratocyte phenotype was induced by substratum-independent pellet culture in serum-free medium containing ascorbate. Gene expression,examined by quantitative RT-PCR,found hES cells co-cultured with PA6 cells for 6 days to upregulate expression of neural crest genes including NGFR,SNAI1,NTRK3,SOX9,and MSX1. Isolated NGFR-expressing cells were free of PA6 feeder cells. After expansion as a monolayer,mRNAs typifying adult stromal stem cells were detected,including BMI1,KIT,NES,NOTCH1,and SIX2. When these cells were cultured as substratum-free pellets keratocyte markers AQP1,B3GNT7,PTDGS,and ALDH3A1 were upregulated. mRNA for keratocan (KERA),a cornea-specific proteoglycan,was upregulated more than 10,000 fold. Culture medium from pellets contained high molecular weight keratocan modified with keratan sulfate,a unique molecular component of corneal stroma. These results show hES cells can be induced to differentiate into keratocytes in vitro. Pluripotent stem cells,therefore,may provide a renewable source of material for development of treatment of corneal stromal opacities. View Publication