The embryonic stem cell test (EST) represents the only validated and accepted in vitro system for the detection and classification of compounds according to their developmental and reproductive teratogenic potency. The widespread implementation of the EST, however, in particular for routine application in pharmaceutical development, has not been achieved so far. Several drawbacks still limit the high-throughput screening of potential drug candidates in this format: The long assay period, the use of non-homogeneous viability assays, the low throughput analysis of marker protein expression and the compatibility of the assay procedures to automation. We have therefore introduced several advancements into the EST workflow: A reduction of the assay period, an introduction of homogeneous viability assays, and a straightforward analysis of marker proteins by flow cytometry and high content imaging to assess the impact of small molecules on differentiation capacity. Most importantly, essential parts of the assay procedure have been adapted to lab automation in 96-well format, thus enabling the interrogation of several compounds in parallel. In addition, extensive investigations were performed to explore the predictive capacity of this next-generation EST, by testing a set of well-known embryotoxicants that encompasses the full range of chemical-inherent embryotoxic potencies possible. Due to these significant improvements, the augmented workflow provides a basis for a sensitive, more rapid, and reproducible high throughput screening compatible platform to predict in vivo developmental toxicity from in vitro data which paves the road towards application in an industrial setting.

•The embryonic stem cell test to predict teratogenicity was made automation-compatible.

•Several key improvements to the assay procedure have been introduced to increase performance.

•The workflow was adapted to human iPS cells and isogenic fibroblast donor cells.

胚胎干细胞测试 (EST) 代表了唯一经过验证和接受的体外系统，用于根据化合物的发育和生殖致畸效力对化合物进行检测和分类。然而，迄今为止，尚未实现 EST 的广泛实施，特别是在药物开发中的常规应用。一些缺点仍然限制了以这种格式对潜在候选药物进行高通量筛选：分析周期长、使用非均质活力分析、标记蛋白表达的低通量分析以及分析程序与自动化的兼容性。因此，我们对 EST 工作流程进行了多项改进：缩短了检测时间，引入了均质活力检测，通过流式细胞术和高内涵成像对标记蛋白进行直接分析，以评估小分子对分化能力的影响。最重要的是，检测程序的基本部分已经适应了 96 孔格式的实验室自动化，从而能够并行询问几种化合物。此外，还进行了广泛的调查，通过测试一组众所周知的胚胎毒性物质，包括所有可能的化学固有胚胎毒性效力，来探索这种下一代 EST 的预测能力。由于这些重大改进，增强的工作流程为更灵敏、更快速、

• 预测致畸性的胚胎干细胞测试与自动化兼容。

• 对化验程序进行了多项关键改进以提高性能。

• 工作流程适用于人类 iPS 细胞和同基因成纤维细胞供体细胞。