(175e) A Multiple Molecular Endpoints HT Screening of Embryotoxic Chemicals Based on ESC-EGFP with Human Gene Promoters Associated with Specific Embryonic Development Pathways

Currently, there are thousands of chemicals in common use; however, only a small number of them have sufficient toxicity evaluation, and even less information for the specific embryo-related toxicity field: embryotoxicity and developmental toxicity. Embryonic stem cell test (EST) is the only accepted in vitro method for assessing embryotoxicity without animal sacrifice. However, the implementation and application of conventional EST for regulatory embryotoxicity screening are impeded by its labor intensity (technical complexity), long testing period (low throughput), and poor robustness (limited differentiation lineage endpoints) for the screening of thousands of drugs and chemicals in use or under development.

A key step in the understanding of embryonic development is to construct a systemic and comprehensive gene regulation network involved in the development-related pathways. In our research, a novel EST with mouse embryonic stem cells (mESCs) expressing enhanced green fluorescent protein (EGFP) driven by human gene promoters, including those associated with survivin in the apoptosis, Oct4 (POU5f1) in pluripotency, and SOX17 and TUBB3 in tissue-specific differentiation pathways, were developed for use in high throughput (HT) screening of potential embryotoxic chemicals and their effects on embryonic development. Our hypothesis was that embryotoxic chemicals would down-regulate these gene promoters and thus decrease EGFP expression. mESCs expressing EGFP driven by the constitutive CMV promoter was used to quantify proliferation and cytotoxicity of tested chemicals. These EGFP-expressing mESCs were cultured in two-dimensional (2D) plate or three-dimensional (3D) fibrous scaffolds in microbioreactors on a multi-well plate, and EGFP fluorescence signals as indicative of cell responses to chemicals were monitored non-invasively using a fluorescent plate reader. The downregulation of these four gene promoters by chemicals with known embryotoxicity were first tested with strongly embryotoxic hydroxyurea and retinoic acid, weakly embryotoxic salicylic acid sodium salt, and non-embryotoxic acrylamide and penicillin. Then, thalidomide, which has not been classified for its embryotoxicity but its past use by pregnant women had caused severe birth defect, was tested. The results from the assay showed that thalidomide down-regulated all four gene promoters, indicating its strong embryotoxicity and damaging effects on a wide range of human embryonic development pathways. The mESC-EGFP assays with these pathway-specific gene promoters as molecular biomarkers thus can be used for HT screening of embryotoxic chemicals. The data obtained from the screening can provide the information needed for the regulation of drugs and chemicals.