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计划的目标是建立组织的高分辨率3-D分子图， 亚细胞水平包括空间基因表达的转录活性分析。迄今 由于技术问题，代表性组织不包括矿化骨骼系统， 排除了HuBMAP计划的要求。我们已经通过一项协议解决了这些问题， 能够进行多模式组织学，包括先进的和重复的原位 针对RNA和蛋白质靶标的杂交，分别称为MERFISH和CODEX。在这 作为一个试点项目，我们将为膝盖的骨骼和软骨结构开发这种技术。那里 是由不同的主管领导的三个部门的工作。协调部门将获得- 从国家疾病研究交流中心鉴定出人类股骨远端样本。一系列 将从关节和软骨中提取核心活组织检查，朝向其来源位置， 通过µCT成像以捕获其矿物结构，并处理成组织学堆栈以创建3D 组织的表现。使用组织学堆栈，矿化组织组件将 使用条形码抗体进行MERFISH原位杂交和免疫组织学， 识别多种细胞类型。数据分析组件将转换由 将这些技术转化为每种细胞类型的靶组织的3D细胞图。从这些数据文件中，我们 背景分子映射程序TOPAS将确定细胞内细胞的3D关系。 软骨、成骨细胞和破骨细胞谱系。 为了实现上述目标，我们需要制定和实施一个高层次的 能够进行MERFISH或CODEX类型实验的通量显微镜系统， 计算机自动化脚本协调杂交的循环， 它扫描、成像并平铺组织的特定区域。这一补充要求资金支付费用 这个仪器。我们的长期目标是，一旦试点项目确认了我们的 膝盖的实验平台，是扩大其使用范围广泛的骨骼组织和更多 复杂的细胞靶点作为一种新的解释性资源，以询问主要的遗传和 影响骨骼健康的治疗挑战。