The embryonic stem cell test (EST) is a validated in vitro alternative test for prediction of embryotoxicity with inhibition of cardiomyocyte differentiation under the microscope as beating areas at day 10 as an endpoint. However, improvements are necessary for regulatory acceptance and application to high-throughput screening. We have previously reported that heart and neural crest derivatives expressed transcript 1 (Hand1), a transcription factor essential for mammalian heart development, and cardiomyopathy associated 1 (Cmya1), an intercalated disk protein implicated in cardiac morphogenesis, are quantitative and objective molecular endpoints for predicting embryotoxicity, detected at day 6 when mouse embryonic stem (ES) cells differentiate into cardiomyocytes. In established stable transgenic ES cells with Hand1 or Cmya1 promoters upstream of luciferase reporter gene, changes in each gene expression were found to be coincident with those in luciferase activities during cardiomyocyte differentiation, suggesting that monitoring might be possible by chemiluminescent determination. In our novel EST, differentiation toxicity and cytotoxicity of test chemicals were here analyzed using ES cells and 3T3 fibroblasts by this approach in 96-microwell plates. Extensive investigations were performed to explore predictive power and validity by comparing a set of 24 well-known test chemicals. The novel EST offers high predictability and accuracy with a reduced test duration and manpower compared with the original EST protocol, thus providing a new rapid and sensitive in vitro method for screening embryotoxicants.

-The Hand1-and Cmya1-ESTs are novel short-term tests for embryotoxic chemicals using genetically engineering mouse ES cells for luciferase reporter gene assays. These ESTs allow convenient determination of differentiation toxicity and cell viability in a short duration with high throughput 96-well microplates for prediction of embryotoxicity of chemicals. To assess the Hand1-EST technical protocol, we firstly compared reporter gene assay and cytotoxicity test data for a representative compound (hydroxyurea) from four different laboratories with tests carried out under the same experimental conditions. Extensive investigations of the Hand1-and Cmya1-ESTs were then performed to explore reproducibility by comparing a set of 6 well-known test chemicals, including hydroxyurea, across the laboratories. The results gave good correspondence in all four laboratories, indicating that transferability, intra-laboratory variability and inter-laboratory variability of the present technical protocols of the ESTs were sufficient to conduct further validation studies.