Hand1-Luc embryonic stem cell test (Hand1-Luc EST): a novel rapid and highly reproducible in vitro test for embryotoxicity by measuring cytotoxicity and differentiation toxicity using engineered mouse ES cells.

Abstract

The embryonic stem cell test (EST) is a promising alternative method for evaluating embryotoxicity of test chemicals by measuring cytotoxicity and differentiation toxicity using mouse ES cells. Differentiation toxicity is analyzed by microscopically counting the beating of embryonic bodies after 10 days of culture. However, improvements are necessary to reduce the laborious manipulations involved and the time required to obtain results. We have previously reported the successful stable transfection of ES cells (ES-D3) with the heart and neural crest derivatives expressed transcript 1 (Hand1) gene and the establishment of a 96-well multi-plate-based new EST with luciferase reporter assay 6 days after treatment with test chemicals. Now, we propose an even more rapid and easier EST, named Hand1-Luc EST. We established another cell line to monitor the Hand1 gene expression via a luciferase reporter gene. By mRNA analysis and luciferase assay, we examined in detail the luciferase activity during cell differentiation, which allowed us to reduce the time of measurement from day 6 to day 5 (120 hr). Furthermore, the protocol was improved, with, among others, the measurement of cytotoxicity and differentiation toxicity taking place in the same 96-well round bottom plate instead of two different plates. With the positive control, 5-fluorouracil (5-FU), and 9 test chemicals, data with high reproducibility and very low variation (CV < 50%) in the relevant endpoints were obtained. This study shows that the Hand1-Luc EST could provide an accurate and sensitive short-term test for prediction of embryotoxicants by measuring cytotoxicity and differentiation toxicity from the same sample.