Development and Evaluation of Rosette Array Technology for HumanNeurodevelopmental Toxicity Screening

项目概要 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（DNT）的评估。 DNT 测试的现行标准要求使用 动物模型吞吐量有限，与人类中枢神经系统 (CNS) 存在显着差异 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 导致取消。当前监管 DNT 测试范式的局限性 促使人们对使用人类多能干进行定量高通量筛选（qHTS）的兴趣增加 基于细胞（hPSC）的方法。在这里，我们建议使用 Rosette Array (RA) 技术来开发 hPSC- 衍生的 qHTS 平台（即 qHTS-RA）用于有效且高效的 DNT 测试。玫瑰花阵列技术 标准化人类神经玫瑰花结组织的体外衍生，这些组织模仿了神经细胞的横向切片 人类神经管，所有中枢神经系统组织的原基。尽管神经玫瑰花结并不是对神经网络的精确再现 体内初级神经形成，它们表现出相同的细胞表型、组织细胞结构，并使用 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 是第一个能够在体外控制神经玫瑰花结出现的空间和时间的技术 微阵列格式。这样可以通过显微镜图像分析进行快速评估。这种高产， 体外神经管类似物的标准化生成能够实现可行的重复性 将基于 hPSC 的 CNS 形态发生读数纳入定量高通量毒理学筛选。 因此，我们假设 qHTS-RA 平台可以提高人类 DNT 风险的吞吐量和准确性 评估，允许整合和扩大商业 DNT 筛查。第一阶段的目标将验证 qHTS-RA 平台的广泛适用性，证实与人类诱导多能干细胞的兼容性 （hiPSC）生产线并建立自动图像采集和批量分析的方法。第二阶段的目标是 使用 100 种化合物 DNT 参考库筛选验证检测适用性，其中自动 AI 图像 利用分析来进一步提高准确性并最大化吞吐量。如果成功，生成的 qHTS-RA 平台可以取代当前的多种 DNT 检测并提高相关毒理学读数的可信度 人类生理学特有的结果。因此，这里提出的工作可能会对 DNT 研究、防止 DNT 暴露的监管工作以及基于 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.