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。当前的体外模型没有 准确反映细胞类型的复杂性以及重要的细胞 - 细胞相互作用和动物 模型无法概括人类状况。还需要更准确和 有86,405种化合物，用于发育神经毒性筛选的可扩展模型 在《有毒物质控制法》上列出的库存17，几乎没有生物学数据，以了解其 风险。干细胞衍生的神经器官的最新进展已导致这些模型使用 研究发展机制，传染病和毒理学应用（18-26和 在27-29中进行了审查），但是它们的成本，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I阶段活动中进行的工作 具有IPSC衍生前体和分化细胞的药物表明复杂的神经 可以在96孔中重复开发包含多种神经亚型的类器官 板上工程水凝胶底物。与在悬浮系统中培养的类器官不同，这些 可以在多孔板中形成，培养和测定器官。 RNA-seq分析 表现出高的类内相关性和较低的变异系数。重要的是，我们 表现出将小胶质细胞掺入器官中，并将其激活作为 神经炎症的模型及其激活或耗竭响应化合物 治疗。为了将这个新颖的模型带入市场，我们提出了以下特定目标 对于II期建议：1）用源自A的细胞优化定时和播种密度 单个IPSC-DONOR源，优化小胶质细胞的掺入以维持可靠的激活 签名但会降低成本并保持数据完整性。比较较低的转录 读出的TEMPO-SEQ S1500人体面板，到我们在I期活动中获得的RNA-seq数据 并验证QPCR面板以进行产品释放质量控制。 2）验证器官 在我们的薄水凝胶涂层上产生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）验证多路复用测定以评估多个 单孔中的反应，包括MEA分析，细胞因子和LDH释放和收获 转录本或蛋白质分析。这项工作将导致第一个商业上可用的神经器官 含有在毒理学和药物中具有广泛适用性的血管细胞和小胶质细胞 发现市场。 。