(36a) An ESC-Based Test for High Throughput Screening of Embryotoxicity of Drugs and Chemicals

Toxicity on embryonic development is a severe adverse reaction of drugs and chemical compounds. About 10% birth defects are caused by mutagenic, embryotoxic, and teratogenic chemicals and drugs taken by pregnant women. Therefore, embryotoxic effects of drugs need to be evaluated before clinical trials. Currently, animal-based test, embryonic stem cells (ESCs) based test (EST), rat whole-embryo culture test, zebrafish embryonic development test and the micro-mass test are applied for assessing drug embryotoxicity. EST using two cell lines is the most promising tool for in vitro embryotoxicity test because no animal is sacrificed. However, large scale application of EST is limited because of the long culture period and need for morphological endpoint analysis which requires experience. To predict embryotoxicity rapidly and precisely, high throughput EST could be achieved by using reporter genes and monitoring their signals from ESCs cultured in microbioreactors. Although luciferase has been used as the reporter gene in previous studies, it requires luciferin as the enzyme substrate and yet can only provide endpoint data. In this study, we have developed an embryotoxicity test based on drug responses of murine embryonic stem cells (mESC) cultured in three-dimensional polymer scaffolds in microbioreactors using enhanced green fluorescent protein (EGFP) as the reporter, which provides a non-invasive and real-time monitoring method for high throughput drug screening. Expressions of EGFP are driven by selected promoters associated with human genes involved in the regulation of proliferation and/or differentiation. Stable mES cell lines expressing EGFP under the control of these promoters were developed and used to evaluate various chemicals with known cytotoxicity and embryotoxicity validated by the European Center for the Validation of Alternative Methods (ECVAM). The results showed that our EST method could accurately predict toxicity and embryotoxicity of tested drugs, demonstrating its application for high throughput embryotoxicity screening.