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.

项目总结