Patterning human forebrain organoids by engineering controlled biochemical microenvironment

项目概要 脑类器官正在成为研究人脑基本机制的强大工具 发育和神经系统疾病，并有潜力用作高通量平台 治疗筛选。然而，目前最先进的大脑类器官的可重复性有限， 可扩展性和结构准确性，从而阻碍了其更广泛的应用。因此，这是一个挑战 研究不同大脑之间不同的细胞生物学、成熟和功能相互作用（电路） 细分。重要的是，当前的大脑类器官缺乏正确的极性和准确的前后（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模拟大脑类器官。在这里，我们的目标是 有效且可重复地将这种形态发生素梯度施加到人类多能干细胞聚集体上 开发两种新型微型设备，可以产生持续且定制的形态发生素浓度 梯度。具体来说，我们将首先开发一种微加工装置来产生 Wnt 的反平行梯度 抑制剂，以及基于局部被动扩散的BMP7和MER/ERK抑制剂（简称LPaD 2.0）。我们 将使用该设备沿着 A-P 轴绘制人类前脑类器官 (hFO) 的图案（目标 1）。那么，我们将 开发一系列水凝胶微针 (HM)，将小分子直接输送到皮肤内部 类器官并将 HM 装置与 LPaD 2.0 装置结合起来生成正交梯度以诱导 hFO 中同时进行 A-P 和 D-V 图案化。除了形态发生素梯度之外，我们还将评估 通过改变设计，类器官形状和营养/氧气呈现对 hFO 发育的影响 HM 设备的。我们将表征源自这些的 hFO 的细胞结构和功能 免疫细胞化学、单细胞 RNA 测序和高密度多电极阵列分析装置。 我们的系统的重现性和产量将与传统方法进行定量比较 衍生的 hFO 和功能状态将以最先进的组装体模型为基准 使用中间神经元迁移测定和丘脑项目测定。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的或区域的类器官的生物制造 具有适当模式的特定大脑类器官。我们的设备有潜力应用于其他类器官 系统。完全图案化的 hFO 提供了一种多功能模型，可以更好地剖析细胞和组织规模 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 药物筛选平台可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.