Mechanistic Study of Developmental Neurotoxicity on 3D Cultured Stem Cell Microarrays

 产品说明：迫切需要改进体外人体毒理学测试，以筛选和鉴定潜在的毒性化合物，它们是致命的水平，并在细胞和分子水平上评估它们对人神经干细胞（NSC）的发育毒性。特别是，理解人类毒性的潜在机制以及包括环境毒物和候选药物的化合物的作用是NIEHS、NIGMS和EPA等联邦机构的主要目标之一，并且对人类健康和疾病预防具有重要意义。尽管动物模型和原代人细胞已被广泛用于此类毒理学研究，但它们的使用受到以下因素的限制：与人类相关性的可预测性很小或不可预测的高物种变异性、原代细胞的不稳定性以及免疫原性的供应不足。 用于高通量筛选的人原代细胞，包括NSC。因此，迫切需要开发体外策略来快速评估化合物诱导的毒性，并准确预测体内不良反应。使用三维（3D）培养的人神经干细胞，结合高通量方法和高内容成像（HCI），我们建议破译毒性模型化合物作用的细胞和分子机制。核心假设是：（i）小型化的3D NSC微阵列可以通过更好地模拟体内微环境来维持高神经元细胞功能;（ii）阻断NSC上的离子通道和转运蛋白可以调节细胞分化和细胞毒性;（iii）生理学上， 化合物的体内作用及其对NSC的机械作用可以通过高通量、高含量的细胞功能分析在体外复制和阐明;和（iv）这种小型化的3D细胞培养系统可以用于促进机械毒理学测定，这反过来可以提高体内毒性的可预测性。本研究的具体目标是：（1）在芯片上以高通量的仿生微环境在3D NSC微阵列上展示高神经元细胞功能，（2）使用高通量、离子通道和转运蛋白测定来研究各种类型的化合物对NSC微阵列的机制作用，（3）在三维神经干细胞微阵列上建立HCI检测方法，研究化合物及其代谢产物的毒性机制。这些信息对于更好地了解药物毒理学对胚胎和成人细胞和组织的机制基础至关重要，并根据其对人类的潜在不良影响优先考虑环境毒物。

 DESCRIPTION: There is a critical need for improved in vitro human toxicology testing to screen and identify potential toxic compounds, the levels at which they are lethal, and evaluate their developmental toxicity on human neural stem cells (NSCs) at the cellular and molecular levels. In particular, understanding mechanisms underlying human toxicity and the role of compounds including environmental toxicants and drug candidates is one of major goals of federal agencies such as NIEHS, NIGMS, and EPA, and has important implications in human health and disease prevention. Although animal models and primary human cells have been extensively used for such toxicology studies, their use is limited by high species variability with little or no predictability of relevance to humans, the instability of primary cells, and insufficient supply of human primary cells including NSCs for high-throughput screening. Therefore, there is an urgent need to develop in vitro strategies to rapidly assess compound-induced toxicity, and accurately predict adverse responses in vivo. Using three-dimensional (3D) cultured, human NSCs, together with high-throughput methodology and high-content imaging (HCI), we propose to decipher the cellular and molecular mechanisms underlying the effects of toxic model compounds. The core hypotheses are: (i) miniaturized 3D NSC microarrays can maintain high neuronal cell functions by better mimicking in vivo microenvironments; (ii) blocking ion channels and transporters on NSCs can modulate cell differentiation and cytotoxicity; (iii) physiological in vivo effects of compounds and their mechanistic actions on NSCs could be replicated and elucidated in vitro via high-throughput, high- content cell function analysis; and (iv) such miniaturized 3D cell culture systems can be used to facilitate mechanistic toxicology assays, which in turn can improve predictability of toxicity in vivo. The specific aims of the proposed work are to (1) demonstrate high neuronal cell functions on 3D NSC microarrays within biomimetic microenvironments in high throughput on the chip, (2) investigate mechanistic actions of various classes of compounds on NSC microarrays using high-throughput, ion channel and transporter assays, and (3) establish HCI assays on 3D NSC microarrays to investigate mechanistic profiles of toxicity by compounds and their metabolites. This information is essential to better understand the mechanistic basis of pharmaceutical toxicology on embryonic and adult human cells and tissues, and prioritize environmental toxicants based on their potential adverse effects on humans.