First Steps in Establishing a Developmental Toxicity Test Method Based on Human Embryonic Stem Cells

The embryonic stem cell test (EST) is currently one of the most promising in vitro developmental toxicity tests. However, it would be promising to adapt the EST to human embryonic stem (hES) cells to avoid false classification of substances due to inter-species variations. The EST consists of following endpoints: IC50 values of fibroblasts and embryonic stem cells as well as the inhibition of differentiation of mES cells into cardiomyocytes. In this study we established a cytotoxicity assay based on hES cells employing two developmental toxicants: 5-fluorouracil (5-FU) and all-trans retinoic acid (RA). The results were compared to historical data from the EST. For 5-FU, no significant differences were obtained between the different cell lines. However, for RA, both test systems produced higher IC50 values for the fibroblasts than for the stem cells, which is a well-known effect of developmental toxicants. Moreover, the reliability of several marker genes was tested. During early differentiation Oct-4, hTert and Dusp6 showed the most reliable results. Brachyury and GATA-4 were found to be best suited to monitor cardiac differentiation. The late cardiac marker gene TNNT2 demonstrated significant results until day 18. Therefore, these marker genes could serve as endpoints for a developmental toxicity test.JRC.I.2-Validation of Alternative Method