Cell-based Drug Discovery Research Articles

Developmental potential of human oocytes reconstructed by transferring somatic cell nuclei into polyspermic zygote cytoplasm

The generation of patient-specific nuclear transfer embryonic stem cells holds huge promise in modern regenerative medicine and cell-based drug discovery. Since human in vivo matured oocytes are not readily available, human therapeutic cloning is developing slowly. Here, we investigated for the first time whether human polyspermic zygotes could support preimplantation development of cloned embryos. Our results showed that polyspermic zygotes could be used as recipients for human somatic cell nuclear transfer (SCNT). The preimplantation developmental potential of SCNT embryos from polyspermic zygotes was limited to the 8-cell stage. Since ES cell lines can be derived from single blastomeres, these results may have important significance for human ES cells derived by SCNT. In addition, confocal images demonstrated that all of the SCNT embryos that failed to cleave showed abnormal microtubule organization. The results of the present study suggest that polyspermic human zygotes could be used as a potential source of recipient cytoplasm for SCNT.

Viral-mediated gene delivery for cell-based assays in drug discovery

Background: Adenovirus, retrovirus and lentivirus-based vectors, originally engineered and optimized for in vivo and ex vivo gene therapy, have become increasingly useful for viral-mediated gene delivery to support in vitro cell-based assays. Viral vectors underpin functional genomics screening of cDNA, shRNA and aptamer libraries, are used for a variety of target validation studies and importantly, for high-throughput cell-based drug discovery and compound profiling assays. The baculovirus/insect cell expression system had gained prevalence as a tool for recombinant protein production when it was observed that recombinant baculovirus vectors too could serve as efficient gene delivery vehicles for a wide range of mammalian cells. Although the use of baculovirus vectors in vivo has lagged behind retroviral, adenoviral and lentiviral vectors, they have gained prominence for development of in vitro cell-based assays due to the ease of generation, broad host range and excellent biosafety profile. There is an increasing emphasis on cell-based assays in high-throughput automated drug discovery laboratories and a variety of commercially available viral-vectors can be used for supporting these assays. Objective: We compare and contrast the current viral-mediated gene delivery vector systems and highlight their suitability for cell-based drug discovery assays. Conclusion: Viral-mediated gene delivery is increasingly being used in support of genome scale target validation studies and cell-based assay development for specific drug target genes such as ion channels, G protein-coupled receptors and intracellular enzymes. The choice of a delivery system over another for a particular application is largely dictated by the cell types and cell lines in use, virus cellular tropism, assay throughput, safety requirements and ease/cost of reagent generation.

Development of Human Cloned Blastocysts Following Somatic Cell Nuclear Transfer with Adult Fibroblasts

Nuclear transfer stem cells hold considerable promise in the field of regenerative medicine and cell-based drug discovery. In this study, a total of 29 oocytes were obtained from three young (20-24 years old) reproductive egg donors who had been successful in previous cycles. These oocytes, deemed by intended parents to be in excess of their reproductive needs, were donated for research without financial compensation by both the egg donor and intended parents after receiving informed consent. All intended parents successfully achieved ongoing pregnancies with the oocytes retained for reproductive purposes. Mature oocytes, obtained within 2 hours following transvaginal aspiration, were enucleated using one of two methods, extrusion or aspiration, after 45 minutes of incubation in cytochalasin B. Rates of oocyte lysis or degeneration did not differ between the two methods. Somatic cell nuclear transfer (SCNT) embryos were constructed using two established adult male fibroblast lines of normal karyotype. High rates of pronuclear formation (66%), early cleavage (47%), and blastocyst (23%) development were observed following incubation in standard in vitro fertilization culture media. One cloned blastocyst was confirmed by DNA and mitochondrial DNA fingerprinting analyses, and DNA fingerprinting of two other cloned blastocysts indicated that they were also generated by SCNT. Blastocysts were also obtained from a limited number of parthenogenetically activated oocytes. This study demonstrates, for the first time, that SCNT can produce human blastocyst-stage embryos using nuclei obtained from differentiated adult cells and provides new information on methods that may be needed for a higher level of efficiency for human nuclear transfer.

High-content single-cell drug screening with phosphospecific flow cytometry

Drug screening is often limited to cell-free assays involving purified enzymes, but it is arguably best applied against systems that represent disease states or complex physiological cellular networks. Here, we describe a high-content, cell-based drug discovery platform based on phosphospecific flow cytometry, or phosphoflow, that enabled screening for inhibitors against multiple endogenous kinase signaling pathways in heterogeneous primary cell populations at the single-cell level. From a library of small-molecule natural products, we identified pathway-selective inhibitors of Jak-Stat and MAP kinase signaling. Dose-response experiments in primary cells confirmed pathway selectivity, but importantly also revealed differential inhibition of cell types and new druggability trends across multiple compounds. Lead compound selectivity was confirmed in vivo in mice. Phosphoflow therefore provides a unique platform that can be applied throughout the drug discovery process, from early compound screening to in vivo testing and clinical monitoring of drug efficacy.

Evaluation of protein kinase activities of cell lysates using peptide microarrays based on surface plasmon resonance imaging

We developed a peptide microarray based on surface plasmon resonance (SPR) imaging for monitoring protein kinase activities in cell lysates. The substrate peptides of kinases were tethered to the microarray surface modified with a self-assembled monolayer of an alkanethiol with triethylene glycol terminus to create a low nonspecific binding surface. The phosphorylation of the substrate peptides immobilized on the surface was detected with the following phosphate specific binders by amplifying SPR signals: anti-phosphotyrosine antibody for tyrosine kinases and Phos-tag biotin (a phosphate-specific ligand with biotin tag) for serine/threonine kinases. Using the microarray, 9 kinds of protein kinases were evaluated as a pattern of phosphorylation of 26 kinds of substrate peptides. The pattern was unique for each protein kinase. The microarray could be used to evaluate the inhibitory activities of kinase inhibitors. The microarray was applied successfully for kinase activity monitoring of cell lysates. The chemical stimuli responsive activity changes of protein kinases in cell lysates could also be monitored by the peptide microarray. Thus, the peptide microarray based on SPR imaging would be applicable to cell-based drug discovery, diagnosis using tissue lysates, and biochemical studies to reveal signal transduction pathways.

A novel mammalian cell-based approach for the discovery of anticancer drugs with reduced cytotoxicity on non-dividing cells.

A key asset of cytotoxic drugs in cancer therapeutics is their ability to discriminate between proliferating and mitotically inert cells and eliminate preferentially neoplastic ones. We have designed a high throughput-compatible mammalian cell-based assay for the discovery of cytotoxic drugs, which selectively kill proliferation-competent target cells. This cytotoxic drug discovery assay is based on a transgenic CHO-K1-derived cell line engineered for a conditional G1-specific growth arrest following tetracycline-responsive overexpression of the human cyclin-dependent kinase inhibitor p27(Kip1). The CHO-derived cell line CHO-p27(Kip1) shows wild type proliferation rates and can be expanded in the presence of tetracycline antibiotics when p27(Kip1) expression is repressed. Upon withdrawal of regulating antibiotics CHO-p27(Kip1) differentiates into a 1:1 mixed population consisting of two different proliferation phenotypes: (i) a G1-arrested cell population induced by heterologous expression of p27(Kip1) which mimics mitotically inactive terminally differentiated cells and (ii) a proliferation-competent cell population which eliminated the p27(Kip1) expression unit and imitates neoplastic cell characteristics. Addition of chemical or metabolic libraries to CHO-p27(Kip1) populations cultivated in tetracycline-free medium followed by scoring for cell viability will reveal cytotoxic drug candidates associated with a high viability ratio of proliferation-competent/arrested populations. We have validated the cell-based cytotoxic drug discovery assay using the clinically licensed cancer drugs mitomycin C, doxorubicin, etoposide and 5-fluorouracil. Comparative proof-of-concept studies showed that these top-prescribed cancer therapeutics preferentially eliminate proliferating cells while showing less interference with the viability of G1-arrested cell populations. These results demonstrate the CHO-p27(Kip1)-based cytotoxic drug finder technology is ready-to-apply for high throughput screenings of chemical as well as metabolic libraries to discover novel cancer therapeutics which show reduced cytotoxicity on terminally differentiated cells.

Cardiovascular tissue engineering

Tissue engineering is a broad term describing a set of tools at the interface of the biomedical and engineering sciences that use living cells or attract endogenous cells to aid tissue formation or regeneration, and thereby produce therapeutic or diagnostic benefit. In the most frequent paradigm, cells are seeded on a scaffold composed of synthetic polymer or natural material (collagen or chemically treated tissue), a tissue is matured in vitro, and the construct is implanted in the appropriate anatomic location as a prosthesis [1–4]. A typical scaffold is a bioresorbable polymer in a porous configuration in the desired geometry for the engineered tissue, often modified to be adhesive for cells, in some cases selective for a specific cell population. Either application-specific differentiated or undifferentiated (stem) cells are used [5,6]. The first phase is the in vitro formation of a tissue construct by placing the chosen cells and scaffold in a metabolically and mechanically supportive environment with growth media (in a bioreactor), in which the cells proliferate and elaborate extracellular matrix (ECM). In the second phase, the construct is implanted in the appropriate anatomic location, where remodeling in vivo is intended to recapitulate the normal functional architecture of an organ or tissue. The key processes occurring during the in vitro and in vivo phases of tissue formation and maturation are (1) cell proliferation, sorting and differentiation; (2) ECM production and organization; (3) degradation of the scaffold; and (4) remodeling and potentially growth of the tissue. The general paradigm of tissue engineering is illustrated in Fig. 1. Since the cells of the implant can contact the surrounding tissues of the recipient, this approach is termed an ‘‘open-system.’’ In contrast, ‘‘closed-system’’ devices have therapeutic cells that are encapsulated or otherwise isolated from the recipient’s blood or tissues by semipermeable polymer membranes. This permits nutrients, wastes, drugs or hormones to pass yet keeps larger molecules such as the recipient’s antibodies or inflammatory/immune cells away, thus protecting the cells from degradation. Closed-system devices are used to deliver biologically active agents (such as drugs) to a restricted anatomic site (localized, controlled drug delivery) or to serve as extracorporeal cell-containing devices for renal, hepatic or pancreatic assist. Some tissue engineering applications use one of various ‘‘incomplete’’ paradigms, in which certain steps in the general paradigm are omitted (Fig. 2). For example, the cell-seeded scaffold model (see Fig. 2A) is exemplified by endothelial seeding of a synthetic or tissue derived vascular graft prior to implantation [7,8]. The cell transplant model (see Fig. 2B) is exemplified by one approach to myocardial tissue engineering in which injected cells (myocytic or with myocyte potential) differentiate and proliferate in or near damaged myocardium [9,10]. Investigators attempting to provide engineered heart valves and vascular grafts have utilized decellularized tissue scaffolds that attract endogenous cells (see Fig. 2C) to repopulate and remodel an altered tissue that preserves architectural and chemical information [11,12]. In vitro tissue ‘‘equivalents’’ (see Fig. 2D) may provide tools to understand normal physiological processes, study pathogenesis of disease and provide diagnostic and therapeutic tools (e.g., patterned cell cultures to probe cellcell and cell-matrix interactions and enhance cell-based drug discovery and target validation, vascular constructs to study contractile responses, and myocardial constructs for studying drug toxicities and responses to tissue injury) [13,13a,13b]. The innovative fabrication of materials and the development of sophisticated methods to repair or regenerate damaged or diseased tissues and to create entire organ replacements requires integration of a diverse array of basic scientific principles and enabling technologies. Thus, tissue engineering requires an understanding of relationships of structure to function in normal and pathological tissues

Molecules for the millennium: how will they look? New drug discovery year 2000.

A new approach to cancer drug discovery targets molecules important in cancer pathogenesis. This approach is thought to be of greater promise than the antiproliferative screens which discovered cytotoxic agents and dominated cancer drug discovery for 60 years. However, one cannot lose sight of the fact that these targets exist in the cellular environment consisting of many additional influences on target function, and that effective drug treatment will take into account drug uptake, metabolism and elimination at the level of the cell as well as the organism. A key goal is to define for the new millennium a path to cancer drug discovery and development which accounts for the cancer cell phenotype in its totality rather than as arising solely from single molecular targets. The US National Cancer Institute maintains a cell-based drug discovery screen which can define a context for drug action in the milieu of more than 300 molecular targets and thousands of gene expression patterns which have been measured in the 60 human tumour cell lines which comprise the screening panel. The challenge of the millennium will be addressed by molecules active against defined targets but with selectivity of action occurring in the milieu of deregulated cancer cell biology in all its aspects. © 2000 Cancer Research Campaign http://www.bjcancer.com

A flow injection renewable surface technique for cell-based drug discovery functional assays.

A novel flow injection-renewable surface (FI-RS) technique is introduced for the execution of automated pharmacology-based assays on living cells. Cells are attached to microcarrier beads, which serve as the disposable and renewable surface with which the assay is performed. The feasibility of this FI-RS technique is demonstrated by performing a functional assay using Chinese hamster ovary cells transfected with the rat muscarinic receptor (M1). The intracellular calcium elevation resulting from the agonist-receptor interaction is measured via a calcium-sensitive fluorescent probe (fura-2) and a fluorescence microscope photometry system. The FI apparatus allows reproducible and precise control of the concentration gradient of chosen muscarinic receptor agonists (carbachol, acetylcholine, pilocarpine) delivered to cells attached to microcarrier beads. The RS methodology eliminates problems associated with diminishing biological response vis-à-vis traditional functional assays that are performed repetitively on the same group of cells. Using this technique, reproducible responses are measured and pharmacologic parameters quantified that compare favorably to literature values. In addition, the use of the FI-RS functional assay as an analytical method for discrimination of agonists based on kinetic parameters is proposed.