Abstract
The embryonic stem cell test (EST) represents the only validated and accepted in vitro system for the detection and classification of compounds according to their developmental and reproductive teratogenic potency. The widespread implementation of the EST, however, in particular for routine application in pharmaceutical development, has not been achieved so far. Several drawbacks still limit the high-throughput screening of potential drug candidates in this format: The long assay period, the use of non-homogeneous viability assays, the low throughput analysis of marker protein expression and the compatibility of the assay procedures to automation. We have therefore introduced several advancements into the EST workflow: A reduction of the assay period, an introduction of homogeneous viability assays, and a straightforward analysis of marker proteins by flow cytometry and high content imaging to assess the impact of small molecules on differentiation capacity. Most importantly, essential parts of the assay procedure have been adapted to lab automation in 96-well format, thus enabling the interrogation of several compounds in parallel. In addition, extensive investigations were performed to explore the predictive capacity of this next-generation EST, by testing a set of well-known embryotoxicants that encompasses the full range of chemical-inherent embryotoxic potencies possible. Due to these significant improvements, the augmented workflow provides a basis for a sensitive, more rapid, and reproducible high throughput screening compatible platform to predict in vivo developmental toxicity from in vitro data which paves the road towards application in an industrial setting.Graphical abstract•The embryonic stem cell test to predict teratogenicity was made automation-compatible.•Several key improvements to the assay procedure have been introduced to increase performance.•The workflow was adapted to human iPS cells and isogenic fibroblast donor cells.
Highlights
In drug discovery, it is mandatory to evaluate drug side effects in early preclinical stages of compound development, in particular, those that impact the physiological development of the fetus and can lead to growth retardation or teratogenesis
The vast number of tissue targets for exogenic induction of malformations during embryonic development is the rationale for toxicity testing of chemicals in highly standardized animal experiments according to Organization for Economic Cooperation and Development (OECD) test guidelines
ID50 results for hydroxyurea and saccharin are in line with data obtained from the conventional beating analysis pluripotent cells
Summary
It is mandatory to evaluate drug side effects in early preclinical stages of compound development, in particular, those that impact the physiological development of the fetus and can lead to growth retardation or teratogenesis. To establish the reliability and feasibility of the EST for industrial application during compound characterization for drug development, several optimizations to the established and validated EST protocol (Seiler and Spielmann 2011) were introduced to (1) shorten the cytotoxicity assessment procedure; (2) utilize state-of-the-art homogeneous assay formats for viability assessment based on ATP; (3) enable automation-compatibility of the workflow, in particular for cell seeding, compound dilution, media exchange, and viability assessment; (4) enable automated EB generation in a 96-well format and EB transfer for downstream applications (e.g., into 96-well imaging microplates); (5) introduce a superior routine for flowcytometric quantification of marker expression; and (6) utilize physiologically more relevant human induced pluripotent stem cells and isogenic primary human fibroblasts for the assay procedure. The proposed workflow was validated using a panel of wellcharacterized non-embryotoxic, weakly embryotoxic, and strongly embryotoxic compounds
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