Cell Toxicology Team, National Food Institute, Technical University of Denmark, Kemitorvet B204, 2800 Kgs, Lyngby, Denmark.
Arch Toxicol. 2024 Dec;98(12):4107-4116. doi: 10.1007/s00204-024-03870-8. Epub 2024 Oct 4.
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.
新的方法学（NAMs）对于生成人类相关数据、减少周转时间和成本以及减轻与动物模型使用相关的伦理问题，迫切需要预测胚胎毒性和发育毒性。我们之前开发了 PluriLum 测定法，这是一种使用基因修饰的人诱导多能干细胞（hiPSCs）的 NKX2.5 报告基因 3D 模型，用于评估化学物质对早期胚胎的不良影响。为了提高 PluriLum 测定法在未来筛选中的预测价值，我们试图在方案中引入标准化步骤，提高 PluriLum 测定法的整体稳健性，并缩短测定法方案。首先，我们表明胚状体（EBs）的初始大小对于适当分化为心肌细胞和测定法的整体重现性至关重要。当 EB 的起始直径超过 500 µm 时，可以预期稳健的分化。在重现性方面，与使用较大初始直径的球体获得的更可靠的浓度反应曲线相比，在较小的初始直径下暴露于杀真菌剂表氯醇会导致衍生数据的更大变化。我们进一步研究了分化方案的理想长度，从而将 PluriLum 测定法缩短了 24 小时至 7 天。在接触致畸剂全反式和 13-顺式视黄酸后，与原始 7 天相比，分化 6 天后记录到心肌细胞收缩和 NKX2.5 衍生的荧光测量的敏感性相似或增加。最后，我们引入了一种有效的 EB 酶解步骤，以在测定结束时终止。这允许甚至分裂单个 EB，并测试除 NKX2.5-荧光素酶报告基因之外的其他附加终点，在这项工作中，通过同时评估 ATP 水平来证明了这一点。总之，我们已经引入了标准化并简化了 PluriLum 测定法方案，以提高其作为用于筛选大量化学物质进行发育毒性测试的 NAM 的适用性。
