The validated embryonic stem cell test to predict embryotoxicity in vitro


In the embryonic stem cell test (EST), differentiation of mouse embryonic stem cells (mESCs) is used as a model to assess embryotoxicity in vitro. The test was successfully validated by the European Center for the Validation of Alternative Methods (ECVAM) and models fundamental mechanisms in embryotoxicity, such as cytotoxicity and differentiation. In addition, differences in sensitivity between differentiated (adult) and embryonic cells are also taken into consideration. To predict the embryotoxic potential of a test substance, three endpoints are assessed: the inhibition of differentiation into beating cardiomyocytes, the cytotoxic effects on stem cells and the cytotoxic effects on 3T3 fibroblasts. A special feature of the EST is that it is solely based on permanent cell lines so that primary embryonic cells and tissues from pregnant animals are not needed. In this protocol, we describe the ECVAM-validated method, in which the morphological assessment of contracting cardiomyocytes is used as an endpoint for differentiation, and the molecular-based FACS-EST method, in which highly predictive protein markers specific for developing heart tissue were selected. With these methods, the embryotoxic potency of a compound can be assessed in vitro within 10 or 7 d, respectively.

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Figure 1: Overview of steps involved in the EST and the approximate time needed.
Figure 2: Pipetting scheme for the cytotoxicity assay.
Figure 3: Stages of mESC development.
Figure 4: Representative concentration-response curves (validated EST protocol).
Figure 5: Molecular FACS-EST (flow cytometry) endpoint.


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We thank A. Visan and R. Buesen for conducting flow cytometry analyses, EST experiments and preparing illustrations, B. Slawik for excellent technical assistance, R. Pirow for excellent statistical support, C. Riebeling for critical reading of the manuscript and for his valuable contribution to the preparation of the figures and G. Friedmann-Marohn for his excellent technical support in preparing high-resolution artwork.

This work was supported in part by the German Federal Ministry of Education and Research BMBF grants 0312312 and 0313070A.

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A.E.M.S. was involved in protocol design, analysis of the experiments and preparation of the manuscript. H.S. was involved in protocol design, analysis of the experiments and preparation of the manuscript.

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Correspondence to Andrea E M Seiler.

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Seiler, A., Spielmann, H. The validated embryonic stem cell test to predict embryotoxicity in vitro. Nat Protoc 6, 961–978 (2011).

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