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

项目摘要/摘要 迫切需要将高级中枢神经系统(CNS)模型转移到筛查中 药物发现和毒理学应用的申请。目前的体外模型不能 准确反映细胞类型的复杂性和重要的细胞-细胞相互作用和动物 模型未能概括人类的状况。还非常需要更准确和更准确的 用于发育神经毒性筛选的可扩展模型有86,405种化合物 列在《有毒物质控制法》清单17上，几乎没有生物学数据来了解它们 风险。干细胞来源的神经有机体的最新进展导致了这些模型的使用 研究发育机制、传染病和毒理学应用(18-26和 在27-29年中进行了审查)，但其成本、复杂性和工作流要求使其具有挑战性 过渡到筛选应用程序。在我们成功的STEM第一阶段活动中所做的工作 药物与IPSC来源的前体细胞和分化细胞已经证明了复杂的神经 含有多种神经亚型的有机化合物可以在96口井中重复开发 工程水凝胶衬底上的板材。与悬浮系统中培养的有机物不同，这些 类有机物可以在多孔板中形成、培养和检测。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 证明了小胶质细胞被掺入到有机体中，并证明了它们的激活为 神经炎症模型及其对化合物反应的激活或耗竭 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 对于第二阶段提案：1)优化时机和播种密度，使用来自 单一的IPSC供体来源，优化小胶质细胞的掺入以保持强劲的激活 签名，但降低了成本并维护了数据完整性。为了比较成本更低的转录 读出Tempo-Seq S1500人体面板，以获得我们在第一阶段活动中获得的RNA-Seq数据 并验证用于产品放行质量控制的定量聚合酶链板。2)验证有机化合物 在我们薄薄的水凝胶涂层上生成，以实现更好的成像选项，微电极阵列 分析和液体处理自动化以及3)验证多路分析以评估多个 单井反应包括MEA分析、细胞因子和乳酸脱氢酶释放及收获 转录本或蛋白质分析。这项工作将导致第一个商业化的神经有机化合物 含有血管细胞和小胶质细胞，在毒理学和药物方面都有广泛的适用性 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. .