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

基本信息

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
项目类别：
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

项目摘要

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：迫切需要改进体外人体毒理学测试，以筛选和识别潜在的有毒化合物、其致死水平，并在细胞和分子水平评估其对人类神经干细胞（NSC）的发育毒性。特别是，了解人类毒性的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，并且对人类健康和疾病预防具有重要影响。和原代人类细胞有已普遍用于此类毒理学研究，但其使用受到物种变异性高、与人类相关性很少或没有可预测性、原代细胞的不稳定性以及供应不足的限制。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，并使用三维 (3D) 培养的人类 NSC 准确预测体内不良反应。结合高通量方法和高内涵成像 (HCI)，我们建议破译有毒模型化合物作用的细胞和分子机制，核心假设是：(i) 小型化 3D NSC 微阵列可以维持高水平。通过更好地模拟体内微环境来调节神经细胞功能；(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； (iv) 这种小型化 3D 细胞培养系统可用于促进机械毒理学测定，从而提高体内毒性的可预测性。拟议工作的具体目标是 (1) 证明 3D NSC 的高神经元细胞功能。高通量仿生微环境中的微阵列(2) 使用高通量、离子通道和转运蛋白分析研究各类化合物在 NSC 微阵列上的机制作用，以及 (3) 在 3D NSC 微阵列上建立 HCI 分析，以研究化合物及其毒性的机制特征这些信息对于更好地了解胚胎和成人细胞和组织的药物毒理学机制基础，并根据环境毒物对人类的潜在不利影响进行优先排序至关重要。