Patterning human forebrain organoids by engineering controlled biochemical microenvironment

项目总结 脑有机化合物正在成为研究人类大脑基本机制的有力工具 发展和神经障碍，并有潜力被用作高通量平台 治疗性筛查。然而，目前最先进的脑有机体存在重复性有限的问题， 可伸缩性和结构的准确性，从而阻止了其更广泛的应用。因此，这是具有挑战性的 研究不同大脑之间不同的细胞生物学、成熟度和功能相互作用(回路) 细分。重要的是，目前的脑器官缺乏正确的极性和准确的前后(A-P) 背-腹侧(D-V)空间构型。最近的研究和我们的初步结果强烈表明， 外在浓度梯度的形态生物质可以有效地构型脑器官。在这里，我们的目标是 通过以下方式将这种形态梯度高效且可重复性地施加到人类多能干细胞聚集体 开发两种可产生持续和定制形态根浓度的新型微器件 渐变。具体地说，我们将首先开发一种微型制造设备来产生反平行的WNT梯度 基于局部被动扩散的BMP7和Mer/ERK抑制剂(简称LPAD 2.0)。我们 将使用这个设备沿着A-P轴对人类前脑有机体(HFO)进行模式(目标1)。那么，我们会 开发一系列水凝胶微针(HM)，将小分子直接输送到 有机化合物，并将HM设备与LPAD 2.0设备相结合，生成正交梯度以诱导 高频电信号的同时A-P和D-V模式。除了形态梯度，我们还将评估 通过改变设计改变有机物形状和营养/氧气呈现对HFOS发展的影响 HM设备的。我们将表征从这些来源的HFO的细胞结构和功能 通过免疫细胞化学、单细胞RNA测序和高密度多电极阵列分析。 我们的系统的重复性和成品率将与传统的方法进行定量比较 推导出的HFO和功能状态将以最先进的集合体模型为基准 采用神经元间迁移实验和丘脑投射实验。这个项目的目标是推进 通过提供易于使用的设备来可重复地生产整个或区域-有机化合物的生物制造- 特定的脑器官，有适当的图案。我们的设备具有应用于其他有机化合物的潜力 系统。全图案的HFO提供了一个通用的模型，以更好地剖析细胞和组织的规模 神经疾病的特征，如精神分裂症和自闭症，并有可能被用作 药物筛选平台，完善针对这些疾病的治疗策略。

PROJECT SUMMARY Brain organoids are becoming powerful tools to study fundamental mechanisms underlying human brain development and neurological disorders and have the potential to be used as a high-throughput platform for therapeutic screening. However, current state-of-the-art brain organoids suffer from limited reproducibility, scalability, and structural accuracy, thereby preventing their broader applications. It is, therefore, challenging to study the diverse cell biology, maturation, and functional interactions (circuitry) among different brain subdivisions. Importantly, current brain organoids lack proper polarity with accurate anterior-posterior (A-P) and dorsal-ventral (D-V) spatial patterning. Recent studies and our preliminary results strongly suggest that the extrinsic concentration gradient of morphogens can effectively pattern brain organoids. Here, we aim to efficiently and reproducibly impose such morphogen gradients to human pluripotent stem cell aggregates by developing two novel microdevices that can generate sustained and customized morphogen concentration gradients. Specifically, we will first develop a microfabricated device to produce antiparallel gradients of Wnt inhibitor, and BMP7 and MER/ERK inhibitor based on localized passive diffusion (referred to as LPaD 2.0). We will use this device to pattern human forebrain organoids (hFOs) along the A-P axis (Aim 1). Then, we will develop an array of Hydrogel Microneedles (HM) to deliver small molecules directly to the inside of the organoids and combine the HM device with the LPaD 2.0 device to generate orthogonal gradients to induce simultaneous A-P and D-V patterning in hFOs. In addition to morphogen gradients, we will also evaluate the effects of organoid shape and nutrient/oxygen presentations on the hFOs development by changing the design of the HM device. We will characterize the cytoarchitecture and function of the hFOs derived from these devices by immunocytochemistry, single-cell RNA sequencing, and high-density multi-electrode array analysis. The reproducibility and yield of our system will be quantitatively compared with conventional methods for deriving hFOs and the functional states will be benchmarked with the state-of-the-art assembloids models using the interneuron migration assay and thalamic project assay. The goal of this project is to advance the biomanufacturing of organoids by providing easy-to-use devices to reproducibly produce complete or region- specific brain organoids with proper patterning. Our devices have the potential to be applied to other organoid systems. The fully patterned hFOs offer a versatile model to better dissect the cellular and tissue scale features of neurological diseases, such as schizophrenia and autism, and have the potential to be used as a drug screening platform to improve the treatment strategies for these diseases.