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测试通常不包括直接 人类发育神经毒性评估(DNT)。当前的DNT测试标准要求使用 吞吐能力有限且与人类中枢神经系统(CNS)有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 因人类DNT而取消的航班。当前监管DNT测试范例的这些限制具有 促使人们对使用人类多能干细胞进行定量高通量筛查(QHTS)产生了越来越大的兴趣 基于细胞(HPSC)的方法。在这里，我们建议使用玫瑰阵列(RA)技术来开发一种hPSC- 派生的qHTS平台(即qHTS-RAS)，用于有效和高效的DNT测试。玫瑰花结阵列技术 标准化人类神经花环组织的体外衍生，这些组织模仿 人类神经管，所有中枢神经组织的原基。尽管神经花环并不是对 体内的初级神经分化，它们表现出相同的细胞表型、组织细胞结构，并且是由 体内神经管形成过程的内源性形态发生信号通路。玫瑰花结阵列 是第一项能够在空间和时间上控制神经花环出现的体外技术 微阵列格式。这使得通过显微镜图像分析进行快速评估成为可能。这种高收益， 标准化的体外神经管类似物的产生使必要的重复性成为可能 将基于hPSC的中枢神经系统形态读数纳入定量高通量毒理学筛选。 因此，我们假设qHTS-RA平台可以增加吞吐量和人类DNT风险的准确性 评估，允许合并和扩大商业DNT筛查。第1阶段AIMS将验证 QHTS-RA平台的广泛适用性，证实与人诱导的多能干细胞相容 (HiPSC)线和建立用于自动图像采集和批处理分析的方法。第二阶段的目标是 使用100个化合物DNT参考文库屏幕验证检测适合性，在屏幕上自动生成AI图像 分析被用来进一步提高精确度和最大化吞吐量。如果成功，则生成的qHTS-RA 平台可以取代目前的多种DNT检测，并增加对相关毒理学读数的信心 人类生理学独有的结果。因此，这里提出的工作可能会对 DNT研究，防止DNT暴露的监管努力，以及基于hPSC的有机化合物未来的可译性 广泛研究人类发展的模型。

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.