An in vitro embryotoxicity assay based on the disturbance of the differentiation of murine embryonic stem cells into endothelial cells. II. Testing of compounds.

The embryonic stem cell test (EST) developed by Spielmann et al. [Spielmann, H., Pohl, I., Doering, B., Liebsch, M., Moldenhauer, F., 1997. The embryonic stem cell test, an in vitro embryotoxicity test using two permanent mouse cell lines: 3T3 fibroblasts and embryonic stem cells. In Vitro. Toxicol. 10, 119-127] is currently the most promising in vitro assay to predict the embryotoxic potential of compounds. In this assay the disturbance of the differentiation of embryonic stem (ES) cells into contracting cardiomyocytes by test compounds as well as the direct cytotoxicity of the test compounds on ES cells and 3T3 fibroblasts is analyzed. On the basis of these results and by applying a biostatistical prediction model (PM) [Genschow, E., Scholz, G., Brown, N., Piersma, A., Brady, M., Clemann, N., Huuskonen, H., Paillard, F., Bremer, S., Becker, K., Spielmann, H., 2000. Development of prediction models for three in vitro embryotoxicity tests in an ECVAM validation study. In Vitr. Mol. Toxicol. 13, 51-66; Genschow, E., Spielmann, H., Scholz, G., Pohl, I., Seiler, A., Clemann, N., Bremer, S., Becker, K., 2004. Validation of the embryonic stem cell test in the international ECVAM validation study on three in vitro embryotoxicity tests. Altern. Lab. Anim. 32, 209-244; Genschow, E., Spielmann, H., Scholz, G., Seiler, A., Brown, N., Piersma, A., Brady, M., Clemann, N., Huuskonen, H., Paillard, F., Bremer, S., Becker, K., 2002. The ECVAM international validation study on in vitro embryotoxicity tests: results of the definitive phase and evaluation of prediction models. European Centre for the Validation of Alternative Methods. Altern. Lab. Anim. 30, 151-176] test compounds can be classified as non-embryotoxic, weakly or strongly embryotoxic. In order to introduce a further endpoint into the EST, the disturbance of vasculogenesis and/or angiogenesis, a protocol to differentiate ES cells into endothelial cells, was established in the accompanying paper. PECAM-1 and VE-Cadherin gene expressions, quantified by real-time TaqMan PCR, were shown to be appropriate molecular markers for the differentiation of ES cells into endothelial cells. In the present study, the disturbance of the differentiation of ES cells into endothelial cells (i.e. the reduction in the expression of PECAM-1 and VE-Cadherin) by six test compounds with known embryotoxic potential was investigated: all-trans-retinoic acid (RA) and 5-fluorouracil (5-FU) are strongly embryotoxic, diphenylhydantoin (DPH) and valproic acid (Val) are weakly embryotoxic and saccharin (Sacch) and penicillin G (Pen G) are non-embryotoxic. In a first step the concentration of the test compound resulting in a 50% inhibition of PECAM-1 and VE-Cadherin gene expression and the concentration leading to a 50% decrease in the viability of ES cells and 3T3 fibroblasts were determined. In a second step and in a first attempt to assess the predictive potential of the newly developed test system the concentration values obtained were applied in the PM of the established EST to classify the selected test compounds. All six test compounds were correctly classified (i.e. the data obtained in vitro correlated with their known embryotoxic potential in vivo). Taken together it can be concluded that the disturbance of the differentiation of murine ES cells into endothelial cells represents a very promising new endpoint in a broadened EST with PECAM-1 and VE-Cadherin as specific differentiation marker genes.