基于 hiPSC 的 PluriLum 试验的标准化和优化,用于检测胚胎和发育毒物。

IF 4.8 2区 医学 Q1 TOXICOLOGY Archives of Toxicology Pub Date : 2024-10-04 DOI:10.1007/s00204-024-03870-8
Andreas Frederik Treschow, Anne Marie Vinggaard, Maria João Valente
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引用次数: 0

摘要

目前迫切需要用于预测胚胎毒性和发育毒性的新方法(NAMs),以生成与人类相关的数据,同时减少周转时间和成本,并减轻与使用动物模型相关的伦理问题。我们之前开发了 PluriLum 试验,这是一种 NKX2.5 报告基因三维模型,使用的是经过基因修饰的人类诱导多能干细胞(hiPSCs),可以评估化学品对早期胚胎的不良影响。为了提高 PluriLum 检测法在未来筛选中的预测价值,我们试图在检测方案中引入标准化步骤,提高 PluriLum 检测法的整体稳健性,并缩短检测方案。首先,我们发现胚状体(EBs)的初始大小对正确分化成心肌细胞和测定的整体可重复性至关重要。当 EBs 的初始直径超过 500 微米时,就可以预期其分化的稳健性。就可重复性而言,与使用初始直径较大的球形体获得的更可靠的浓度-反应曲线相比,初始直径较小的球形体暴露于杀真菌剂环唑醇会导致得出的数据变化较大。我们进一步研究了分化方案的理想长度,结果是将 PluriLum 试验从 24 小时缩短到 7 天。在暴露于致畸剂全反式和 13 顺式维甲酸后,与原来的 7 天相比,分化 6 天后记录的心肌细胞收缩和 NKX2.5 衍生发光的灵敏度相似或更高。最后,我们还引入了一个高效步骤,用于在检测终止时酶解 EB。这样就可以均匀拆分单个 EB,并测试 NKX2.5-luciferase 报告器以外的其他终点,这项工作通过同时评估 ATP 水平证明了这一点。总之,我们对 PluriLum 检测方案进行了标准化和简化,使其更适合作为一种 NAM 用于筛选大量化学物质以进行发育毒性测试。
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Standardization and optimization of the hiPSC-based PluriLum assay for detection of embryonic and developmental toxicants

New approach methodologies (NAMs) for predicting embryotoxicity and developmental toxicity are urgently needed for generating human relevant data, while reducing turnover time and costs, and alleviating ethical concerns related to the use of animal models. We have previously developed the PluriLum assay, a NKX2.5-reporter gene 3D model using human-induced pluripotent stem cells (hiPSCs) that are genetically modified to enable the assessment of adverse effects of chemicals on the early-stage embryo. Aiming at improving the predictive value of the PluriLum assay for future screening purposes, we sought to introduce standardization steps to the protocol, improving the overall robustness of the PluriLum assay, as well as a shortening of the assay protocol. First, we showed that the initial size of embryoid bodies (EBs) is crucial for a proper differentiation into cardiomyocytes and overall reproducibility of the assay. When the starting diameter of the EBs exceeds 500 µm, robust differentiation can be anticipated. In terms of reproducibility, exposure to the fungicide epoxiconazole at smaller initial diameters resulted in a larger variation of the derived data, compared to more reliable concentration–response curves obtained using spheroids with larger initial diameters. We further investigated the ideal length of the differentiation protocol, resulting in a shortening of the PluriLum assay by 24 h to 7 days. Following exposure to the teratogens all-trans and 13-cis retinoic acid, both cardiomyocyte contraction and measurement of NKX2.5-derived luminescence were recorded with a similar or increased sensitivity after 6 days of differentiation when compared to the original 7 days. Finally, we have introduced an efficient step for enzymatic dissociation of the EBs at assay termination. This allows for an even splitting of the individual EBs and testing of additional endpoints other than the NKX2.5-luciferase reporter, which was demonstrated in this work by the simultaneous assessment of ATP levels. In conclusion, we have introduced standardizations and streamlined the PluriLum assay protocol to improve its suitability as a NAM for screening of a large number of chemicals for developmental toxicity testing.

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来源期刊
Archives of Toxicology
Archives of Toxicology 医学-毒理学
CiteScore
11.60
自引率
4.90%
发文量
218
审稿时长
1.5 months
期刊介绍: Archives of Toxicology provides up-to-date information on the latest advances in toxicology. The journal places particular emphasis on studies relating to defined effects of chemicals and mechanisms of toxicity, including toxic activities at the molecular level, in humans and experimental animals. Coverage includes new insights into analysis and toxicokinetics and into forensic toxicology. Review articles of general interest to toxicologists are an additional important feature of the journal.
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