In vitro tests are increasingly applied in chemical hazard assessment. Basic culture conditions may affect the outcome of in vitro tests and should be optimised to reduce false predictions. The neural embryonic stem cell test (ESTn) can predict early neurodevelopmental effects of chemicals, as it mimics the differentiation of stem cells towards the neuroectodermal lineage. Normal early neural differentiation depends crucially on folic acid (FA) and methionine (MET), both elements of the one-carbon (1C) cycle. The aim of this study was to assess the concentration-dependent influence of FA and MET on neural differentiation in the ESTn, and its consequences for assay sensitivity to methotrexate (MTX), a compound that interferes with the 1C cycle. Neural differentiation was inhibited below 0.007 mM and above 0.22 mM FA, while both stem cell viability (< 0.097 mM, > 1.52 mM) and neural differentiation (< 0.181 mM, > 1.35 mM) were affected when changing MET concentrations. A 10-day exposure to 13 nM MTX inhibited neural differentiation, especially in FA- and MET-deficient conditions. However, a 24-hour exposure to 39 nM MTX decreased neural cell and neural crest cell differentiation markers only when the concentration of FA in the medium was three times the standard concentration, which was expected to have a protective effect against MTX. These results show the importance of nutrient concentrations, exposure scenarios and timing of read-outs for cell differentiation and compound sensitivity in the ESTn. Caution should be taken when interpreting results from a single in vitro test, especially when extrapolating to effects on complex morphogenetic processes, like neural tube development.

中文翻译：

---

培养条件影响体外化学诱导的发育毒性：神经胚胎干细胞试验中叶酸、蛋氨酸和甲氨蝶呤的案例

体外测试越来越多地应用于化学危害评估。基本培养条件可能会影响体外测试的结果，应进行优化以减少错误预测。神经胚胎干细胞测试 (ESTn) 可以预测化学物质对早期神经发育的影响，因为它模拟了干细胞向神经外胚层谱系的分化。正常的早期神经分化主要取决于叶酸 (FA) 和甲硫氨酸 (MET)，这两种元素都是一碳 (1C) 循环的元素。本研究的目的是评估 FA 和 MET 对 ESTn 神经分化的浓度依赖性影响，及其对甲氨蝶呤 (MTX)（一种干扰 1C 循环的化合物）检测灵敏度的影响。神经分化在低于 0.007 mM 和高于 0.22 mM FA 时被抑制，而当改变 MET 浓度时，干细胞活力（< 0.097 mM，> 1.52 mM）和神经分化（< 0.181 mM，> 1.35 mM）都会受到影响。10 天暴露于 13 nM MTX 会抑制神经分化，尤其是在 FA 和 MET 缺乏的情况下。然而，只有当培养基中的 FA 浓度是标准浓度的三倍时，24 小时暴露于 39 nM MTX 才能降低神经细胞和神经嵴细胞分化标志物，预计这对 MTX 具有保护作用。这些结果显示了营养浓度、暴露场景和读数时间对 ESTn 中细胞分化和化合物敏感性的重要性。解释单个体外试验的结果时应谨慎，