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

3D 培养干细胞微阵列的发育神经毒性机制研究

• 批准号: 8944604
• 负责人: MOO-YEAL LEE
• 金额: $ 32.45万
• 依托单位: CLEVELAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8944604
• 关键词: ABCB1 gene; ABCG2 gene; Acute; Adult; Adverse effects; Affect; Animal Model; Apoptosis; Astrocytes; Binding; Biological Assay; Biological Markers; Biomimetics; Brain; Cell Culture System; Cell Culture Techniques; Cell Differentiation process; Cell Surface Receptors; Cell physiology; Cells; Cellular Morphology; Cytochrome P450; Cytochromes; Data Set; Development; Embryo; Encapsulated; Enzymes; Extracellular Matrix; Glutathione; Goals; Growth; Health; Hepatocyte; Human; Hydrogels; Image; In Vitro; Individual; Ion Channel; Lead; Measures; Membrane Potentials; Metabolism; Methodology; Modeling; Molecular; National Institute of Environmental Health Sciences; National Institute of General Medical Sciences; Necrosis; Neurites; Neurons; Nuclear; Outcome; Oxidative Stress; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Physiological; Poison; Recombinants; Research; Robotics; Role; Shapes; Staging; Stem Cell Development; Stem cells; Subfamily lentivirinae; System; Technology; Testing; Therapeutic; Time; Tissues; Toxic Environmental Substances; Toxic effect; Toxicokinetics; Toxicology; Work; base; biochip; cytotoxic; cytotoxicity; developmental disease; developmental neurotoxicity; developmental toxicity; developmental toxicology; disorder prevention; drug candidate; drug discovery; high throughput screening; improved; in vivo; inhibitor/antagonist; miniaturize; mitochondrial membrane; nerve stem cell; neurogenesis; neuronal cell body; neurotoxicity; neurotoxicology; oligodendrocyte lineage; preclinical evaluation; public health relevance; receptor; response; screening; tool; toxicant; trait

 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.

 描述：迫切需要改进的体外人体毒理学测试来筛选和识别潜在的有毒化合物，确定它们的致死水平，并在细胞和分子水平上评估它们对人类神经干细胞(NSCs)的发育毒性。特别是，了解潜在的人类毒性机制以及包括环境毒物和候选药物在内的化合物的作用是NIEHS、NIGMS和EPA等联邦机构的主要目标之一，并对人类健康和疾病预防具有重要意义。虽然动物模型和原代人类细胞已被广泛用于此类毒理学研究，但它们的使用受到高度物种变异性的限制，与人类相关的可预测性很小或根本没有可预测性，原代细胞的不稳定性，以及 包括神经干细胞在内的人类原代细胞用于高通量筛选。因此，迫切需要发展体外策略来快速评估化合物的毒性，并准确地预测体内的不良反应。使用三维(3D)培养的人类神经干细胞，结合高通量方法和高内容成像(HCI)，我们建议破译有毒模型化合物影响的细胞和分子机制。核心假设是：(I)微型3D神经干细胞微阵列可以通过更好地模拟体内微环境来维持高神经细胞功能；(Ii)阻断神经干细胞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)生理功能 通过高通量、高含量的细胞功能分析，可以在体外复制和阐明化合物对神经干细胞的体内效应及其机制作用；以及(Iv)这种微型化的3D细胞培养系统可以用于促进机械性毒理学分析，这反过来可以提高体内毒性的可预测性。建议工作的具体目标是(1)在芯片上高通量地展示仿生微环境中3D NSC微阵列上的高神经细胞功能，(2)使用高通量、离子通道和转运体分析来研究各类化合物对NSC微阵列的机制作用，以及(3)在3D NSC微阵列上建立HCI分析，以研究化合物及其代谢物毒性的机制。这些信息对于更好地了解药物毒理学对胚胎和成人人类细胞和组织的机制基础，以及根据环境毒物对人类的潜在不利影响确定优先顺序是至关重要的。