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首页> 外文期刊>BMC Neuroscience >High yield derivation of enriched glutamatergic neurons from suspension-cultured mouse ESCs for neurotoxicology research
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High yield derivation of enriched glutamatergic neurons from suspension-cultured mouse ESCs for neurotoxicology research

机译：从悬浮培养的小鼠胚胎干细胞中高产地衍生出丰富的谷氨酸能神经元，用于神经毒理学研究

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Background Recently, there has been a strong emphasis on identifying an in vitro model for neurotoxicity research that combines the biological relevance of primary neurons with the scalability, reproducibility and genetic tractability of continuous cell lines. Derived neurons should be homotypic, exhibit neuron-specific gene expression and morphology, form functioning synapses and consistently respond to neurotoxins in a fashion indistinguishable from primary neurons. However, efficient methods to produce neuronal populations that are suitable alternatives to primary neurons have not been available. Methods With the objective of developing a more facile, robust and efficient method to generate enriched glutamatergic neuronal cultures, we evaluated the neurogenic capacity of three mouse embryonic stem cell (ESC) lines (R1, C57BL/6 and D3) adapted to feeder-independent suspension culture. Neurogenesis and neuronal maturation were characterized as a function of time in culture using immunological, genomic, morphological and functional metrics. The functional responses of ESNs to neurotropic toxins with distinctly different targets and mechanisms of toxicity, such as glutamate, α-latrotoxin (LTX), and botulinum neurotoxin (BoNT), were also evaluated. Results Suspension-adapted ESCs expressed markers of pluripotency through at least 30 passages, and differentiation produced 97×106 neural progenitor cells (NPCs) per 10-cm dish. Greater than 99% of embryonic stem cell-derived neurons (ESNs) expressed neuron-specific markers by 96 h after plating and rapidly developed complex axodendritic arbors and appropriate compartmentalization of neurotypic proteins. Expression profiling demonstrated the presence of transcripts necessary for neuronal function and confirmed that ESN populations were predominantly glutamatergic. Furthermore, ESNs were functionally receptive to all toxins with sensitivities and responses consistent with primary neurons. Conclusions These findings demonstrate a cost-effective, scalable and flexible method to produce a highly enriched glutamatergic neuron population. The functional characterization of pathophysiological responses to neurotropic toxins and the compatibility with multi-well plating formats were used to demonstrate the suitability of ESNs as a discovery platform for molecular mechanisms of action, moderate-throughput analytical approaches and diagnostic screening. Furthermore, for the first time we demonstrate a cell-based model that is sensitive to all seven BoNT serotypes with EC50 values comparable to those reported in primary neuron populations. These data providing compelling evidence that ESNs offer a neuromimetic platform suitable for the evaluation of molecular mechanisms of neurotoxicity.
机译：背景技术近来，一直非常重视鉴定用于神经毒性研究的体外模型，该模型将原代神经元的生物学相关性与连续细胞系的可扩展性，可再现性和遗传易处理性相结合。衍生的神经元应该是同型的，表现出神经元特异性的基因表达和形态，形成功能性突触，并以与原代神经元无法区分的方式持续响应神经毒素。但是，还没有有效的方法来产生神经元种群，而神经元种群是原代神经元的合适替代者。方法为了开发一种更简便，可靠且有效的方法来生成丰富的谷氨酸能神经元培养物，我们评估了三种适于饲养者非依赖性的小鼠胚胎干细胞（ESC）系（R1，C57BL / 6和D3）的神经生成能力。悬浮培养。使用免疫学，基因组学，形态学和功能指标将神经发生和神经元成熟表征为培养中时间的函数。还评估了ESN对具有明显不同的靶标和毒性机制的神经毒素的功能响应，例如谷氨酸，α-拉托毒素（LTX）和肉毒杆菌神经毒素（BoNT）。结果悬浮液适应的ESCs通过至少30代表达多能性标记，并且每10厘米培养皿的分化产生97×10 6 神经祖细胞（NPC）。接种后96小时，超过99％的胚胎干细胞衍生神经元（ESNs）表达了神经元特异性标记，并迅速形成复杂的轴突树突和适当的神经型蛋白区室。表达谱表明存在神经元功能所必需的转录本，并证实ESN群体主要为谷氨酸能。此外，ESN在功能上可以接受所有毒素，其敏感性和反应与原代神经元一致。结论这些发现证明了生产高浓缩谷氨酸能神经元群体的经济有效，可扩展和灵活的方法。对神经营养毒素的病理生理反应的功能表征以及与多孔平板格式的兼容性被用来证明ESN作为分子机理，中通量分析方法和诊断筛选的发现平台的适用性。此外，我们首次展示了一种基于细胞的模型，该模型对所有七个BoNT血清型均敏感，其EC 50 值可与原代神经元群体中报道的值相媲美。这些数据提供了令人信服的证据，表明ESNs提供了适合模拟神经毒性分子机制的仿神经平台。

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