Development and Evaluation of Rosette Array Technology for HumanNeurodevelopmental Toxicity Screening

项目摘要 新药和化学品的临床前和现场前毒理学试验通常不包括直接 人类发育神经毒性（DNT）。DNT测试的现行标准要求使用 具有有限通量和与人类中枢神经系统（CNS）显著差异的动物模型 发展因此，许多化学产品需要批准后（EPA/FDA）限制或 由于人类DNT而取消。当前DNT测试规范的这种局限性 促使人们对使用人类多能干细胞进行定量高通量筛选（qHTS）的兴趣增加 细胞（hPSC）的方法。在这里，我们提出使用玫瑰花阵列（RA）技术来开发hPSC- 导出的qHTS平台（即，qHTS-RAs）进行有效和高效的DNT测试。玫瑰阵列技术 在体外衍生的人神经组织是模仿的横切片， 人类神经管，所有中枢神经系统组织的原基。虽然神经元的再生并不是对神经元的精确再现， 在体内初级神经形成中，它们表现出相同的细胞表型、组织细胞结构，并且是使用 在体内神经管形成过程中内源性的形态发生信号传导途径。玫瑰花阵列 是第一个在体外技术，使空间和时间控制神经玫瑰花出现在一个 微阵列格式。这使得能够通过显微镜图像分析进行快速评估。这种高产量， 体外神经管类似物的标准化生成使得能够实现可行地 将基于hPSC CNS形态发生读数并入定量高通量毒理学筛选。 因此，我们假设qHTS-RA平台可以增加人类DNT风险的通量和准确性， 评估，从而可以巩固和扩大商业DNT筛查。第一阶段的目标是验证 qHTS-RA平台的广泛适用性，证实了与人诱导多能干细胞的相容性 （hiPSC）线和建立用于自动化图像采集和批量分析的方法。第二阶段的目标是 用100个化合物DNT参考文库筛选验证测定适合性，其中自动AI图像 利用分析来进一步提高准确性并使吞吐量最大化。如果成功，则产生的qHTS-RA 该平台可以取代目前的多种DNT检测方法，并增加相关毒理学读数的置信度。 人类生理学的独特结果。因此，这里提出的工作可能对以下方面产生变革性影响： DNT研究，防止DNT暴露的监管努力以及基于hPSC的类器官的未来可翻译性 广泛研究人类发展的模式。

Project Summary Pre-clinical and pre-field toxicology testing of new drugs and chemicals does not routinely include direct assessment of human developmental neurotoxicity (DNT). Current standards for DNT testing require use of animal models with limited throughput and significant differences from human central nervous system (CNS) development. As such, many chemical products have required post-approval (EPA/FDA) restrictions or cancellations due to human DNT. Such limitations of the current regulatory DNT testing paradigm have prompted increased interest in quantitative high-throughput screening (qHTS) using human pluripotent stem cell (hPSC)-based approaches. Here, we propose to use Rosette Array (RA) technology to develop an hPSC- derived qHTS platform (i.e., qHTS-RAs) for effective and efficient DNT testing. Rosette Array technology standardizes in vitro derivation of human neural rosettes tissues that are mimetic of transverse slices of the human neural tube, the anlage of all CNS tissue. Although neural rosettes are not an exact recapitulation of in vivo primary neurulation, they exhibit the same cell phenotypes, tissue cytoarchitecture, and are derived using morphogenetic signaling pathways endogenous to the in vivo neural tube formation process. Rosette arrays are the first in vitro technology to enable spatial and temporal control of neural rosette emergence in a microarray format. This enables rapid assessment via microscope image analysis. This high-yield, standardized generation of in vitro neural tube analogs enables the repeatability necessary to feasibly incorporate hPSC-based CNS morphogenic readouts into quantitative high-throughput toxicology screening. Thus, we hypothesize that the qHTS-RA platform could increase throughput and accuracy of human DNT risk assessment, allowing consolidation and scale-up of commercial DNT screening. Phase 1 Aims will validate the broad applicability of the qHTS-RA platform, confirming compatibility with human induced pluripotent stem cell (hiPSC) lines and establishing methods for automated image acquisition and batch analysis. Phase 2 Aims to validate assay fitness with a 100 compound DNT reference library screen, in which automated AI image analysis is utilized to further increase accuracy and maximize throughput. If successful, the resulting qHTS-RA platform could replace multiple current DNT assays and increase confidence in toxicological readouts relevant to outcomes unique to human physiology. Thus, the work proposed here could have a transformative effect on DNT research, regulatory efforts to prevent DNT exposure, and future translatability of hPSC-based organoid models for broad study of human development.