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

用于神经毒性应用的水凝胶自组装人脑类器官

• 批准号: 10374175
• 负责人: Connie S Lebakken
• 金额: $ 78.35万
• 依托单位: STEM PHARM, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-15 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10374175
• 关键词: Address; Animal Model; Animals; Automation; Biological; Biological Assay; Biological Models; Biological Products; Bioreactors; Blood Vessels; Brain; Cell Communication; Cell Density; Cell Differentiation process; Cells; Cerebrovascular system; Characteristics; Chemicals; Clinical; Coculture Techniques; Communicable Diseases; Complex; Contracts; Cost Analysis; Data; Data Set; Development; Disease; Drug Modelings; Embryo; Endothelial Cells; Engineering; Extracellular Matrix; Failure; Gene Expression; Gene Expression Profiling; Genes; Genetic Transcription; Goals; Harvest; Human; Hydrogels; Image; Immune; Immunofluorescence Immunologic; Inflammation; International; Laboratories; Liquid substance; Manuals; Methods; Microelectrodes; Microglia; Modeling; Monitor; Neuraxis; Neurons; Organoids; Output; Pathology; Pharmaceutical Preparations; Phase; Poison; Protein Analysis; Publishing; Quality Control; Reproducibility; Risk; Safety; Signal Transduction; Small Business Innovation Research Grant; Source; Suspensions; System; Techniques; Testing; Thinness; Time; Toxic effect; Toxicology; Toxin; Transcript; Tumor-Derived; Universities; Validation; Variant; Wisconsin; Work; archive data; archived data; cell type; cost; cost effective; cytokine; data integrity; density; developmental neurotoxicity; drug candidate; drug discovery; experimental study; feeding; human embryonic stem cell; in vitro Model; induced pluripotent stem cell; multi-electrode arrays; multimodality; multiplex assay; nerve stem cell; neural model; neuroinflammation; neurotoxicity; novel; pre-clinical; relating to nervous system; response; screening; single-cell RNA sequencing; small molecule; stem; stem cells; transcriptome sequencing

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. .

项目概要/摘要 迫切需要将先进的中枢神经系统 (CNS) 模型进行筛选 药物发现和毒理学应用的应用。目前的体外模型没有 准确反映细胞类型的复杂性和重要的细胞与细胞相互作用以及动物 模型无法概括人类的状况。还非常需要更准确和 用于发育神经毒性筛选的可扩展模型，共有 86,405 种化合物 被列入《有毒物质控制法》清单17，但几乎没有生物学数据来了解其 风险。干细胞衍生的神经类器官的最新进展导致这些模型的使用 研究发育机制、传染病和毒理学应用（18-26 和 在 27-29 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。我们在 Stem 成功的第一阶段活动中所做的工作 使用 iPSC 衍生的前体细胞和分化细胞进行的药物研究表明，复杂的神经 含有多种神经亚型的类器官可以在 96 孔板中重复开发 板在工程水凝胶基底上。与悬浮系统中培养的类器官不同，这些 类器官可以在多孔板中形成、培养和分析。 RNA测序分析 表现出较高的组内相关性和较低的变异系数。重要的是，我们 证明小胶质细胞融入类器官并证明它们的激活 神经炎症模型及其响应化合物的激活或消耗 治疗。为了将这种新颖的模型推向市场，我们提出以下具体目标 对于第二阶段提案：1）优化源自细胞的时间和接种密度 单一 iPSC 供体来源，优化小胶质细胞的掺入以维持稳健的激活 签名，但可以降低成本并保持数据完整性。比较成本较低的转录 读出 TempO-Seq S1500 人类面板，以获取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 qPCR 面板。 2）验证类器官 在我们的薄水凝胶涂层上生成，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) 验证多重测定以评估多种 单孔反应，包括 MEA 分析、细胞因子和 LDH 释放以及收获 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。 。