Patterning human forebrain organoids by engineering controlled biochemical microenvironment

项目摘要 脑类器官正在成为研究人脑基本机制的有力工具 发展和神经系统疾病，并有可能被用作高通量平台， 治疗筛选然而，目前最先进的脑类器官的再现性有限， 可扩展性和结构准确性，从而阻止了它们的更广泛应用。因此， 研究不同的细胞生物学，成熟和不同大脑之间的功能相互作用（电路） 细分。重要的是，目前的脑类器官缺乏正确的极性，缺乏准确的前后（A-P） 和背腹（D-V）空间模式。最近的研究和我们的初步结果强烈表明， 形态发生素的外在浓度梯度可以有效地对脑类器官进行图案化。在这里，我们的目标是 有效地和可重复地将这样的形态发生梯度施加到人多能干细胞聚集体， 开发了两种新型的微型装置，可以产生持续的和定制的形态原浓度， 梯度。具体来说，我们将首先开发一种微加工设备，以产生Wnt的反平行梯度 抑制剂和基于局部被动扩散的BMP 7和MER/ERK抑制剂（称为LPaD 2.0）。我们 将使用该设备沿A-P轴（Aim 1）对人类前脑类器官（hFO）进行沿着图案化。那就 开发一系列水凝胶微针（HM），将小分子直接输送到 类器官和联合收割机将HM设备与LPaD 2.0设备结合，以产生正交梯度， hFO中的同时A-P和D-V图案化。除了形态梯度，我们还将评估 通过改变设计，类器官形状和营养/氧呈现对hFO发育的影响 的HM设备。我们将描述这些衍生的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， 我们的系统的再现性和产量将与常规方法进行定量比较， 推导hFO和功能状态将使用最先进的抛物面模型进行基准测试 使用中间神经元迁移分析和丘脑投射分析。该项目的目标是推进 通过提供易于使用的设备来可重复地生产完整或区域的类器官的生物制造， 特定的脑类器官有适当的模式。我们的设备有可能应用于其他类器官 系统.完全图案化的hFO提供了一个多功能模型，以更好地解剖细胞和组织规模 神经系统疾病的特征，如精神分裂症和自闭症，并有可能被用作 药物筛选平台，以改善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.