In Vitro Toxicology and Biomedicine, Department Inaugurated By the Doerenkamp-Zbinden Chair Foundation, University of Konstanz, Box 657, 78457, Konstanz, Germany.
Department of Statistics, TU Dortmund, 44221, Dortmund, Germany.
Arch Toxicol. 2020 Jan;94(1):151-171. doi: 10.1007/s00204-019-02612-5. Epub 2019 Nov 11.
The first in vitro tests for developmental toxicity made use of rodent cells. Newer teratology tests, e.g. developed during the ESNATS project, use human cells and measure mechanistic endpoints (such as transcriptome changes). However, the toxicological implications of mechanistic parameters are hard to judge, without functional/morphological endpoints. To address this issue, we developed a new version of the human stem cell-based test STOP-tox. For this purpose, the capacity of the cells to self-organize to neural rosettes was assessed as functional endpoint: pluripotent stem cells were allowed to differentiate into neuroepithelial cells for 6 days in the presence or absence of toxicants. Then, both transcriptome changes were measured (standard STOP-tox) and cells were allowed to form rosettes. After optimization of staining methods, an imaging algorithm for rosette quantification was implemented and used for an automated rosette formation assay (RoFA). Neural tube toxicants (like valproic acid), which are known to disturb human development at stages when rosette-forming cells are present, were used as positive controls. Established toxicants led to distinctly different tissue organization and differentiation stages. RoFA outcome and transcript changes largely correlated concerning (1) the concentration-dependence, (2) the time dependence, and (3) the set of positive hits identified amongst 24 potential toxicants. Using such comparative data, a prediction model for the RoFA was developed. The comparative analysis was also used to identify gene dysregulations that are particularly predictive for disturbed rosette formation. This 'RoFA predictor gene set' may be used for a simplified and less costly setup of the STOP-tox assay.
体外发育毒性的首批测试利用了啮齿动物细胞。较新的致畸学测试，例如在 ESNATS 项目期间开发的测试，使用人类细胞并测量机制终点（例如转录组变化）。然而，如果没有功能/形态终点，判断机制参数的毒理学意义是很困难的。为了解决这个问题，我们开发了一种基于人类干细胞的新型 STOP-tox 测试版本。为此，将细胞自我组织成神经玫瑰花结的能力评估为功能终点：多能干细胞在存在或不存在毒物的情况下分化为神经上皮细胞 6 天。然后，测量转录组变化（标准 STOP-tox）并允许细胞形成玫瑰花结。在优化染色方法后，实施了用于自动玫瑰花结形成测定法（RoFA）的玫瑰花结定量成像算法。神经管毒物（如丙戊酸）已知会在存在形成玫瑰花结的细胞的阶段干扰人类发育，被用作阳性对照。已建立的毒物导致明显不同的组织组织和分化阶段。RoFA 结果和转录变化在（1）浓度依赖性、（2）时间依赖性和（3）在 24 种潜在毒物中确定的阳性命中集方面与转录变化密切相关。使用这种比较数据，开发了 RoFA 的预测模型。比较分析还用于识别对扰乱玫瑰花结形成具有特别预测性的基因失调。这个“RoFA 预测基因集”可用于简化和降低 STOP-tox 测定的成本。
