High-Throughput Platform for Identifying Stem Cell Toxicity

描述（由申请人提供）：药物，候选药物，环境化学品等的表征和安全性评估需要广泛的资源，特别是当使用动物研究时。因此，有必要开发新的体外技术，以预测哪些化合物对人类健康构成更大的威胁，因此应优先进行进一步筛选和评价。由于自我更新的干细胞在组织功能和体内平衡中起着关键作用，因此保留健康的成体干细胞对人类健康至关重要。然而，与终末分化细胞类型（如原代和转化细胞系）相比，人类干细胞对药物、候选药物和化学物质的不同反应仅得到很少的研究。这些资料对于查明这类化学品对人体和特定器官的毒性作用是至关重要的。我们假设干细胞的行为可能与通常用于毒性研究的分化细胞有根本不同，我们将通过使用适当的模型系统来解决这一假设。因此，这项研究的一个结果是，毒性研究必须扩大到包括干细胞，否则药物将在缺乏甚至误导毒性信息的情况下进行试验。为了验证这一假设，我们建议开发一种高通量、微尺度的3D细胞筛选工具，该工具将能够快速、高度定量地获得药物、候选药物和化学毒物对人类干细胞的影响；特别是神经干细胞（NSCs）与成人原代和转化神经细胞以及人间充质干细胞（MSCs）的比较。拟议工作的具体目标是：1。建立健全的三维细胞培养芯片平台，培养和分化NSCs和MSCs；2. 进行组合体外细胞外基质开发；3. 开发对NSCs和MSCs增殖（自我更新）和分化至关重要的关键信号通路的高通量芯片分析；4. 使用一系列参考化合物验证3D细胞培养微阵列平台对干细胞的细胞毒性，并将其与终末分化的原代细胞和转化细胞系进行比较。这些信息对于开发药物发现的高通量预测毒理学筛选和根据其潜在的人体毒性确定环境化学品的优先级至关重要。

DESCRIPTION (provided by applicant): The characterization and safety assessment of drugs, drug candidates, environmental chemicals, etc. requires extensive resources, particularly when animal studies are employed. Thus, there is a need to develop new in vitro techniques to predict which compounds pose an increased threat to human health and should therefore be prioritized for further screening and evaluation. Because of the critical role self-renewing stem cells play in tissue function and homeostasis, retention of healthy adult stem cells is crucial for human health. Nevertheless, the different responses of human stem cells compared with terminally differentiated cell types (e.g., primary and transformed cell lines) against drugs, drug candidates, and chemicals have been only sparsely studied. Such information would be vital to identify the toxic effects of such chemicals on the human body and in specific organs. We hypothesize that stem cells may behave in a fundamentally different way from the differentiated cells often used for toxicity studies, and we will address this hypothesis through the use of appropriate model systems. An outcome of this study, then, is that toxicity studies must be expanded to include stem cells, or drugs will proceed into trials with missing and even misleading toxicity information. To test this hypothesis, we propose to develop a high-throughput, microscale 3D cell-based screening tool that will enable rapid and highly quantitative information to be obtained on the effects of drugs, drug candidates, and chemical toxicants on human stem cells; specifically neural stem cells (NSCs) in comparison to adult primary and transformed neural cells, and human mesenchymal stem cells (MSCs). The specific aims of the proposed work are to: 1. Establish a robust 3D cell culture chip platform to grow and differentiate NSCs and MSCs; 2. Perform combinatorial in vitro extracellular matrix development; 3. Develop high-throughput on-chip assays of key signaling pathways in NSCs and MSCs that are critical in proliferation (self-renewal) and differentiation; 4. Validate the 3D cell culture microarray platform for cytotoxicity of stem cells in comparison to terminally differentiated primary cells and transformed cell lines using a series of reference compounds. This information is essential for the development of high throughput predictive toxicology screens for drug discovery and the prioritization of environmental chemicals based on their potential human toxicity.