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，神经系统疾病给美国经济造成的损失超过1.5万亿美元 占国内生产总值的8.8%。这种身体、情感和经济负担凸显了 开发创新平台研究大脑发育、生理学和 相关双目人类诱导多能干细胞（hiPSC）衍生的3D皮质类器官的出现 文化作为一种模式系统已经显示出很大的希望，但仍然存在一些技术限制， 已经阻碍了它们重现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）、颅骨和轴向形态原的外在影响 这些梯度共同帮助塑造和多样化发育中的大脑区域。 目前，标准的类器官方案涉及将类器官包埋在基质胶液滴中或悬浮浴中， 培养，这阻止了细胞外环境的可再现的用户控制。这限制了创造的能力 形态发生梯度，拓扑学地覆盖干细胞，或者问分子和物理性质如何 脑实质外的ECM引导神经发育。为了应对这些挑战，我们建议 开发3D生物打印的皮质类器官构建体，重现原生微环境的关键线索 脑组织该项目建立在我们最近的技术成就之上，使嵌入式生物打印成为可能。 高空间分辨率（20 µm）的定制组织构建体，专为长期类器官培养而设计。 我们的目标是将大脑皮质类器官生物打印成3D支架， 物理力学成分脑类器官和生物打印技术的协同作用提供了一个近 操纵复杂多细胞人类模型系统的外在发育线索的无限潜力。 我们将追求两个综合的具体目标，使用多PI领导机制，以联合收割机互补 斯隆（神经发育）和Serpooshan（组织工程）的研究技能和专业知识 laboratories.在目标1中，我们将解耦的分子组成和物理刚度参数的 ECM，并询问这些因素如何影响神经发育，分化，成熟和架构。在 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，我们将使用它来诱导背侧（苍白）和腹侧的类器官内极化 （subpallial）区域身份。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，并迎来新一代的神经发育建模。

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.