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Abstract：
Human induced pluripotent stem cell (hiPSC)-derived neural stem cells (NSCs) and their differentiated neuronal/glial derivatives have been recently considered suitable to assess in vitro developmental neurotoxicity (DNT) triggered by exposure to environmental chemicals. The use of human-relevant test systems combined with in vitro assays specific for different neurodevelopmental events, enables a mechanistic understanding of the possible impact of environmental chemicals on the developing brain, avoiding extrapolation uncertainties associated with in vivo studies. Currently proposed in vitro battery for regulatory DNT testing accounts for several assays suitable to study key neurodevelopmental processes, including NSC proliferation and apoptosis, differentiation into neurons and glia, neuronal migration, synaptogenesis, and neuronal network formation. However, assays suitable to measure interference of compounds with neurotransmitter release or clearance are at present not included, which represents a clear gap of the biological applicability domain of such a testing battery. Here we applied a HPLC-based methodology to measure the release of neurotransmitters in a previously characterized hiPSC-derived NSC model undergoing differentiation towards neurons and glia. Glutamate release was assessed in control cultures and upon depolarization, as well as in cultures repeatedly exposed to some known neurotoxicants (BDE47 and lead) and chemical mixtures. Obtained data indicate that these cells have the ability to release glutamate in a vesicular manner, and that both glutamate clearance and vesicular release concur in the maintenance of extracellular glutamate levels. In conclusion, analysis of neurotransmitter release is a sensitive readout that should be included in the envisioned battery of in vitro assays for DNT testing.
摘要：
人类诱导的多能干细胞(hiPSC)衍生的神经干细胞(NSC)及其分化的神经元/神经胶质衍生物最近被认为适合于评估由暴露于环境化学品引发的体外发育神经毒性(DNT)。使用与人类相关的测试系统结合针对不同神经发育事件的体外测定，能够机械地理解环境化学物质对发育中的大脑的可能影响，避免与体内研究相关的外推不确定性。目前提出的用于调节性DNT测试的体外电池说明了几种适合研究关键神经发育过程的测定法，包括NSC增殖和凋亡，分化为神经元和神经胶质，神经元迁移，突触发生,和神经元网络的形成。然而,目前不包括适合测量化合物对神经递质释放或清除的干扰的测定，这代表了这种测试电池的生物适用性领域的明显差距。在这里，我们应用了基于HPLC的方法来测量先前表征的hiPSC衍生的NSC模型中神经递质的释放，该模型正在向神经元和神经胶质分化。在对照培养物中和去极化时评估谷氨酸释放，以及在反复暴露于某些已知神经毒物（BDE47和铅）和化学混合物的培养物中。获得的数据表明，这些细胞具有以囊泡方式释放谷氨酸的能力，谷氨酸清除和囊泡释放都有助于维持细胞外谷氨酸水平。总之,神经递质释放的分析是一个灵敏的读数，应包括在预期的DNT测试体外试验中。
