The Use of Pluripotent Cells in Developmental Toxicity Testing

The development of new in vitro tests is urgently required due to the new chemical policy of the European Union, which requests toxicological risk assessment for about 30.000 chemicals. One of the needs for this risk assessment are high throughput test systems that provide data for the classification of chemicals with regard to their developmental toxicological potential.Pluripotent cell lines offer an excellent tool for monitoring early embryonic development and thus, the interaction of chemicals during this process in vitro. These cell lines are able to differentiate into derivatives of all three germ layers. Therefore, also effects of chemicals on specific target tissues can be detected using these types of cells. In this study the different potentials of three different pluripotent cell lines: murine embryonic teratocarcinoma cells (P19); murine embryonic stem cells (D3); and human embryonic stem cells (H1) were compared.Accordingly, a new in vitro test system based on transgenic mTert_GFP/P19 cells was developed in order to detect differentiation inducing potentials of chemicals. The known in vivo ranking of the three employed retinoids: all-trans retinoic acid, 9-cis retinoic acid and 13-cis retinoic acid was confirmed using the mTert_GFP expression as an endpoint. Furthermore, it was shown that this new endpoint was up to 28 times more sensitive than the cytotoxicity test for differentiation inducing substances.In addition, threshold concentrations for the use of the solvents DMSO and ethanol were determined for their application in developmental toxicity testing. Especially, the effects of these substances on the differentiation of embryonic stem cells were explored. Unexpectedly, the concentrations used in the EST were already affecting differentiation on a molecular level in the mES cells. It was shown that mES cells could pick up differentiation inducing as well as inhibiting effects using the marker genes mTert and Oct-4 while the transgenic P19 cells are better suited to pick up differentiation inducing substances.Additionally, the possibility of adapting the murine EST to hES cells was explored. Therefore, a cytotoxicity test based on hES cells and the adult fibroblast cell line MRC-5 was established using the two well-known embryotoxic compounds 5-FU and RA. It was shown that the human system was able to reproduce the previously obtained results from the mouse system for these two chemicals. In addition, the expression of marker genes, which are involved in early and cardiac differentiation, were analysed. Among the early differentiation markers, the genes Oct-4, hTert and Dusp6 showed significant differences in comparison to the undifferentiated cells of day 0. Although an optimum medium composition for the induction of the mesoderm layer formation was determined, the yield of beating cardiomyocytes was overall very poor. Therefore, different marker genes involved in the cardiac differentiation were tested for their suitability as endpoints. Among all marker genes only the mesodermal marker gene Brachyury, the cardiac precursor marker GATA-4 and the late cardiac marker TNNT2 produced significant results until day 18.Overall, all cell lines showed advantages and disadvantages. P19 cells are well suited for the detection of differentiation inducing substances. Nonetheless, the relevance of results obtained from cancer derived cells is questionable. This problem does not arise with the use of embryonic stem cells. Murine ES cells have the advantage that they are very well explored and culture conditions are well standardised. However, these cells originate from mouse and therefore, some human embryotoxicants might not be detected by such a test method. For this reason, human embryonic stem cells seem to be best suited for developmental toxicity testing. Employing these cells, the problem of inter-species differences can be overcome and extrapolations from one species to the other become unnecessary. Nonetheless, test methods based on hES cells are difficult to standardise and high variances are typical.However, only a combination of tests will be able to pick up the whole range of possible interactions of chemicals during human development. Therefore, the advantages of different cell lines could be combined in a testing strategy consisting of different specific tests arranged in a hierarchical order. Despite the problems in standardising test methods based on hES cells, we could show in this study that these cells have the potential to provide an excellent tool for toxicological testing