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

During the metastatic cascade,cancer cells travel through the bloodstream as circulating tumor cells (CTCs) to a secondary site. Clustered CTCs have greater shear stress and treatment resistance,yet their biology remains poorly understood. We therefore engineered a tunable superhydrophobic array device (SHArD). The SHArD-C was applied to culture a clinically relevant model of CTC clusters. Using our device,we cultured a model of cancer cell aggregates of various sizes with immortalized cancer cell lines. These exhibited higher E-cadherin expression and are significantly more capable of surviving high fluid shear stress-related forces compared to single cells and model clusters grown using the control method,helping to explain why clustering may provide a metastatic advantage. Additionally,the SHArD-S,when compared with the AggreWell 800 method,provides a more consistent spheroid-forming device culturing reproducible sizes of spheroids for multiple cancer cell lines. Overall,we designed,fabricated,and validated an easily tunable engineered device which grows physiologically relevant three-dimensional (3D) cancer models containing tens to thousands of cells. View Publication

Inhibitory receptors (IRs) function as critical regulators of immune responses by tempering T cell activity. In humans,several persisting viruses as well as cancers exploit IR signaling by upregulating IR ligands,resulting in suppression of T cell function (i.e.,exhaustion). This allows escape from immune surveillance and continuation of disease. Here,we report the design,implementation,and results of a phenotypic high-throughput screen for molecules that modulate CD8+ T cell activity. We identify 19 compounds from the ReFRAME drug-repurposing collection that restore cytokine production and enhance the proliferation of exhausted T cells. Analysis of our top hit,ingenol mebutate,a protein kinase C (PKC) inducing diterpene ester,reveals a role for this molecule in overriding the suppressive signaling cascade mediated by IR signaling on T cells. Collectively,these results demonstrate a disease-relevant methodology for identifying modulators of T cell function and reveal new targets for immunotherapy. View Publication

The recent approvals of anticancer therapeutic agents targeting the histone deacetylases and DNA methyltransferases have highlighted the important role that epigenetics plays in human diseases,and suggested that the factors controlling gene expression are novel drug targets. Protein arginine deiminase 4 (PAD4) is one such target because its effects on gene expression parallel those observed for the histone deacetylases. We demonstrated that F- and Cl-amidine,two potent PAD4 inhibitors,display micromolar cytotoxic effects towards several cancerous cell lines (HL-60,MCF7 and HT-29); no effect was observed in noncancerous lines (NIH 3T3 and HL-60 granulocytes). These compounds also induced the differentiation of HL-60 and HT29 cells. Finally,these compounds synergistically potentiated the cell killing effects of doxorubicin. Taken together,these findings suggest PAD4 inhibition as a novel epigenetic approach for the treatment of cancer,and suggest that F- and Cl-amidine are candidate therapeutic agents for this disease. View Publication

In the present investigation,we studied the modulating effects of several tea catechins and bioflavonoids on DNA methylation catalyzed by prokaryotic SssI DNA methyltransferase (DNMT) and human DNMT1. We found that each of the tea polyphenols [catechin,epicatechin,and (-)-epigallocatechin-3-O-gallate (EGCG)] and bioflavonoids (quercetin,fisetin,and myricetin) inhibited SssI DNMT- and DNMT1-mediated DNA methylation in a concentration-dependent manner. The IC(50) values for catechin,epicatechin,and various flavonoids ranged from 1.0 to 8.4 microM,but EGCG was a more potent inhibitor,with IC(50) values ranging from 0.21 to 0.47 microM. When epicatechin was used as a model inhibitor,kinetic analyses showed that this catechol-containing dietary polyphenol inhibited enzymatic DNA methylation in vitro largely by increasing the formation of S-adenosyl-L-homocysteine (a potent noncompetitive inhibitor of DNMTs) during the catechol-O-methyltransferase-mediated O-methylation of this dietary catechol. In comparison,the strong inhibitory effect of EGCG on DNMT-mediated DNA methylation was independent of its own methylation and was largely due to its direct inhibition of the DNMTs. This inhibition is strongly enhanced by Mg(2+). Computational modeling studies showed that the gallic acid moiety of EGCG plays a crucial role in its high-affinity,direct inhibitory interaction with the catalytic site of the human DNMT1,and its binding with the enzyme is stabilized by Mg(2+). The modeling data on the precise molecular mode of EGCG's inhibitory interaction with human DNMT1 agrees perfectly with our experimental finding. View Publication

Histone lysine methylation has important roles in the organization of chromatin domains and the regulation of gene expression. To analyze its function and modulate its activity,we screened for specific inhibitors against histone lysine methyltransferases (HMTases) using recombinant G9a as the target enzyme. From a chemical library comprising 125,000 preselected compounds,seven hits were identified. Of those,one inhibitor,BIX-01294 (diazepin-quinazolin-amine derivative),does not compete with the cofactor S-adenosyl-methionine,and selectively impairs the G9a HMTase and the generation of H3K9me2 in vitro. In cellular assays,transient incubation of several cell lines with BIX-01294 lowers bulk H3K9me2 levels that are restored upon removal of the inhibitor. Importantly,chromatin immunoprecipitation at several G9a target genes demonstrates reversible reduction of promoter-proximal H3K9me2 in inhibitor-treated mouse ES cells and fibroblasts. Our data identify a biologically active HMTase inhibitor that allows for the transient modulation of H3K9me2 marks in mammalian chromatin. View Publication

We have previously reported the genetic correction of Huntington's disease (HD) patient-derived induced pluripotent stem cells using traditional homologous recombination (HR) approaches. To extend this work,we have adopted a CRISPR-based genome editing approach to improve the efficiency of recombination in order to generate allelic isogenic HD models in human cells. Incorporation of a rapid antibody-based screening approach to measure recombination provides a powerful method to determine relative efficiency of genome editing for modeling polyglutamine diseases or understanding factors that modulate CRISPR/Cas9 HR. View Publication

Human pluripotent stem (hPS) cells are a potential source of cells for medical therapy and an ideal system to study fate decisions in early development. However,hPS cells cultured in vitro exhibit a high degree of heterogeneity,presenting an obstacle to clinical translation. hPS cells grow in spatially patterned colony structures,necessitating quantitative single-cell image analysis. We offer a tool for analyzing the spatial population context of hPS cells that integrates automated fluorescent microscopy with an analysis pipeline. It enables high-throughput detection of colonies at low resolution,with single-cellular and sub-cellular analysis at high resolutions,generating seamless in situ maps of single-cellular data organized by colony. We demonstrate the tool's utility by analyzing inter- and intra-colony heterogeneity of hPS cell cycle regulation and pluripotency marker expression. We measured the heterogeneity within individual colonies by analyzing cell cycle as a function of distance. Cells loosely associated with the outside of the colony are more likely to be in G1,reflecting a less pluripotent state,while cells within the first pluripotent layer are more likely to be in G2,possibly reflecting a G2/M block. Our multi-scale analysis tool groups colony regions into density classes,and cells belonging to those classes have distinct distributions of pluripotency markers and respond differently to DNA damage induction. Lastly,we demonstrate that our pipeline can robustly handle high-content,high-resolution single molecular mRNA FISH data by using novel image processing techniques. Overall,the imaging informatics pipeline presented offers a novel approach to the analysis of hPS cells that includes not only single cell features but also colony wide,and more generally,multi-scale spatial configuration. View Publication

The enteric pathogen Lawsonia intracellularis is one of the main causes of diarrhea and compromised weight gain in pigs worldwide. Traditional cell-line cultures have been used to study L. intracellularis pathogenesis. However,these systems fail to reproduce the epithelial changes observed in the intestines of L. intracellularis-infected pigs,specifically,the changes in intestinal cell constitution and gene expression. A more physiologically accurate and state-of-the-art model is provided by swine enteroids derived from stem cell-containing crypts from healthy pigs. The objective of this study was to verify the feasibility of two-dimensional swine enteroids as in vitro models for L. intracellularis infection. We established both three- and two-dimensional swine enteroid cultures derived from intestinal crypts. The two-dimensional swine enteroids were infected by L. intracellularis in four independent experiments. Enteroid-infected samples were collected 3 and 7 d postinfection for analysis using real-time quantitative PCR and L. intracellularis immunohistochemistry. In this study,we show that L. intracellularis is capable of infecting and replicating intracellularly in two-dimensional swine enteroids derived from ileum. View Publication

Recent research in breast biology has provided support for the cancer stem-cell hypothesis. Two important components of this hypothesis are that tumors originate in mammary stem or progenitor cells as a result of dysregulation of the normally tightly regulated process of self-renewal. As a result,tumors contain and are driven by a cellular subcomponent that retains key stem-cell properties including self-renewal,which drives tumorigenesis and differentiation that contributes to cellular heterogeneity. Advances in stem-cell technology have led to the identification of stem cells in normal and malignant breast tissue. The study of these stem cells has helped to elucidate the origin of the molecular complexity of human breast cancer. The cancer stem-cell hypothesis has important implications for early detection,prevention,and treatment of breast cancer. Both hereditary and sporadic breast cancers may develop through dysregulation of stem-cell self-renewal pathways. These aberrant stem cells may provide targets for the development of cancer prevention strategies. Furthermore,because breast cancer stem cells may be highly resistant to radiation and chemotherapy,the development of more effective therapies for this disease may require the effective targeting of this cell population. View Publication

Microglial cells play a dynamic role in the brain beyond their established function of immune surveillance. Activated microglia play key roles in neural development,neuroinflammation,neural repair and neurotoxicity. They are particularly important in several neurodegenerative diseases in which sustained microglial activation contributes to the progression of neurodegenerative processes. Consequently,understanding microglial function in CNS health and disease has become an area of active research in recent years. However,a significant obstacle to progress in this field has been the inherent difficulties in obtaining large amounts of primary microglial cells to routinely perform mechanistic studies and characterize signaling pathways regulating the dynamics of microglial activation. Herein,we describe a novel column-free magnetic separation protocol for high-yield isolation of primary microglia from mouse postnatal mixed glial cultures. The procedure is based on optimized culture conditions that enable high microglial cell densities in confluent mixed glial cultures followed by highly efficient recovery of pure microglia by magnetic separation. The novel column-free magnetic separation system utilizes tetrameric antibody complexes (TAC) with dual specificity for CD11b-PE labeled microglia and dextran magnetic nanoparticles. An FcR blocker (anti-CD16/32) is added to enhance the purity of the microglial separation by preventing non-specific labeling of other cell types. This procedure yields on average textgreater3×10�?� microglial cells per mouse pup,with a remarkable purity of 97% and recovery of around 87% of microglia from the mixed glial population. Importantly,the microglia obtained by this method are fully functional and respond like cells obtained by conventional isolation techniques. View Publication

IntroductionExposure to teratogenic compounds during pregnancy can lead to significant birth defects. Given the considerable variation in drug responses across species,along with the financial and ethical challenges associated with animal testing,the development of advanced human-based in vitro assays is imperative for effectively identifying potential human teratogens. Previously,we developed a human induced pluripotent stem cells (hiPSCs)-based biomarker assay,ReproTracker,that follows the differentiation of hiPSCs into hepatocytes and cardiomyocytes. The assay combines morphological profiling with the assessment of time-dependent expression patterns of cell-specific biomarkers to detect developmental toxicity responses.MethodsTo further increase the predictability of the assay in identifying potential teratogens,we added differentiation of hiPSCs towards neural rosette-like cells. We evaluated the performance of the extended assay with a set of 51 well-known in vivo teratogens and non-teratogens,including the compounds listed in the ICH S5 reference list.ResultsThe optimized assay correctly identified (neuro)developmental toxicants that were not detected in the hepatocyte and cardiomyocyte differentiation assays. These compounds selectively downregulated gene and protein expression of the neuroectodermal marker PAX6 and/or neural rosette marker NESTIN in a concentration-dependent manner and disrupted the differentiation of hiPSCs towards neural rosette-like cells. Overall,based on the current dataset,the addition of neural commitment improved the assay accuracy (from 72.55% to 86.27%) and sensitivity (from 67.50% to 87.50%),when compared to the previously described assay.DiscussionIn summary,trilineage differentiation expanded the spectrum of teratogenic agents detectable by ReproTracker,making the assay an invaluable tool for early in vitro teratogenicity screening. View Publication

HiBiT is an engineered luciferase’s 11-amino-acid component that can be introduced as a tag at either terminus of a protein of interest. When the LgBiT component and a substrate are present,HiBiT and LgBiT dimerize forming a functional luciferase. The HiBiT technology has been extensively used for high-throughput protein turnover studies in cells. Here,we have adapted the use of the HiBiT technology to quantify messenger RNA (mRNA) translation temporally in vitro in the rabbit reticulocyte system and in cellulo in HEK293 cells constitutively expressing LgBiT. The assay system can uniquely detect differences in cap,5?UTR,modified nucleotide composition,coding sequence optimization and poly(A) length,and their effects on mRNA translation over time. Importantly,using these assays we established the optimal mRNA composition varied depending on the encoded protein of interest,highlighting the importance of screening methods tailored to the protein of interest,and not reliant on reporter proteins. Our findings demonstrated that HiBiT can be easily and readily adapted to monitor real-time mRNA translation in live cells and offers a novel and highly favourable method for the development of mRNA-based therapeutics. View Publication