Improving Brain Organoid Models by Mediating Metabolic Dysregulation

项目摘要 目前存在对人脑的精确模型系统的未满足的需求，以研究其细胞和组织结构。 分子特征大脑皮层控制着我们的认知能力，而细胞的多样性，电路 形成和功能，建立这种潜力，在很大程度上仍然是一个谜。人类的大脑皮层 与其他物种相比，它包含更多的细胞多样性和丰度，使模式生物有限 用于翻译研究。脑类器官提供了获得人类细胞的途径，用于实验操作和最近的研究。 研究表明，类器官模型可以作为人类大脑的代理， 发育轨迹、电路和各种复杂的神经系统疾病。然而，使用单细胞 RNA测序我们进行了皮质类器官模型与原代人类皮质的验证研究 我们发现了类器官细胞中的几个显著缺陷。首先，类器官不能产生相同的 在正常皮层发育过程中观察到的具有清晰基因表达程序的精细细胞类型。他们还 缺乏内源性分子成熟网络。由于类器官细胞不制造特定的、精细的 我们在人脑中观察到的细胞类型，这些模型在旨在模拟电路活动时受到严重限制 和疾病回路由高度特化的神经元亚型组成，它们相互瞄准， 定义的连接，并最终产生适当的生理活动。我们需要器官模型， 使特定的细胞类型，可以成熟到足以形成标准电路的构建块，使 神经连接性的研究取得进展。除了细胞类型缺陷外，检测的所有类器官， 无论推导方法如何，都表达了高水平的代谢应激。通过移植研究，我们 发现细胞应激与体外环境有关，它对细胞类型的同一性产生负面影响， 可以通过从培养条件中去除来逆转。我所提出的研究将直接解决目前的问题， 类器官模型的技术限制，通过干预代谢失调来改善 细胞类型特化和成熟。首先，我将针对糖酵解和内质网应激途径， 使用小分子来抑制这些过程，并通过调节葡萄糖和氧气浓度。我会 还利用人类皮层发育中相关信号通路的知识，例如LIF信号通路， 途径，这是一个重要的调节人富集群体的皮质干细胞。第二，我会 致力于确定内源性正常代谢调节所需的相关非神经细胞， 在类器官模型中不存在。为了实现这一目标，我将使用人类类器官来源的细胞移植， 将相关的血管和免疫细胞移植到小鼠皮质类器官中， 体外总之，这些研究将改善目前在代谢和细胞类型损伤方面的技术限制， 确保类器官更准确地反映人类皮层的细胞多样性，并提供易于处理的 人类神经科学研究的模型系统。

Project Summary There is currently an unmet need for accurate model systems of the human brain to study its cellular and molecular features. The cerebral cortex regulates our cognitive capacity, yet the cellular diversity, circuit formation, and function that establish this potential, largely remains a mystery. The cortex is expanded in humans compared to other species; it contains more cellular diversity and abundance, making model organisms limited for translational studies. Brain organoids provide access to human cells for experimental manipulation and recent studies have suggested that organoid models may act as a proxy for the human brain when studying developmental trajectories, circuitry, and a variety of complex neurological diseases. However, using single cell RNA sequencing we performed validation studies of cortical organoid models compared to primary human cortex cells and we discovered several significant deficiencies in organoid cells. First, organoids do not make the same refined cell types with clear gene expression programs observed during normal cortex development. They also lack molecular maturation networks observed endogenously. As organoid cells do not make the specific, refined cell types we observe in the human brain, these models are severely limited when aiming to model circuit activity and disease. Circuits are comprised of highly specialized neuronal subtypes that target one another to wire defined connections and ultimately produce appropriate physiological activity. We require organoid models that make specific cell types that can mature enough to form the building blocks of canonical circuits to enable the study of neural connectivity moving forward. In addition to cell type deficiencies, all organoids assayed, regardless of derivation method, express high levels of metabolic stress. Using transplantation studies, we identified that cellular stress is linked to the in vitro environment, it negatively impacts cell type identity, and it can be reversed by removal from culture conditions. My proposed studies will directly address the current technical limitations of organoid models by intervening in the metabolic dysregulation with the goal of improving cell type specification and maturation. First, I will target glycolytic and endoplasmic reticulum stress pathways, using small molecules to inhibit these processes and by modulating glucose and oxygen concentrations. I will also utilize knowledge of relevant signaling pathways in human cortex development, such as the LIF signaling pathway, which is an important regulator of a human-enriched population of cortical stem cells. Second, I will work to identify the relevant non-neural cells required for normal metabolic regulation endogenously, which are absent from organoid models. To work toward this aim I will use transplantation of human organoid-derived cells into the mouse cortex environment and transplant relevant vascular and immune cells into cortical organoids in vitro. Together, these studies will improve current technical limitations in metabolism and cell type impairment to ensure that organoids more accurately reflect the cellular diversity of the human cortex and provide a tractable model system for the study of human neuroscience.