High-throughput Screening in Embryonic Stem Cell-derived Neurons Identifies Potentiators of α-Amino-3-hydroxyl-5-methyl-4-isoxazolepropionate-type Glutamate Receptors

Stem cell biology offers advantages to investigators seeking to identify new therapeutic molecules. Specifically, stem cells are genetically stable, scalable for molecular screening, and function in cellular assays for drug efficacy and safety. A key hurdle for drug discoverers of central nervous system disease is a lack of high quality neuronal cells. In the central nervous system, α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) subtype glutamate receptors mediate the vast majority of excitatory neurotransmissions. Embryonic stem (ES) cell protocols were developed to differentiate into neuronal subtypes that express AMPA receptors and were pharmacologically responsive to standard compounds for AMPA potentiation. Therefore, we hypothesized that stem cell-derived neurons should be predictive in high-throughput screens (HTSs). Here, we describe a murine ES cell-based HTS of a 2.4 × 10⁶ compound library, the identification of novel chemical “hits” for AMPA potentiation, structure function relationship of compounds and receptors, and validation of chemical leads in secondary assays using human ES cell-derived neurons. This reporting of murine ES cell derivatives being formatted to deliver HTS of greater than 10⁶ compounds for a specific drug target conclusively demonstrates a new application for stem cells in drug discovery. In the future new molecular entities may be screened directly in human ES or induced pluripotent stem cell derivatives.     

蛋白酪氨酸磷酸酶1B抑制剂高通量筛选模型建立和应用

本文以蛋白酪氨酸磷酸酶1B（protein tyrosine phosphatase,PTP1B）作为靶点,采用分子克隆GST融合蛋白的方法,重组表达获得PTP1B,结合自动化操作技术和比色分析,建立了一种PTP1B抑制剂的高通量筛选模型.经在96孔板优化各种反应条件并对17940个样品的筛选结果表明,靶向PTP1B建立的高通量筛选模型具有微量、快速、特异、灵敏等特点,平均日筛选量可达15000样次以上,为寻找新的抗糖尿病药物和先导化合物提供了一种先进的手段.     

Bioreactor-Based Online Recovery of Human Progenitor Cells with Uncompromised Regenerative Potential: A Bone Tissue Engineering Perspective

The use of a 3D perfusion culture environment for stem cell expansion has been shown to be beneficial for maintenance of the original cell functionality but due to several system inherent characteristics such as the presence of extracellular matrix, the continued development and implementation of 3D perfusion bioreactor technologies is hampered. Therefore, this study developed a methodology for harvesting a progenitor cell population from a 3D open porous culture surface after expansion in a perfusion bioreactor and performed a functional characterization of the expanded cells. An initial screening showed collagenase to be the most interesting reagent to release the cells from the 3D culture surface as it resulted in high yields without compromising cell viability. Subsequently a Design of Experiment approach was used to obtain optimized 3D harvest conditions by assessing the interplay of flow rate, collagenase concentration and incubation time on the harvest efficiency, viability and single cell fraction. Cells that were recovered with the optimized harvest protocol, by perfusing a 880 U/ml collagenase solution for 7 hours at a flow rate of 4 ml/min, were thereafter functionally analyzed for their characteristics as expanded progenitor cell population. As both the in vitro tri-lineage differentiation capacity and the in vivo bone forming potential were maintained after 3D perfusion bioreactor expansion we concluded that the developed seeding, culture and harvest processes did not significantly compromise the viability and potency of the cells and can contribute to the future development of integrated bioprocesses for stem cell expansion.     

High-throughput screening: update on practices and success

High-throughput screening (HTS) has become an important part of drug discovery at most pharmaceutical and many biotechnology companies worldwide, and use of HTS technologies is expanding into new areas. Target validation, assay development, secondary screening, ADME/Tox, and lead optimization are among the areas in which there is an increasing use of HTS technologies. It is becoming fully integrated within drug discovery, both upstream and downstream, which includes increasing use of cell-based assays and high-content screening (HCS) technologies to achieve more physiologically relevant results and to find higher quality leads. In addition, HTS laboratories are continually evaluating new technologies as they struggle to increase their success rate for finding drug candidates. The material in this article is based on a 900-page HTS industry report involving 54 HTS directors representing 58 HTS laboratories and 34 suppliers.     

Bioprocessing of embryonic stem cells for drug discovery

Embryonic stem cells provide a potential resource for research and drug screening. To make such a resource feasible, it is necessary to generate cells of sufficient quality and quantity. The challenge is to expand cell numbers while maintaining the fidelity of phenotype and to control and direct differentiation to produce the cell type of interest in a format that is suitable for drug screening. At present, large-scale culturing of human ES cell lines is problematic and provides substantial challenges. This article provides an overview of current bioprocessing techniques that could be used to generate cells for drug discovery applications. This will generate further technical expertise that can be applied in the production of cells for potential therapeutic applications.     

A novel embryotoxic estimation method of VPA using ES cells differentiation system

Valproic acid (VPA), which has a wide range of therapeutic applications, is known as a potent teratogen that induces neural tube defects in vertebrates. Here, we have characterized the tissue-specific, embryotoxic effects of VPA on developmental processes using a novel system with differentiating mouse ES cells. Under our cultivating condition, ES cells differentiated into cardiomyocytes, although various cell types can be differentiated. VPA affected cell viability and differentiation from undifferentiated ES cells to cardiomyocytes in a dose-dependent manner. The analysis of tissue-specific markers also revealed that VPA potently inhibited mesodermal and endodermal development but promoted neuronal differentiation in a lineage-specific manner. Taking the in vivo teratogenicity of VPA into account, this assay system could be useful in predicting the degree of embryotoxicity of VPA. We, thus, propose that the in vivo embryotoxic effects of various medicines can be estimated fast and accurately using this in vitro cell differentiation system.     

Aptamers as reagents for high-throughput screening

The identification of new drug candidates from chemical libraries is a major component of discovery research in many pharmaceutical companies. Given the large size of many conventional and combinatorial libraries and the rapid increase in the number of possible therapeutic targets, the speed with which efficient high-throughput screening (HTS) assays can be developed can be a rate-limiting step in the discovery process. We show here that aptamers, nucleic acids that bind other molecules with high affinity, can be used as versatile reagents in competition binding HTS assays to identify and optimize small-molecule ligands to protein targets. To illustrate this application, we have used labeled aptamers to platelet-derived growth factor B-chain and wheat germ agglutinin to screen two sets of potential small-molecule ligands. In both cases, binding affinities of all ligands tested (small molecules and aptamers) were strongly correlated with their inhibitory potencies in functional assays. The major advantages of using aptamers in HTS assays are speed of aptamer identification, high affinity of aptamers for protein targets, relatively large aptamer-protein interaction surfaces, and compatibility with various labeling/detection strategies. Aptamers may be particularly useful in HTS assays with protein targets that have no known binding partners such as orphan receptors. Since aptamers that bind to proteins are often specific and potent antagonists of protein function, the use of aptamers for target validation can be coupled with their subsequent use in HTS.     

Kinetic and metabolic analysis of mouse embryonic stem cell expansion under serum-free conditions

There is a need for a deeper understanding of the biochemical events affecting embryonic stem (ES) cell culture by analyzing the expansion of mouse ES cells in terms of both cell growth and metabolic kinetics. The influence of the initial cell density on cell expansion was assessed. Concomitantly, the biochemical profile of the culture was evaluated, which allowed measuring the consumption of important substrates, such as glucose and glutamine, and the production of metabolic byproducts, like lactate. The results suggest a more efficient cell metabolism in serum-free conditions and a preferential use of glutaminolysis as an energy source during cell expansion at low seeding densities. This work contributes to the development of fully-controlled bioprocesses to produce relevant numbers of ES cells for cell therapies and high-throughput drug screening.     

Efficient establishment of human embryonic stem cell lines and long-term maintenance with stable karyotype by enzymatic bulk passage

Human ES (hES) cell lines are considered to be a valuable resource for medical research and for applications in cell therapy and drug discovery. For such utilization of hES cells to be realized, however, protocols involved in the use of hES cells, such as those for establishment, propagation, and cryopreservation, have still to be improved. Here, we report on an efficient method for the establishment of hES cell lines and its detailed characterization. Additionally, we developed a new bulk-passaging technique that preserves the karyotypic integrity of hES cell lines when maintained in culture for up to 2 years. Finally, we show that a simplified vitrification cryopreservation technique is vastly superior to standard slow-cooling methods with respect to cell viability. These results provide valuable information that will assist in achieving the goal of the large-scale hES cell culture required for the application of hES cells to disease therapy.     

A low-volume platform for cell-respirometric screening based on quenched-luminescence oxygen sensing

Cell viability assays represent an important technology in modern cell biology, drug discovery and biotechnology, where currently there is a high demand for simple, sensitive and cost-effective screening methods. We have developed a new methodology and associated tools for cell-based screening assays, which are based on the measurement of the rates of oxygen uptake in cells by luminescence quenching. Sealable microchamber devices matching the footprint of a standard 96-well plate were developed and used in conjunction with long-decay phosphorescent oxygen probes. These devices permit cell non-invasive, real-time monitoring of cellular respiration and a rapid, one-step, kinetic assessment of multiple samples for cell viability, drug/effector action. These assays can be carried out on conventional fluorescence plate readers, they are suitable for different types of cells, including adherent and slow-respiring cells, require small sample volumes and cell numbers, and are amenable for high throughput screening. Monitoring of as little as 300 mammalian cells in 3 microl volume has been demonstrated.     

Multiparametric Analysis of Tissue Spheroids Fabricated from Different Types of Cells

Reproducible, scalable, and cost effective fabrication and versatile characterization of tissue spheroids (TS) is highly demanded by 3D bioprinting and drug discovery. Consistent geometry, defined mechanical properties, optimal viability, appropriate extracellular matrix/cell organization are required for cell aggregates aimed for application in these fields. A straightforward procedure for fabrication and systematic multiparametric characterization of TS with defined properties and uniform predictable geometry employing non‐adhesive technology is suggested. Applying immortalized and primary cells, the reproducibility of spheroid generation, the strong correlation of ultimate spheroid diameter, and growth pattern with cell type and initial seeding concentration are demonstrated. Spheroids viability and mechanical properties are governed by cell derivation. In this study, a new decision procedure to apply for any cell type one starts to work with to prepare and typify TS meeting high quality standards in biofabrication and drug discovery is suggested.     

Formation of germ-line chimeras from embryonic stem cells maintained with recombinant leukemia inhibitory factor

Murine embryonic stem (ES) cells can be maintained as stem cells in vitro only in the presence of feeder cells or a soluble factor produced by a number of cell lines. We have previously demonstrated that leukemia inhibitory factor (LIF) is the molecule which prevents ES cell differentiation in culture. In this report we demonstrate that recombinant LIF can substitute for feeder cells in maintaining the full developmental potential of ES cells. The totipotent D3 ES cell line, previously isolated and maintained on growth-arrested primary embryo fibroblasts, was transferred to media supplemented with 1000 U/ml (10 ng/ml) recombinant LIF. In the presence of LIF the ES cells were maintained for over 2 months as undifferentiated cells in the absence of any feeder cells. When injected into blastocysts the ES cells which had been maintained in LIF-supplemented media efficiently formed germ-line chimeras.     

Joint effects of acetochlor and urea on germinating characteristics of crop seeds

Neuraminidase (NA) is one of the most important targets to screen the drugs of anti-influenza virus A and B. After virtual screening approaches were applied to a compound database which possesses more than 10000 compound structures, 160 compounds were selected for bioactivity assay, then a High Throughput Screening (HTS) model established for influenza virus NA inhibitors was applied to detect these compounds. Finally, three compounds among them displayed higher inhibitory activities, the range of their IC5o was from 0.1 μmol/L to 3 μmol/L. Their structural scaffolds are novel and different from those of NA inhibitors approved for influenza treatment, and will be useful for the design and research of new NA inhibitors. The result indicated that the combination of virtual screening with HTS was very significant to drug screening and drug discovery.     

Drug screening for influenza neuraminidase inhibitors

Neuraminidase (NA) is one of the most important targets to screen the drugs of anti-influenza virus A and B. After virtual screening approaches were applied to a compound database which possesses more than 10000 compound structures, 160 compounds were selected for bioactivity assay, then a High Throughput Screening (HTS) model established for influenza virus NA inhibitors was applied to detect these compounds. Finally, three compounds among them displayed higher inhibitory activities, the range of their IC50 was from 0.1 μmol/L to 3μmol/L. Their structural scaffolds are novel and different from those of NA inhibitors approved for influenza treatment, and will be useful for the design and research of new NA inhibitors. The resuit indicated that the combination of virtual screening with HTS was very significant to drug screening and drug discovery.     

Evaluation of cellular adhesion and organization in different microporous polymeric scaffolds

The lack of prediction accuracy during drug development and screening risks complications during human trials, such as drug‐induced liver injury (DILI), and has led to a demand for robust, human cell‐based, in vitro assays for drug discovery. Microporous polymer‐based scaffolds offer an alternative to the gold standard flat tissue culture plastic (2D TCPS) and other 3D cell culture platforms as the porous material entraps cells, making it advantageous for automated liquid handlers and high‐throughput screening (HTS). In this study, we optimized the surface treatment, pore size, and choice of scaffold material with respect to cellular adhesion, tissue organization, and expression of complex physiologically relevant (CPR) outcomes such as the presence of bile canaliculi‐like structures. Poly‐l‐ lysine and fibronectin (FN) coatings have been shown to encourage cell attachment to the underlying substrate. Treatment of the scaffold surface with NaOH followed with a coating of FN improved cell attachment and penetration into pores. Of the two pore sizes we investigated (A: 104 ± 4 μm; B: 175 ± 6 μm), the larger pore size better promoted cell penetration while limiting tissue growth from reaching the hypoxia threshold. Finally, polystyrene (PS) proved to be conducive to cell growth, penetration into the scaffold, and yielded CPR outcomes while being a cost‐effective choice for HTS applications. These observations provide a foundation for optimizing microporous polymer‐based scaffolds suitable for drug discovery. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:505–514, 2018     

A fluorometric assay for high-throughput screening targeting nicotinamide phosphoribosyltransferase

Nicotinamide adenine dinucleotide (NAD) plays a crucial role in many cellular processes. As the rate-limiting enzyme of the predominant NAD biosynthesis pathway in mammals, nicotinamide phosphoribosyltransferase (Nampt) regulates the cellular NAD level. Tumor cells are more sensitive to the NAD levels, making them more susceptible to Nampt inhibition than their nontumorigenic counterparts. Experimental evidence has indicated that Nampt might have proangiogenic activity and supports the growth of some tumors, so Nampt inhibitors may be promising as antitumor agents. However, only four Nampt inhibitors have been reported, and no high-throughput screening (HTS) strategy for Nampt has been proposed to date, largely limiting the drug discovery targeting Nampt. Therefore, the development of a robust HTS strategy for Nampt is both imperative and significant. Here we developed a fluorometric method for a Nampt activity assay by measuring the fluorescence of nicotinamide mononucleotide (NMN) derivative resulting from the enzymatic product NMN through simple chemical reactions. Then we set up an HTS system after thorough optimizations of this method and validated that it is feasible and effective through a pilot screening on a small library. This HTS system should expedite the discovery of Nampt inhibitors as antitumor drug candidates.     

High-density seeding of myocyte cells for cardiac tissue engineering

Tissue engineering of 1- to 5-mm-thick, functional constructs based on cells that cannot tolerate hypoxia for prolonged time periods (e.g., cardiac myocytes) critically depends on our ability to seed the cells at a high and spatially uniform initial density and to maintain their viability and function. We hypothesized that rapid gel-cell inoculation in conjunction with direct medium perfusion through the seeded scaffold would increase the rate, yield, viability, and uniformity of cell seeding. Two cell types were studied: neonatal rat cardiomyocytes for feasibility studies of seeding and cultivation with direct medium perfusion, and C2C12 cells (a murine myoblast cell line) for detailed seeding studies. Cells were seeded at densities corresponding to those normally present in the adult rat heart ([0.5-1] x 10(8) cells/cm(3)), into collagen sponges (13 mm x 3 mm discs), using Matrigel as a vehicle for rapid cell delivery. Scaffolds inoculated with cell-gel suspension were seeded either in perfused cartridges with alternating medium flow or in orbitally mixed Petri dishes. The effects of seeding time (1.5 or 4.5 h), initial cell number (6 or 12 million cells per scaffold), and seeding set-up (medium perfusion at 0.5 and 1.5 mL/min; orbitally mixed dishes) were investigated using a randomized three-factor factorial experimental design with two or three levels and three replicates. The seeding cell yield was consistently high (over 80%), and it appeared to be determined by the rapid gel inoculation. The decrease in cell viability was markedly lower for perfused cartridges than for orbitally mixed dishes (e.g., 8.8 +/- 0.8% and 56.3 +/- 4%, respectively, for 12 million cells at 4.5 h post-seeding). Spatially uniform cell distributions were observed in perfused constructs, whereas cells were mainly located within a thin (100-200 microm) surface layer in dish seeded constructs. Over 7 days of cultivation, medium perfusion maintained the viability and differentiated function of cardiac myocytes, and the constructs contracted synchronously in response to electrical stimulation. Direct perfusion can thus enable seeding of hypoxia-sensitive cells at physiologically high and spatially uniform initial densities and maintain cell viability and function.