Federal Institute for Risk Assessment (BfR), Center for Alternative Methods to Animal Experiments (ZEBET), Berlin, Germany.
Nat Protoc. 2011 Jun 16;6(7):961-78. doi: 10.1038/nprot.2011.348.
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.
在胚胎干细胞测试（EST）中，使用小鼠胚胎干细胞（mESCs）的分化作为体外评估胚胎毒性的模型。该测试已被欧洲替代方法验证中心（ECVAM）成功验证，并模拟了胚胎毒性的基本机制，如细胞毒性和分化。此外，还考虑了分化（成人）和胚胎细胞之间敏感性的差异。为了预测测试物质的胚胎毒性潜力，评估了三个终点：抑制分化为搏动心肌细胞、对干细胞的细胞毒性作用以及对 3T3 成纤维细胞的细胞毒性作用。EST 的一个特点是它仅基于永久细胞系，因此不需要来自怀孕动物的原代胚胎细胞和组织。在本方案中，我们描述了 ECVAM 验证的方法，其中收缩心肌细胞的形态评估用作分化的终点，以及基于分子的 FACS-EST 方法，其中选择了针对发育中的心脏组织的高度预测性蛋白标记物。使用这些方法，可以在 10 或 7 天内分别在体外评估化合物的胚胎毒性效力。
