当前位置: X-MOL 学术Arch. Toxicol. › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
Development of a neural rosette formation assay (RoFA) to identify neurodevelopmental toxicants and to characterize their transcriptome disturbances.
Archives of Toxicology ( IF 6.1 ) Pub Date : 2019-11-11 , DOI: 10.1007/s00204-019-02612-5
Nadine Dreser 1 , Katrin Madjar 2 , Anna-Katharina Holzer 1 , Marion Kapitza 1 , Christopher Scholz 1 , Petra Kranaster 1, 3 , Simon Gutbier 1, 4 , Stefanie Klima 1 , David Kolb 5, 6 , Christian Dietz 5, 6 , Timo Trefzer 1, 7 , Johannes Meisig 8 , Christoph van Thriel 9 , Margit Henry 10 , Michael R Berthold 5 , Nils Blüthgen 8 , Agapios Sachinidis 10 , Jörg Rahnenführer 2 , Jan G Hengstler 9 , Tanja Waldmann 1 , Marcel Leist 1
Affiliation  

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(UKN). 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(UKN)) 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(UKN) assay.

中文翻译:

开发神经玫瑰花结形成测定法(RoFA),以鉴定神经发育毒物并表征其转录组紊乱。

发育毒性的第一个体外测试利用了啮齿动物细胞。例如在ESNATS项目期间开发的较新的术语学测试使用人体细胞并测量机械终点(例如转录组变化)。但是,如果没有功能/形态学终点,则很难判断机械参数的毒理学含义。为了解决此问题,我们开发了基于人类干细胞的测试STOP-tox(UKN)的新版本。为此,将细胞自组织为神经玫瑰花结的能力评估为功能终点:在有毒或无毒的情况下,使多能干细胞分化为神经上皮细胞达6天。然后,测量两个转录组的变化(标准STOP-tox(UKN)),并使细胞形成玫瑰花结。优化染色方法后,实施了用于玫瑰花定量的成像算法,并将其用于自动化玫瑰花形成测定(RoFA)。已知在存在玫瑰花结形成细胞的阶段会干扰人类发育的神经管有毒物质(如丙戊酸)被用作阳性对照。既定的有毒物质导致明显不同的组织组织和分化阶段。RoFA结果和转录物变化与(1)浓度依赖性,(2)时间依赖性和(3)在24种潜在有毒物质中鉴定出的一组阳性结果之间存在很大的相关性。使用这些比较数据,开发了RoFA的预测模型。比较分析还用于确定基因失调,这些失调特别可预测玫瑰花结的形成。这个“ RoFA预测基因集”
更新日期:2019-11-11
down
wechat
bug