Leveraging 3D bioprinted organoid constructs to pattern and model human brain development

项目摘要 除了严重的人类痛苦之外，神经疾病还给美国经济造成了超过1.5万亿美元的损失 每年--占国内生产总值的8.8%。这种身体、情感和经济上的负担突显了 开发创新平台来研究大脑发育、生理学和 关联的命令。人类诱导多能干细胞(HiPSC)衍生的三维皮质器官的出现 文化已经显示出作为模型系统的巨大前景，但仍然存在许多技术限制， 阻碍了他们概括人类大脑发育的关键非细胞自主方面的能力。 这些成分包括细胞外基质(ECM)、颅骨和轴向形态的外在影响。 这些梯度共同帮助塑造和多样化发育中的大脑区域。 目前，标准的类有机物方案包括将类有机物嵌入Matrigel液滴或悬浮浴中。 培养，这阻止了用户对细胞外环境的可重复控制。这限制了创作的能力 形态梯度，或询问分子和物理性质 脑实质外的细胞外基质引导神经发育。为了应对这些挑战，我们建议 开发3D生物打印皮质有机结构，概括自然环境的关键微环境线索 脑组织。该项目建立在我们最新的技术成果基础上，使嵌入式生物打印成为可能 高空间分辨率(20微米)的定制组织结构专为长期有机培养而设计。 我们的目标是将皮质脑器官生物打印到3D支架中，这种支架具有可定制的分子和 物理力学成分。脑有机体和生物打印技术的协同作用提供了近 操纵复杂的多细胞人体模型系统的外部发育线索的无限潜力。 我们将追求两个综合的具体目标，利用多元PI领导机制将互补结合起来 斯隆(神经发育)和瑟波山(组织工程)的研究技能和专业知识 实验室。在目标1中，我们将解耦分子组成和物理刚性参数 ECM，并询问这些因素如何影响神经发育、分化、成熟和结构。在……里面 目标2，我们将使用三种不同的方法在生物打印中产生稳定的形态梯度 结构，我们将用它来诱导背侧(掌侧)和腹侧的器质内极化 (亚于苍白的)地区认同。总之，这些方法为3D干细胞建模提供了一个新的平台 可以广泛应用于许多系统，并迎来新一代神经发育建模。

Project Summary In addition to significant human distress, neurological disorders cost the U.S. economy more than $1.5 trillion per year—8.8 percent of the gross domestic product. This physical, emotional and financial burden underscores the potential benefit from developing innovative platforms to study brain development, physiology, and associated diorders. The advent of human induced pluripotent stem cell (hiPSC)-derived 3D cortical organoid cultures has shown great promise as a model system, yet there remain a number of technical limitations that have stymied their ability to recapitulate critical non-cell autonomous aspects of human brain development. These components include extrinsic influences of the extracellular matrix (ECM), skull, and axial morphogen gradients that together help shape and diversify regions of the developing brain. Currently, standard organoid protocols involve embedding organoids in Matrigel droplets or in suspended bath culture, which prevents reproducible user-control of the extracellular environment. This limits the ability to create morphogen gradients that topographically polarize stem cells, or to ask how molecular and physical properties of the ECM outside the brain parenchyma guide neurodevelopment. To address these challenges, we propose developing 3D bioprinted cortical organoid constructs that recapitulate key microenvironmental cues of native brain tissue. This project builds upon our recent technological achievements, enabling embedded bioprinting of custom tissue constructs at high spatial resolution (20 µm) specifically designed for long-term organoid culture. Our goal is to bioprint cortical brain organoids into 3D scaffolds with customizable molecular and physiomechanical compositions. The synergy of brain organoid and bioprinting technologies provides a nearly unlimited potential to manipulate extrinsic developmental cues of a complex multicellular human model system. We will pursue two integrated Specific Aims using the multi-PI leadership mechanism to combine complementary research skills and expertise in the Sloan (neurodevelopment) and Serpooshan (tissue engineering) laboratories. In Aim 1, we will decouple the molecular composition and physical stiffness paramaters of the ECM, and ask how these factors influence neural development, differentiation, maturation and architecture. In Aim 2, we will use three separate approaches to generate a stable morphogen gradient within bioprinted constructs, which we will use to induce intra-organoid polarization of both dorsal (pallial) and ventral (subpallial) regional identities. Together, these approaches offer a novel platform for 3D stem cell modeling that could be applied broadly to numerous systems and usher a new generation of neurodevelopmental modeling.