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

项目摘要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，需要一种创新的 生物工程方法用于改进人脑的体外器官型模型。干细胞研究进展 基于细胞的神经类器官能够形成类神经元，类神经元是代表神经元的类器官的融合体。 不同的大脑区域。然而，目前的方法在正确概括天然的生物学特性方面仍然有限。 这些类器官内的脑细胞结构和成熟，也会导致大的异质性， 缺乏明确的矩阵。拟议的研究旨在通过创建一个可靠的 以及用于神经类器官培养的可重复的体外环境，以研究神经发育的各个方面， 目前的平台难以实现的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）定义基质应激的作用 松弛和限制类器官生长;以及3）利用工程化水凝胶平台来研究 22q11.2缺失综合征（22 q11 DS）疾病模型中受损的中间神经元迁移。我假设 在我的建议中描述的实验将表明，基质衍生的生物化学和生物物理信号 将允许更稳健的神经类器官培养，其更好地再现神经细胞的结构和成熟。 与传统的神经类器官模型相比，人脑。我还假设22 q11 DS神经融合 工程水凝胶内的类器官将有力地证明神经元间迁移介导的失调 通过删除DCGR 8。值得注意的是，中间神经元迁移是一种在小鼠系统中不发生的现象 因此不能使用常规的鼠模型进行研究。为了验证这个假设，我将使用一个最小的 基质（HELP）来培养源自诱导多能干细胞的脑区域特异性神经类器官 （iPSCs）来自健康和22 q11 DS患者。我将对神经类器官结构进行强有力的表征， 成熟和响应于调节基质生物化学和生物物理性质的生长速率。我也会 评估22 q11 DS患者的中间神经元迁移到背侧前脑的能力， 背腹前脑类神经病模型。总之，这些结果将是至关重要的工程 这是一个既允许又有益于强大而有效的神经类器官培养的平台。除了 扩展我的科学技术技能，我的培训计划包括发展导师制，科学写作， 和演讲技巧;研究道德培训;通过一系列 校内课程、工作坊和研讨会以及校外会议。总之，这项研究 建议将使我成为一个独立的，富有成效的研究科学家，因为我利用自我- 干细胞的组织能力和工程材料的可调能力，以开发更多的人类- 相关的神经疾病模型。

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.