3D Cellular and Molecular Mapping within Skeletal Tissue

骨骼组织内的 3D 细胞和分子图谱

• 批准号: 10355748
• 负责人: PETER MAYE
• 金额: $ 10万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-21 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10355748
• 关键词: 3-Dimensional; Affect; Analysis of Variance; Antibodies; Automation; Back; Bar Codes; Biopsy; Bone Marrow; Cadaver; Cartilage; Cell Lineage; Cell Nucleus; Cells; Cessation of life; Clinical; Collaborations; Complex; Computers; Core Biopsy; Cryoultramicrotomy; DNA; Data; Data Analyses; Data Files; Data Set; Databases; Disease; Distal; Ensure; Epitopes; Extracellular Matrix; Femur; Fluorescence; Funding; Gene Expression Profiling; Gene Proteins; Genetic; Genetic Transcription; Goals; Health; Histologic; Histological Techniques; Histology; Hour; Human; Human BioMolecular Atlas Program; Image; In Situ Hybridization; Knee; Lead; Link; Liquid substance; Location; Maps; Methods; Microscope; Minerals; Molecular; Molecular Target; Mus; Myelogenous; Oligonucleotides; Osteoblasts; Osteoclasts; Pathway Analysis; Pilot Projects; Positioning Attribute; Procedures; Process; Proteins; Protocols documentation; RNA; Recording of previous events; Research; Resolution; Resources; Sampling; Sampling Studies; Scanning; Series; Signaling Protein; Site; Skeletal system; Source; Specimen; Structure; System; Systems Analysis; Techniques; Technology; Therapeutic; Time; Tissue Donors; Tissue Sample; Tissues; Translating; base; bone; cell type; cellular targeting; cost; design; experimental study; genetic signature; imaging probe; imaging system; instrumentation; member; men; multimodality; programs; reconstruction; single cell analysis; single-cell RNA sequencing; skeletal; skeletal tissue; skills; visual map

The goal of the HuBMAP program is to build high resolution 3-D molecular maps of tissues at the subcellular level including spatial gene expression analysis of transcriptional activity. To date the representative tissues have not included the mineralized skeletal system due to technical issues that preclude the requirements of the HuBMAP program. We have solved those issues with a protocol that is capable of performing multimodal histology that include methods for advanced and repetitive in situ hybridization for both RNA and protein targets, known as MERFISH and CODEX, respectively. In this pilot project, we will develop this technology for the bone and cartilage structures of the knee. There are three divisions of effort led by separate directors. The Coordination Component will acquire de- identified human distal femur samples from the National Disease Research Interchange. A series of core biopsies will be extracted from the articular and enthenic cartilage, oriented to its source location, imaged by µCT to capture its mineral structure and processed into a histological stack to create a 3D representation of the tissue. Using the histological stack, the Mineralized Tissue Component will perform both MERFISH in situ hybridization and immunohistology using bar coded antibodies to identify multiple cell types. The Data Analysis Component will translate the image files generated by these techniques into 3D cellular maps of the target tissue of each cell type. From those data files, our contextual molecular mapping program, TOPAS, will identify the 3D relationship of cells within the cartilage, osteoblast and osteoclast lineages. Essential to the objectives underlined above, we need to assemble and implement a high throughput microscope system capable of performing MERFISH or CODEX types of experiments in which a computer automation script coordinates cycles of hybridization and washes with a microscope which scans, images and tiles specific regions of tissue. This supplement request funds to cover cost of this instrumentation. Our long-term goal, once the pilot program affirms the validity of our experimental platform for the knee, is to expand its use to a broad range of skeletal tissues and more complex cellular targets as a new investigatory resource to interrogate the major genetic and therapeutic challenges affecting skeletal health.

Hubmap程序的目的是建立高分辨率3-D分子图的组织 亚细胞水平包括转录活性的空间基因表达分析。迄今为止 由于技术问题 排除Hubmap程序的要求。我们已经通过协议解决了这些问题 能够执行包括高级和重复原位方法的多模式组织学 RNA和蛋白质靶标的杂交分别称为Merfish和Codex。在这个 试点项目，我们将开发这项技术，用于膝盖的骨骼和软骨结构。那里 是由单独的董事领导的三个努力。协调部分将获得 从国家疾病研究互换中确定了人类股骨远端样本。一系列 核心活检将从关节和令人陶醉的软骨中提取，定向到其源位置， 由µCT成像以捕获其矿物结构并加工成组织学堆栈以创建3D 组织的表示。使用组织学堆栈，矿化组织成分将 使用条形图 识别多种单元格类型。数据分析组件将翻译由 这些技术进入了每种细胞类型的目标组织的3D细胞图。从这些数据文件，我们的 上下文分子映射程序TOPA将识别细胞内的3D关系 软骨，成骨细胞和破骨细胞谱系。 对于上述强调的对象，我们需要组装并实施高 吞吐量显微镜系统，能够在 计算机自动化脚本协调杂交周期并用显微镜洗涤 扫描，图像和图块特定的组织区域。此补充要求资金支付成本 该仪器。我们的长期目标，一旦飞行员计划肯定了我们的有效性 膝盖的实验平台是将其用途扩展到广泛的骨骼组织等 复杂的细胞靶标作为一种新的研究资源，以询问主要遗传和 影响骨骼健康的治疗挑战。