Synthetic hydrogels for biomanufacturing of iPSC-derived neural cells for precision medicine

用于精准医学 iPSC 衍生神经细胞生物制造的合成水凝胶

• 批准号: 10237392
• 负责人: Connie S Lebakken
• 金额: $ 67.86万
• 依托单位: STEM PHARM, INC.
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10237392
• 关键词: Animals; Biochemical; Biological Assay; Biomanufacturing; Bioreactors; Cell Adhesion; Cell Differentiation process; Cell model; Cells; Chemicals; Coupled; Cultured Cells; Data; Development; Disease; Disease model; Elasticity; Environment; Environmental Exposure; Environmental Risk Factor; Etiology; Feasibility Studies; Generations; Genetic Transcription; Goals; Growth Factor; Human; Hydration status; Hydrogels; Luciferases; Major Depressive Disorder; Measurement; Mechanics; Methodology; Methods; Microelectrodes; Mitotic; Modeling; Morphology; Neurodevelopmental Disorder; Neurons; Output; Patients; Pharmaceutical Preparations; Phase; Phenotype; Polystyrenes; Precision therapeutics; Protocols documentation; Quality Control; Reporter; Reproducibility; Sampling; Surface; System; Techniques; Technology; Therapeutic; Thick; Thinness; Time; Tissues; Toxic Environmental Substances; Toxin; Validation; Work; base; cell type; cellular imaging; commercialization; comparative; cost; design; experimental study; improved; induced pluripotent stem cell; innovation; mechanical properties; nano; nerve stem cell; novel; phase 1 study; physical property; polymerization; precision medicine; public health relevance; relating to nervous system; response; screening; self assembly; stem; time use; tissue culture; tool; treatment strategy

Human neural cells manufactured using patient-derived induced pluripotent stem cells (iPSCs) hold great promise for modeling neurodevelopmental disorders, discovering new precision therapies, and screening for potential risks from environmental toxins 1-4. There have been significant advances in the last decade in protocols and commercial media systems developed for differentiation into specific neural cell types 5-8. However, there remain significant technical challenges to overcome in their generation, manufacturing and assay workflows. iPSCs are typically differentiated on animal-derived substrates that introduce intrinsic variability and lack control over mechanical stiffness and biochemical composition. This often results in low yields and high variability, which may be more pronounced when generating cellular models of diseases. There is a critical need to develop commercial tools that promote differentiation of iPSCs into mature neural cells in a controlled, efficient, and reproducible fashion and that eliminate animal derived products. The resulting cells, associated cell-based assays and cellular therapeutics will have a transformative impact on neural disease modeling, drug and therapeutic discovery and toxin screening. Our Phase I study identified chemically defined and robust synthetic hydrogels for efficient differentiation of iPSC-derived neural progenitor cells (NPCs) into cortical neurons and subsequent maturation to post-mitotic, functionally mature neurons. The highly innovative aspects of this work are that the substrates are employed as thin hydrogel coatings using our proprietary surface-localized polymerization methods which provides several technical and commercialization advantages. In order to bring these novel substrates to market we propose the following specific aims for our Phase II proposal: Specific Aim 1 will further validate the work that demonstrated our optimized synthetic thin hydrogel coatings support neural differentiation and maturation. Including further functional characterization of cells cultured on the substrates by employing microelectrode array analysis and differential transcriptional analysis to compare cells cultured on the substrate. We will characterize of the physical and mechanical properties of the optimized thin hydrogels and develop methods for coating plates using automated systems. Specific Aim 2 will apply the substrates in a Proof-of-Concept demonstration utilizing the substrates to assess cortical neurons from Major Depressive Disorder patient-derived samples compared with controls. Specific Aim 3 will expand the technology platform by optimizing coating techniques on microcarriers suitable for bioreactor scaling, which is a critical step to demonstrate these substrates are applicable to biomanufacturing applications. This work is significant, as there is a critical need for better tools to optimize yields and reduce variability in the differentiation of iPSCs to defined neural subtypes, support their long-term culture, reduce the time needed to reach functional maturity and eliminate animal-derived products in the workflow.

使用患者来源的诱导多能干细胞 (iPSC) 制造的人类神经细胞具有良好的前景 有望模拟神经发育障碍、发现新的精准疗法以及筛查 环境毒素的潜在风险1-4。过去十年协议取得了重大进展 以及为分化成特定神经细胞类型 5-8 而开发的商业培养基系统。然而，有 在其生成、制造和分析工作流程中仍然存在需要克服的重大技术挑战。 iPSC 通常在动物源性基质上分化，从而引入内在变异性且缺乏控制 超过机械刚度和生化成分。这通常会导致低产量和高变异性， 当生成疾病的细胞模型时可能更加明显。迫切需要开发 促进 iPSC 以受控、高效和稳定的方式分化为成熟神经细胞的商业工具 可复制的时尚并消除动物衍生产品。产生的细胞，相关的基于细胞的 检测和细胞疗法将对神经疾病模型、药物和治疗产生变革性影响 治疗发现和毒素筛选。 我们的第一阶段研究确定了化学成分明确且坚固的合成水凝胶，可实现有效分化 将 iPSC 衍生的神经祖细胞 (NPC) 转化为皮质神经元，并随后成熟至有丝分裂后， 功能成熟的神经元。这项工作的高度创新之处在于基材被用作 使用我们专有的表面局部聚合方法形成薄水凝胶涂层，可提供多种 技术和商业化优势。为了将这些新颖的基材推向市场，我们建议 我们的第二阶段提案的具体目标如下： 具体目标 1 将进一步验证所展示的工作 我们优化的合成薄水凝胶涂层支持神经分化和成熟。进一步包括 通过微电极阵列分析对基质上培养的细胞进行功能表征 差异转录分析以比较在基质上培养的细胞。我们将表征物理 和优化的薄水凝胶的机械性能，并开发使用涂层板的方法 自动化系统。具体目标 2 将利用以下技术在概念验证演示中应用基板 与来自重度抑郁症患者的样本相比，评估皮层神经元的底物 控制。 Specific Aim 3 将通过优化微载体上的涂层技术来扩展技术平台 适用于生物反应器规模化，这是证明这些底物适用于的关键步骤 生物制造应用。这项工作意义重大，因为迫切需要更好的工具来优化产量 并减少 iPSC 分化为定义的神经亚型的变异性，支持其长期培养， 减少达到功能成熟所需的时间并消除工作流程中的动物衍生产品。