An Engineered Hydrogel Platform to Improve Neural Organoid Reproducibility for a Multi-Organoid Disease Model of 22q11.2 Deletion Syndrome

一种工程水凝胶平台，可提高 22q11.2 缺失综合征多器官疾病模型的神经类器官再现性

• 批准号: 10679749
• 负责人: Michelle S Huang
• 金额: $ 4.56万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-16 至 2025-11-15
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679749
• 关键词: 22q11.2; 3-Dimensional; Address; Affect; Age; Architecture; Behavior; Biochemical; Biochemistry; Biocompatible Materials; Biological Models; Biomechanics; Biomedical Engineering; Biophysics; Brain; Brain region; Cells; Chemicals; Child; Collaborations; Complex; Cues; DNA; Defect; Development; DiGeorge Syndrome; Disease; Disease model; Dorsal; Drug Screening; Educational workshop; Elasticity; Encapsulated; Engineering; Environment; Extracellular Matrix; Gel; Gene Deletion; Genetic Screening; Growth; Heterogeneity; Human; Hyaluronic Acid; Hydrogels; Impairment; In Vitro; Interneurons; Label; Laminin; Ligands; Liquid substance; Manuals; Mechanics; Mediating; Mentorship; Modeling; Monitor; Mus; Neurodevelopmental Disorder; Organoids; Outcome; Patients; Pattern; Phenotype; PicoGreen; Positioning Attribute; Process; Productivity; Proliferating; Property; Prosencephalon; Protocols documentation; Recombinants; Relaxation; Reproducibility; Research; Research Ethics; Research Proposals; Role; Scientist; Series; Signal Transduction; Stress; Structure; Supine Position; Suspensions; System; Technical Expertise; Testing; Time; Tissues; Training; Transfection; United States; Western Blotting; Work; Writing; biophysical properties; brain tissue; covalent bond; crosslink; design; empowerment; experience; experimental study; human model; human stem cells; immunocytochemistry; improved; in vitro Model; induced pluripotent stem cell; innovation; insight; interdisciplinary approach; live cell microscopy; matrigel; migration; mouse model; nervous system disorder; neural; neurodevelopment; novel; overexpression; permissiveness; prevent; progenitor; protein crosslink; response; sarcoma; scale up; self organization; single-cell RNA sequencing; skills; stem cells; symposium; viscoelasticity

Project Summary l The complexity of the human brain and lack of adequate models severely hinders our ability to understand mechanisms guiding neurodevelopment and neurodevelopmental disorders (NDDs), necessitating an innovative bioengineered approach for improving in vitro organotypic models of the human brain. Recent advances in stem cell-based neural organoids enable the formation of assembloids, which are fusions of organoids representing different brain regions. However, current approaches remain limited in the ability to properly recapitulate native brain cytoarchitecture and maturation within these organoids and also result in large heterogeneity due to the lack of a well-defined matrix. The proposed research seeks to resolve these critical issues by creating a reliable and reproducible in vitro environment for neural organoid culture to study aspects of neurodevelopment and NDDs that have been difficult to achieve with current platforms. To do this, I will 1) assess the effect of matrix biochemical cues for improving neural organoid architecture and maturation; 2) define the role of matrix stress relaxation and confinement on organoid growth; and 3) leverage the engineered hydrogel platform to study impaired interneuron migration in a disease model of 22q11.2 deletion syndrome (22q11DS). I hypothesize that the experiments described in my proposal will show that matrix-derived biochemical and biophysical signaling will allow for more robust neural organoid culture that better recapitulates the architecture and maturation of the human brain compared to conventional neural organoid models. I also hypothesize that fusion of 22q11DS neural organoids within engineered hydrogels will robustly demonstrate dysregulated interneuron migration mediated by the deletion of DCGR8. Of note, interneuron migration is a phenomenon that does not occur in murine systems and therefore cannot be studied using conventional murine models. To test this hypothesis, I will utilize a minimal matrix (HELP) to culture brain region-specific neural organoids derived from induced pluripotent stem cells (iPSCs) from healthy and 22q11DS patients. I will perform robust characterization of neural organoid architecture, maturation, and growth rate in response to tuning matrix biochemical and biophysical properties. I will also assess the ability of interneurons from 22q11DS patients to migrate into the dorsal forebrain by establishing a dorsal–ventral forebrain assembloid disease model. Together, these results will be critical for engineering a platform that is both permissive and instructive for robust and efficient neural organoid culture. In addition to expanding my scientific technical skills, my training plan includes development of mentorship, scientific writing, and presentation skills; training in research ethics; and enhancement of collaboration skills through a series of on-campus courses, workshops, and seminars as well as off-campus conferences. Altogether, this research proposal will empower me to become an independent, productive research scientist as I leverage the self- organizing capacity of stem cells and the tunable capacity of engineered materials to develop more human- relevant neural disease models.

项目概要 l 人脑的复杂性和缺乏足够的模型严重阻碍了我们的理解能力 指导神经发育和神经发育障碍（NDD）的机制，需要创新 改善人脑体外器官模型的生物工程方法。干细胞的最新进展。 基于细胞的神经类器官能够形成组合体，这些类器官是代表 然而，目前的方法在正确再现本机的能力方面仍然有限。 这些类器官内的脑细胞结构和成熟，也导致了巨大的异质性，因为 缺乏明确定义的矩阵。拟议的研究旨在通过创建可靠的模型来解决这些关键问题。 以及用于神经类器官培养的可重复的体外环境，以研究神经发育和 当前平台难以实现的 NDD 为此，我将 1) 评估矩阵的效果。 改善神经类器官结构和成熟的生化线索2）定义基质应力的作用； 对类器官生长的松弛和限制；3）利用工程水凝胶平台进行研究 22q11.2 缺失综合征 (22q11DS) 疾病模型中神经元迁移受损。 我的提案中描述的实验将表明基质衍生的生化和生物物理信号传导 将允许更强大的神经类器官培养，更好地概括神经类器官的结构和成熟 人类大脑与传统神经类器官模型的比较我也遵循了 22q11DS 神经的融合。 工程水凝胶内的类器官将有力地证明中间神经元迁移介导的失调 值得注意的是，中间神经元迁移是一种在小鼠系统中不会发生的现象。 因此不能使用传统的小鼠模型进行研究 为了检验这个假设，我将使用最小的模型。 基质（HELP）培养源自诱导多能干细胞的大脑区域特异性神经类器官 （iPSC）来自健康和 22q11DS 患者，我将对神经类器官结构进行稳健的表征， 我还将根据基质的生化和生物物理特性来响应成熟度和生长率。 通过建立一个模型来评估 22q11DS 患者的中间神经元迁移到背侧前脑的能力 总之，这些结果对于设计一个前脑背腹组装疾病模型至关重要。 该平台对于稳健且高效的神经类器官培养既具有包容性又具有指导性。 扩展我的科学技术技能，我的培训计划包括指导、科学写作、 和演讲技巧；研究道德培训；以及通过一系列的合作技能提高 校内课程、讲习班、研讨会以及校外会议。 当我利用自我能力时，提案将使我成为一名独立、富有成效的研究科学家。 干细胞的组织能力和工程材料的可调节能力，以开发更多的人类 相关神经疾病模型。