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  • A novel, immortal, and multipotent human neural stem cell line generating functional neurons and oligodendrocytes. 17556596

    The discovery and study of neural stem cells have revolutionized our understanding of the neurogenetic process, and their inherent ability to adopt expansive growth behavior in vitro is of paramount importance for the development of novel therapeutics based on neural cell replacement. Recent advances in high-throughput assays for drug development and gene discovery dictate the need for rapid, reproducible, long-term expansion of human neural stem cells (hNSCs). In this view, the complement of wild-type cell lines currently available is insufficient. Here we report the establishment of a stable human neural stem cell line (immortalized human NSCs [IhNSCs]) by v-myc-mediated immortalization of previously derived wild-type hNSCs. These cells demonstrate three- to fourfold faster proliferation than wild-type cells in response to growth factors but retain rather similar properties, including multipotentiality. By molecular biology, biochemistry, immunocytochemistry, fluorescence microscopy, and electrophysiology, we show that upon growth factor removal, IhNSCs completely downregulate v-myc expression, cease proliferation, and differentiate terminally into three major neural lineages: astrocytes, oligodendrocytes, and neurons. The latter are functional, mature cells displaying clear-cut morphological and physiological features of terminally differentiated neurons, encompassing mostly the GABAergic, glutamatergic, and cholinergic phenotypes. Finally, IhNSCs produce bona fide oligodendrocytes in fractions up to 20% of total cell number. This is in contrast to the negligible propensity of hNSCs to generate oligodendroglia reported so far. Thus, we describe an immortalized hNSC line endowed with the properties of normal hNSCs and suitable for developing the novel, reliable assays and reproducible high-throughput gene and drug screening that are essential in both diagnostics and cell therapy studies.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • A plate-based assay to measure cellular ERK substrate phosphorylation: utility for drug discovery of the MAPK-signaling cascade. 20482377

    The Ras, Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) signaling cascade is critically involved in cellular signaling with activating mutations in Ras and Raf present in many human tumors. Each constituent of this pathway is considered an important target for pharmaceutical intervention. The terminal kinase ERK is known to phosphorylate p90RSK among myriad substrates, yet robust plate-based high-throughput cellular assays monitoring such activity are not commercially available. In this study, we have utilized the Meso Scale Discovery platform to develop a plate-based assay to monitor the level of phosphorylation of p90RSK. This method is highly robust and can be used to evaluate a large number of inhibitors of ERK, MEK, or Raf in a variety of cellular backgrounds. Furthermore, this assay can be used to quantify the level of phospho-p90RSK in tumor lysates to function as a valuable pharmacodynamic readout.
    Document Type:
    Reference
    Product Catalog Number:
    04-419
  • A promoter-proximal transcript targeted by genetic polymorphism controls E-cadherin silencing in human cancers 28555645

    Long noncoding RNAs are emerging players in the epigenetic machinery with key roles in development and diseases. Here we uncover a complex network comprising a promoter-associated noncoding RNA (paRNA), microRNA and epigenetic regulators that controls transcription of the tumour suppressor E-cadherin in epithelial cancers. E-cadherin silencing relies on the formation of a complex between the paRNA and microRNA-guided Argonaute 1 that, together, recruit SUV39H1 and induce repressive chromatin modifications in the gene promoter. A single nucleotide polymorphism (rs16260) linked to increased cancer risk alters the secondary structure of the paRNA, with the risk allele facilitating the assembly of the microRNA-guided Argonaute 1 complex and gene silencing. Collectively, these data demonstrate the role of a paRNA in E-cadherin regulation and the impact of a noncoding genetic variant on its function. Deregulation of paRNA-based epigenetic networks may contribute to cancer and other diseases making them promising targets for drug discovery.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
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    Multiple
  • A quantitative homogeneous assay for fragile X mental retardation 1 protein. 23548045

    Hypermethylation of the fragile X mental retardation 1 gene FMR1 results in decreased expression of FMR1 protein FMRP, which is the underlying cause of Fragile X syndrome - an incurable neurological disorder characterized by mental retardation, anxiety, epileptic episodes and autism. Disease-modifying therapies for Fragile X syndrome are thus aimed at treatments that increase the FMRP expression levels in the brain. We describe the development and characterization of two assays for simple and quantitative detection of FMRP protein.Antibodies coupled to fluorophores that can be employed for time-resolved Förster's resonance energy transfer were used for the development of homogeneous, one-step immunodetection. Purified recombinant human FMRP and patient cells were used as control samples for assay development.The assays require small sample amounts, display high stability and reproducibility and can be used to quantify endogenous FMRP in human fibroblasts and peripheral blood mononuclear cells. Application of the assays to FXS patient cells showed that the methods can be used both for the characterization of clinical FXS patient samples as well as primary readouts in drug-discovery screens aimed at increasing endogenous FMRP levels in human cells.This study provides novel quantitative detection methods for FMRP in FXS patient cells. Importantly, due to the simplicity of the assay protocol, the method is suited to be used in screening applications to identify compounds or genetic interventions that result in increased FMRP levels in human cells.
    Document Type:
    Reference
    Product Catalog Number:
    MAB2160
    Product Catalog Name:
    Anti-Fragile X Mental Retardation Protein Antibody, clone 1C3
  • A selective HDAC 1/2 inhibitor modulates chromatin and gene expression in brain and alters mouse behavior in two mood-related tests. 23967191

    Psychiatric diseases, including schizophrenia, bipolar disorder and major depression, are projected to lead global disease burden within the next decade. Pharmacotherapy, the primary--albeit often ineffective--treatment method, has remained largely unchanged over the past 50 years, highlighting the need for novel target discovery and improved mechanism-based treatments. Here, we examined in wild type mice the impact of chronic, systemic treatment with Compound 60 (Cpd-60), a slow-binding, benzamide-based inhibitor of the class I histone deacetylase (HDAC) family members, HDAC1 and HDAC2, in mood-related behavioral assays responsive to clinically effective drugs. Cpd-60 treatment for one week was associated with attenuated locomotor activity following acute amphetamine challenge. Further, treated mice demonstrated decreased immobility in the forced swim test. These changes are consistent with established effects of clinical mood stabilizers and antidepressants, respectively. Whole-genome expression profiling of specific brain regions (prefrontal cortex, nucleus accumbens, hippocampus) from mice treated with Cpd-60 identified gene expression changes, including a small subset of transcripts that significantly overlapped those previously reported in lithium-treated mice. HDAC inhibition in brain was confirmed by increased histone acetylation both globally and, using chromatin immunoprecipitation, at the promoter regions of upregulated transcripts, a finding consistent with in vivo engagement of HDAC targets. In contrast, treatment with suberoylanilide hydroxamic acid (SAHA), a non-selective fast-binding, hydroxamic acid HDAC 1/2/3/6 inhibitor, was sufficient to increase histone acetylation in brain, but did not alter mood-related behaviors and had dissimilar transcriptional regulatory effects compared to Cpd-60. These results provide evidence that selective inhibition of HDAC1 and HDAC2 in brain may provide an epigenetic-based target for developing improved treatments for mood disorders and other brain disorders with altered chromatin-mediated neuroplasticity.
    Document Type:
    Reference
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    Multiple
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    Multiple
  • A single-cell and feeder-free culture system for monkey embryonic stem cells. 24505480

    Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
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    Multiple
  • Alveolar epithelial differentiation of human induced pluripotent stem cells in a rotating bioreactor. 24144903

    Traditional stem cell differentiation protocols make use of a variety of cytokines including growth factors (GFs) and inhibitors in an effort to provide appropriate signals for tissue specific differentiation. In this study, iPSC-derived type II pneumocytes (iPSC-ATII) as well as native isolated human type II pneumocytes (hATII) were differentiated toward a type I phenotype using a unique air-liquid interface (ALI) system that relies on a rotating apparatus that mimics in vivo respiratory conditions. A relatively homogenous population of alveolar type II-like cells from iPSC was first generated (iPSC-ATII cells), which had phenotypic properties similar to mature human alveolar type II cells. iPSC-ATII cells were then cultured in a specially designed rotating culture apparatus. The effectiveness of the ALI bioreactor was compared with the effectiveness of small molecule-based differentiation of type II pneumocytes toward type 1 pneumocytes. The dynamics of differentiation were examined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), flow cytometry and immunocytochemistry. iPSC-ATII and hATII cells cultured in the ALI bioreactor had higher levels of type I markers, including aquaporin-5(AQ5), caveolin-1, and T1α, at both the RNA and protein levels as compared with the flask-grown iPSC-ATII and hATII that had been treated with small molecules to induce differentiation. In summary, this study demonstrates that a rotating bioreactor culture system that provides an air-liquid interface is a potent inducer of type I epithelial differentiation for both iPS-ATII cells and hATII cells, and provides a method for large-scale production of alveolar epithelium for tissue engineering and drug discovery.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
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    Multiple
  • An H2A histone isotype regulates estrogen receptor target genes by mediating enhancer-promoter-3'-UTR interactions in breast cancer cells. 24371278

    A replication-dependent histone H2A isotype, H2ac, is upregulated in MCF-7 cells and in estrogen receptor-positive clinical breast cancer tissues. Cellular depletion of this H2A isotype leads to defective estrogen signaling, loss of cell proliferation and cell cycle arrest at G0/G1 phase. H2ac mediates regulation of estrogen receptor target genes, particularly BCL2 and c-MYC, by recruiting estrogen receptor alpha through its HAR domain and facilitating the formation of a chromatin loop between the promoter, enhancer and 3'-untranslated region of the respective genes. These findings reveal a new role for histone isotypes in the regulation of gene expression in cancer cells, and suggest that these molecules may be targeted for anti-cancer drug discovery.
    Document Type:
    Reference
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    Multiple
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    Multiple
  • Angiogenic factors stimulate growth of adult neural stem cells. 20195471

    The ability to grow a uniform cell type from the adult central nervous system (CNS) is valuable for developing cell therapies and new strategies for drug discovery. The adult mammalian brain is a source of neural stem cells (NSC) found in both neurogenic and non-neurogenic zones but difficulties in culturing these hinders their use as research tools.Here we show that NSCs can be efficiently grown in adherent cell cultures when angiogenic signals are included in the medium. These signals include both anti-angiogenic factors (the soluble form of the Notch receptor ligand, Dll4) and pro-angiogenic factors (the Tie-2 receptor ligand, Angiopoietin 2). These treatments support the self renewal state of cultured NSCs and expression of the transcription factor Hes3, which also identifies the cancer stem cell population in human tumors. In an organotypic slice model, angiogenic factors maintain vascular structure and increase the density of dopamine neuron processes.We demonstrate new properties of adult NSCs and a method to generate efficient adult NSC cultures from various central nervous system areas. These findings will help establish cellular models relevant to cancer and regeneration.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple