(174bz) High Throughput Embryotoxicity Assay of Drugs and Chemicals Based on Embryonic Stem Cell-EGFP Reporter System

Embryos as the early stage of development are susceptible to teratogenic chemicals during rapid cell division and differentiation. Exposure to drugs and chemicals of pregnant women may cause the embryo exposure of teratogenic chemicals by the cross of placenta, which may result in malformation. Around 3% developmental defects are caused by environmental factors such as chemicals and radiation. 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. Embryonic stem cell test (EST) is the only generally accepted in vitro method for assessing embryotoxicity without animal sacrifice. However, the implementation and application of EST for regulatory embryotoxicity screening are impeded by its technical complexity, long testing period, and limited endpoint data. Real-time PCR helps to improve the embryotoxicity prediction by molecular biological endpoints. However, monoclonal antibodies and more sophisticated detection methods such as FACS are required, which are expensive to use in high throughput screening.

In our research, a high throughput (HTS) embryotoxicity screening based on mouse embryonic stem cells (mESCs) expressing enhanced green fluorescent protein (EGFP) driven by human key genes promoters and cytomegalovirus (CMV) promoter are developed. These EGFP expressing mESCs are cultured in three-dimensional (3D) fibrous scaffolds in microbioreactors on a multiwell plate with EGFP fluorescence signals as cell responses to chemicals monitored non-invasively in a high throughput manner. The reliability of this mESC model for embryotoxicity screening of drugs and chemicals were tested with strongly, weakly and non embryotoxic chemicals validated by European Center for the Validation of Alternative Methods (ECVAM). In addition, chemicals and drugs with unknown embryotoxicity including several Chinese herbal medicines were tested using this mESC model. Our results demonstrated that the mESC-EGFP system can be used for high-throughput screening of embryotoxic potential of chemicals with a high accuracy.