Patterning human forebrain organoids by engineering controlled biochemical microenvironment

通过工程控制的生化微环境来图案化人类前脑类器官

• 批准号: 10508548
• 负责人: Yubing Sun
• 金额: $ 18.59万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10508548
• 关键词: 3-Dimensional; Agonist; Anterior; BMP7 gene; Basal Ganglia; Benchmarking; Biochemical; Biological Assay; Biomanufacturing; Biomedical Engineering; Brain; Brain region; Cell Line; Cells; Cellular biology; Chemicals; Coupled; Custom; Data; Development; Devices; Diffusion; Dimensions; Disease; Dorsal; Drug Screening; Effectiveness; Electrophysiology (science); Engineering; Environment; Forebrain Development; Foundations; Germ Layers; Goals; Grant; Growth; Growth Factor; Human; Hydrogels; Hypothalamic structure; In Vitro; Individual; Insulin; Interneurons; Methodology; Methods; Migration Assay; Modeling; Necrosis; Nutrient; Organoids; Oxygen; Pattern; Penetration; Pharmaceutical Preparations; Physiology; Prosencephalon; Proto-Oncogene Proteins c-akt; Regenerative Medicine; Reproducibility; Research; SHH gene; Schizophrenia; Series; Shapes; Side; Signal Transduction; Standardization; System; Technology; Telencephalon; Testing; Thalamic structure; Therapeutic; Time; Tissues; Toxin; Variant; autism spectrum disorder; base; cell type; density; design; diencephalon; human pluripotent stem cell; immunocytochemistry; improved; in vivo; inhibitor; insight; microdevice; migration; morphogens; multi-electrode arrays; nervous system disorder; neuropsychiatric disorder; next generation; novel; prevent; rational design; regenerative therapy; screening; single-cell RNA sequencing; small molecule; stem cell biology; therapeutic development; tool; treatment strategy

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.

项目摘要 脑器官正在成为研究人脑的基本机制的强大工具 发育和神经系统疾病，并有可能被用作高通量平台 治疗筛查。但是，当前最新的脑器官的可重复性有限， 可扩展性和结构性准确性，从而阻止其更广泛的应用。因此，这具有挑战性 研究不同大脑之间的各种细胞生物学，成熟和功能相互作用（电路） 细分。重要的是，当前的脑器官缺乏适当的极性，精确的前后后方（A-P） 和背腹侧（D-V）空间图案。最近的研究和我们的初步结果强烈表明 形态剂的外在浓度梯度可以有效地对大脑器官进行构图。在这里，我们的目标是 通过 开发两个可产生持续和定制的形态浓度的新型微型设计 梯度。具体而言，我们将首先开发一个微生物的装置，以产生Wnt的抗平行梯度 抑制剂，BMP7和MER/ERK抑制剂基于局部被动扩散（称为LPAD 2.0）。我们 将使用此设备沿A-P轴对人前脑前脑器官（HFOS）进行模板（AIM 1）。然后，我们会的 开发一系列水凝胶微针（HM），将小分子直接传递到其内部 器官并将HM设备与LPAD 2.0设备相结合，以生成正交梯度以诱导 同时在HFOS中同时进行A-P和D-V图案。除形态梯度外，我们还将评估 器官形状和养分/氧气表现对HFOS开发的影响通过更改设计 HM设备。我们将表征从这些的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的设备。 与常规方法相比，我们系统的可重复性和产量将被定量 得出HFO和功能状态将通过最新的组合模型进行基准测试 使用中间神经元迁移测定法和丘脑项目测定法。该项目的目的是促进 通过提供易于使用的设备来可重复生产完整或区域 - 特定的脑器官具有适当的图案。我们的设备有可能应用于其他类器官 系统。完全模式的HFO提供了一种多功能模型，以更好地剖析细胞和组织量表 神经系统疾病的特征，例如精神分裂症和自闭症，并且有可能被用作 用于改善这些疾病治疗策略的药物筛查平台。