Hydrogel-enabled self-assembled human brain organoids for neurotoxicity applications

项目概要/摘要 迫切需要将先进的中枢神经系统（CNS）模型用于筛选 用于药物发现和毒理学应用。目前的体外模型不 准确反映细胞类型的复杂性和重要的细胞间相互作用， 模型无法概括人类的状况。此外，还需要更准确和更可靠的信息。 可扩展模型用于发育神经毒性筛选，因为有86，405种化合物 在有毒物质控制法清单17上列出的生物数据很少，以了解其 风险干细胞衍生的神经类器官的最新进展已经导致使用这些模型来 研究发育机制，传染病和毒理学应用（18-26和 在27 -29中进行了审查），但它们的成本、复杂性和工作流程要求使它们具有挑战性， 过渡到筛选应用程序。在我们成功的第一阶段活动中所做的工作 iPSC衍生的前体细胞和分化细胞的Pharm已经证明， 含有各种神经亚型的类器官可以在96孔板中可重复地开发， 在工程化水凝胶基质上涂布。与悬浮系统中培养的类器官不同， 可在多孔板中形成、培养和测定类器官。RNA-seq分析 表现出高的组内相关性和低的变异系数。重要的是我们 证明了小胶质细胞掺入类器官中，并证明了它们的激活， 神经炎症以及它们响应于化合物的活化或耗竭的模型 治疗为了将这一新模式推向市场，我们提出了以下具体目标 对于第二阶段的建议：1）为了优化时间和接种密度与细胞来源于一个 单一iPSC供体来源，优化小胶质细胞的掺入以维持稳健的活化 签名，但降低了成本，并保持数据的完整性。为了比较一个成本较低的转录 读出，TempO-Seq S1500人类面板，我们在I期活动中获得的RNA-seq数据 并验证用于产品放行质量控制的qPCR面板。2)验证类器官 在我们的薄水凝胶涂层上产生，以实现更好的成像选择，微电极阵列 分析和液体处理自动化以及3）用于评估多种 单个威尔斯孔中的响应，包括MEA分析、细胞因子和LDH释放和收获， 转录物或蛋白质分析。这项工作将导致第一个商业化的神经类器官 含有血管细胞和小胶质细胞，在毒理学和药物方面具有广泛的适用性 发现市场。 .

Project Summary/Abstract There is a critical need to move advanced Central Nervous System (CNS) models into screening applications for drug discovery and toxicology applications. Current in vitro models do not accurately reflect the complexity of cell types and important cell-cell interactions and animal models fail to recapitulate the human condition. There is also a great need for more accurate and scalable models for developmental neurotoxicity screenings as there are 86,405 compounds listed on the Toxic Substance Control Act inventory17 with little biological data to understand their risks. Recent advances in stem-cell derived neural organoids have led to use of these models to study developmental mechanisms, infectious diseases, and toxicology applications (18-26 and reviewed in27-29), but their cost, complexity, and workflow requirements make them challenging to transition to screening applications. Work performed in our successful Phase I activities at Stem Pharm with iPSC-derived precursor and differentiated cells has demonstrated that complex neural organoids containing a variety of neural subtypes can be developed reproducibly in a 96-well plate on engineered hydrogel substrates. Unlike organoids cultured in suspension systems, these organoids can be formed, cultured, and assayed in multi-well plates. RNA-seq analysis demonstrated high intraclass correlation and low coefficients of variation. Importantly, we demonstrated incorporation of microglia into the organoids and demonstrated their activation as a model of neural inflammation as well as their activation or depletion in response to compound treatment. In order to bring this novel model to the market we propose the following specific aims for the Phase II proposal: 1) To optimize timing and seeding densities with cells derived from a single iPSC-donor source, optimize incorporation of microglia to maintain robust activation signatures but decrease cost and maintain data integrity. To compare a less-costly transcriptional read-out, the TempO-Seq S1500 human panel, to our RNA-seq data obtained in Phase I activities and to validate a qPCR panel for product release quality control. 2) To validate organoids generated on our thin hydrogel coatings to enable better imaging options, microelectrode array analysis and liquid handling automation and 3) Validate multiplexed assays to assess multiple responses in single wells including MEA analysis, cytokine and LDH release and harvest for transcript or protein analysis. This work will lead to the first commercially available neural organoid containing vascular cells and microglia with broad applicability in both toxicology and drug discovery markets. .