- Functional Comparison of Neuronal Cells Differentiated from Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Different Oxygen and Medium Conditions. [Journal Article]
- JBJ Biomol Screen 2016; 21(10):1054-1064
- Because neurons are difficult to obtain from humans, generating functional neurons from human induced pluripotent stem cells (hiPSCs) is important for establishing physiological or disease-relevant s...
Because neurons are difficult to obtain from humans, generating functional neurons from human induced pluripotent stem cells (hiPSCs) is important for establishing physiological or disease-relevant screening systems for drug discovery. To examine the culture conditions leading to efficient differentiation of functional neural cells, we investigated the effects of oxygen stress (2% or 20% O2) and differentiation medium (DMEM/F12:Neurobasal-based [DN] or commercial [PhoenixSongs Biologicals; PS]) on the expression of genes related to neural differentiation, glutamate receptor function, and the formation of networks of neurons differentiated from hiPSCs (201B7) via long-term self-renewing neuroepithelial-like stem (lt-NES) cells. Expression of genes related to neural differentiation occurred more quickly in PS and/or 2% O2 than in DN and/or 20% O2, resulting in high responsiveness of neural cells to glutamate, N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and ( S)-3,5-dihydroxyphenylglycine (an agonist for mGluR1/5), as revealed by calcium imaging assays. NMDA receptors, AMPA receptors, mGluR1, and mGluR5 were functionally validated by using the specific antagonists MK-801, NBQX, JNJ16259685, and 2-methyl-6-(phenylethynyl)-pyridine, respectively. Multielectrode array analysis showed that spontaneous firing occurred earlier in cells cultured in 2% O2 than in 20% O2. Optimization of O2 tension and culture medium for neural differentiation of hiPSCs can efficiently generate physiologically relevant cells for screening systems.
- Development of an In Vitro Model to Screen CYP1B1-Targeted Anticancer Prodrugs. [Journal Article]
- JBJ Biomol Screen 2016; 21(10):1090-1099
- Cytochrome P450 1B1 (CYP1B1) is an anticancer therapeutic target due to its overexpression in a number of steroid hormone-related cancers. One anticancer drug discovery strategy is to develop prodrug...
Cytochrome P450 1B1 (CYP1B1) is an anticancer therapeutic target due to its overexpression in a number of steroid hormone-related cancers. One anticancer drug discovery strategy is to develop prodrugs specifically activated by CYP1B1 in malignant tissues to cytotoxic metabolites. Here, we aimed to develop an in vitro screening model for CYP1B1-targeted anticancer prodrugs using the KLE human endometrial carcinoma cell line. KLE cells demonstrated superior stability of CYP1B1 expression relative to transiently transfected cells and did not express any appreciable amount of cognate CYP1A1 or CYP1A2, which would have compromised the specificity of the screening assay. The effect of two CYP1B1-targeted probe prodrugs on KLE cells was evaluated in the absence and presence of a CYP1B1 inhibitor to chemically "knock out" CYP1B1 activity (CYP1B1 inhibited). Both probe prodrugs were more toxic to KLE cells than to CYP1B1-inhibited KLE cells and significantly induced G0/G1 arrest and decreased the S phase in KLE cells. They also exhibited pro-apoptotic effects in KLE cells, which were attenuated in CYP1B1-inhibited KLE cells. In summary, a KLE cell-based model has been characterized to be suitable for identifying CYP1B1-targeted anticancer prodrugs and should be further developed and employed for screening chemical libraries.
- Enabling 1536-Well High-Throughput Cell-Based Screening through the Application of Novel Centrifugal Plate Washing. [Journal Article]
- JBJ Biomol Screen 2016 Dec 01; :2472555216683650
- Cell-based assays have long been important within hit discovery paradigms; however, improving the disease relevance of the assay system can positively affect the translation of small-molecule drug di...
Cell-based assays have long been important within hit discovery paradigms; however, improving the disease relevance of the assay system can positively affect the translation of small-molecule drug discovery, especially if adopted in the initial hit identification assay. Consequently, there is an increasing need for disease-relevant assay systems capable of running at large scale, including the use of induced pluripotent stem cells and donor-derived primary cells. Major hurdles to adopting these assays for high-throughput screening are the cost, availability of cells, and complex protocols. Miniaturization of such assays to 1536-well format is an approach that can reduce costs and increase throughput. Adaptation of these complex cell assays to 1536-well format brings major challenges in liquid handling for high-content assays requiring washing steps and coating of plates. In addition, problematic edge effects and reduced assay quality are frequently encountered. In this study, we describe the novel application of a centrifugal plate washer to facilitate miniaturization of a range of 1536-well cell assays and techniques to reduce edge effects, all of which improved throughput and data quality. Cell assays currently limited in throughput because of cost and complex protocols may be enabled by the techniques presented in this study.
- Induced Pluripotent HD Monkey Stem Cells Derived Neural Cells for Drug Discovery. [Journal Article]
- JBJ Biomol Screen 2016 Dec 01; :2472555216685044
- Huntington's disease (HD) is a neurodegenerative disease caused by an expansion of CAG trinucleotide repeat (polyglutamine [polyQ]) in the huntingtin ( HTT) gene, which leads to the formation of muta...
Huntington's disease (HD) is a neurodegenerative disease caused by an expansion of CAG trinucleotide repeat (polyglutamine [polyQ]) in the huntingtin ( HTT) gene, which leads to the formation of mutant HTT (mHTT) protein aggregates. In the nervous system, an accumulation of mHTT protein results in glutamate-mediated excitotoxicity, proteosome instability, and apoptosis. Although HD pathogenesis has been extensively studied, effective treatment of HD has yet to be developed. Therapeutic discovery research in HD has been reported using yeast, cells derived from transgenic animal models and HD patients, and induced pluripotent stem cells from patients. A transgenic nonhuman primate model of HD (HD monkey) shows neuropathological, behavioral, and molecular changes similar to an HD patient. In addition, neural progenitor cells (NPCs) derived from HD monkeys can be maintained in culture and differentiated to neural cells with distinct HD cellular phenotypes including the formation of mHTT aggregates, intranuclear inclusions, and increased susceptibility to oxidative stress. Here, we evaluated the potential application of HD monkey NPCs and neural cells as an in vitro model for HD drug discovery research.
- A Systematic Investigation of the Best Buffers for Use in Screening by MALDI-Mass Spectrometry. [Journal Article]
- JBJ Biomol Screen 2016 Dec 08
- Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) offers a label-free alternative for the screening of biochemical targets in both 1536- and 6144-assay formats, as well as pote...
Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) offers a label-free alternative for the screening of biochemical targets in both 1536- and 6144-assay formats, as well as potentially providing increased sensitivity, reproducibility, and the simultaneous detection of multiple assay components within a specified m/z range. Ion suppression effects are one of the principal limitations reported for MS analysis. Within MALDI-MS screening, it has been identified that certain biochemical components incorporated into the assay (e.g., the buffers used to preserve the physiological conditions of the enzyme, salts, and other additives) induce suppression of the analyte ion signals monitored. This poorly understood phenomenon of ion suppression is a key reason the screening community has been reluctant to shift their investigations toward MS methods with reduced sample cleanup. Using acetylcholine as an assay substrate mimic, we have generated robust data to quantify the degree to which the most highly used components (base buffers, additional components, detergents, cell culture media, and other additives) within current screening assays are compatible with MALDI-MS. Here, the most suitable buffers and components, along with their identified optimal concentrations in terms of limiting ion suppression effects, are proposed for use in screening assays measured by MALDI-MS.
- HtrA3 Isoform-Specific ELISAs for Early Detection of Preeclampsia. [Journal Article]
- JBJ Biomol Screen 2016 Dec 08
- Preeclampsia is a serious disorder of human pregnancy occurring after 20 weeks of gestation. It can be divided into subtypes of early onset (<34 weeks of gestation) and late onset (>34 weeks). Presym...
Preeclampsia is a serious disorder of human pregnancy occurring after 20 weeks of gestation. It can be divided into subtypes of early onset (<34 weeks of gestation) and late onset (>34 weeks). Presymptomatic detection to identify those at high risk is important for managing this disease. HtrA3, a serine protease with high expression in the developing placenta, exists in long (HtrA3-L) and short (HtrA3-S) isoforms. They are identical, except HtrA3-S lacks the C-terminal PDZ domain. We have previously shown by Western blot analysis that serum HtrA3 levels at the end of the first trimester are significantly higher in women who later develop preeclampsia than in controls. In this study, using highly specific HtrA3 monoclonal antibodies, we established and fully validated two enzyme-linked immunosorbent assays to detect both HtrA3 isoforms together (HtrA3-T) and HtrA3-L alone in the human serum. We then determined serum HtrA3 at 11 to 13 weeks of gestation in a cohort of singleton pregnancies that proceeded without complications or developed preeclampsia in the third trimester. Compared with controls, those who developed late-onset preeclampsia had significantly higher levels of HtrA3-L, whereas those who developed early-onset preeclampsia had significantly lower ratios of HtrA3-L/HtrA3-T. These data support a potential utility of these HtrA3 ELISAs for early detection of preeclampsia.
- Identification of Chemical Compounds That Inhibit Protein Synthesis in Pseudomonas aeruginosa. [Journal Article]
- JBJ Biomol Screen 2016 Nov 21
- Four inhibitory compounds were identified using a poly-uridylic acid (polyU) mRNA-directed aminoacylation/translation (A/T) protein synthesis system composed of phenylalanyl-tRNA synthetases (PheRS),...
Four inhibitory compounds were identified using a poly-uridylic acid (polyU) mRNA-directed aminoacylation/translation (A/T) protein synthesis system composed of phenylalanyl-tRNA synthetases (PheRS), ribosomes, and ribosomal factors from Pseudomonas aeruginosa in an in vitro screen of a synthetic compound library. The compounds were specific for inhibition of bacterial protein synthesis. In enzymatic assays, the compounds inhibited protein synthesis with IC50 values ranging from 20 to 60 μM. Minimum inhibitory concentrations (MICs) were determined in cultures for a panel of pathogenic organisms, including Enterococcus faecalis, Escherichia coli, Haemophilus influenzae, P. aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae All the compounds were observed to have broad-spectrum activity and inhibited an efflux pump mutant strain of P. aeruginosa with MICs of 0.5-16 μg/mL. The molecular target of two compounds was determined to be PheRS. These two compounds were bacteriostatic against both Gram-positive and Gram-negative pathogens. In competition assays, they were not observed to compete with the natural substrates ATP or phenylalanine for active site binding. The other two compounds directly inhibited the ribosome and were bactericidal against both Gram-positive and Gram-negative pathogens. In cytotoxicity MTT testing in human cell lines, the compounds were shown to be from 2500- to 30,000-fold less active than the control staurosporine.
- High-Throughput Platform for Identifying Molecular Factors Involved in Phenotypic Stabilization of Primary Human Hepatocytes In Vitro. [Journal Article]
- JBJ Biomol Screen 2016; 21(9):897-911
- Liver disease is a leading cause of morbidity worldwide and treatment options are limited, with organ transplantation being the only form of definitive management. Cell-based therapies have long held...
Liver disease is a leading cause of morbidity worldwide and treatment options are limited, with organ transplantation being the only form of definitive management. Cell-based therapies have long held promise as alternatives to whole-organ transplantation but have been hindered by the rapid loss of liver-specific functions over a period of days in cultured hepatocytes. Hypothesis-driven studies have identified a handful of factors that modulate hepatocyte functions in vitro, but our understanding of the mechanisms involved remains incomplete. We thus report here the development of a high-throughput platform to enable systematic interrogation of liver biology in vitro. The platform is currently configured to enable genetic knockdown screens and includes an enzyme-linked immunosorbent assay-based functional assay to quantify albumin output as a surrogate marker for hepatocyte synthetic functions as well as an image-based viability assay that counts hepatocyte nuclei. Using this platform, we identified 12 gene products that may be important for hepatocyte viability and/or liver identity in vitro. These results represent important first steps in the elucidation of mechanisms instrumental to the phenotypic maintenance of hepatocytes in vitro, and we hope that the tools reported here will empower additional studies in various fields of liver research.
- SLAS Europe High-Content Screening Conference in Dresden: A Glimpse of the Future? [Editorial]
- JBJ Biomol Screen 2016; 21(9):883-6
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- One-Step Seeding of Neural Stem Cells with Vitronectin-Supplemented Medium for High-Throughput Screening Assays. [Journal Article]
- JBJ Biomol Screen 2016; 21(10):1112-1124
- Human neuronal cells differentiated from induced pluripotent cells have emerged as a new model system for the study of disease pathophysiology and evaluation of drug efficacy. Differentiated neuronal...
Human neuronal cells differentiated from induced pluripotent cells have emerged as a new model system for the study of disease pathophysiology and evaluation of drug efficacy. Differentiated neuronal cells are more similar in genetics and biological content to human brain cells than other animal disease models. However, culture of neuronal cells in assay plates requires a labor-intensive procedure of plate precoating, hampering its applications in high-throughput screening (HTS). We developed a simplified method with one-step seeding of neural stem cells in assay plates by supplementing the medium with a recombinant human vitronectin (VTN), thus avoiding plate precoating. Robust results were obtained from cell viability, calcium response, and neurite outgrowth assays using this new method. Our data demonstrate that this approach greatly simplifies high-throughput assays using neuronal cells differentiated from human stem cells for translational research.