3D Cellular and Molecular Mapping within Skeletal Tissue

HuBMAP计划的目标是在以下位置建立高分辨率的组织三维分子图谱 亚细胞水平包括转录活性的空间基因表达分析。到目前为止 由于技术问题，典型的组织没有包括矿化的骨骼系统 排除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方法 RNA和蛋白质靶标的杂交，分别称为MerFish和Codex。在这 在试点项目中，我们将为膝关节的骨骼和软骨结构开发这项技术。那里 由不同的董事领导的三个工作部门。协调部分将获得 鉴定了来自国家疾病研究交换中心的人类股骨远端样本。一系列 核心活组织检查将从关节软骨和增厚软骨中提取，定位于其来源位置， 由µCT成像以捕捉其矿物结构并处理到组织学堆栈中以创建3D 组织的代表。利用组织学堆叠，矿化组织成分将 使用条形码抗体进行MerFish原位杂交和免疫组织学检查 识别多种单元格类型。数据分析组件将转换由生成的图像文件 将这些技术转化为每种细胞类型的目标组织的3D细胞图。从这些数据文件中，我们的 上下文分子作图程序TOPAS将识别细胞内细胞的3D关系 软骨、成骨细胞和破骨细胞谱系。 对于上述强调的目标来说，至关重要的是，我们需要集合和实施一项高 一种能够进行MerFish或Codex类型的实验的吞吐量显微镜系统 计算机自动化脚本协调杂交周期并用显微镜洗涤 它扫描、成像和平铺组织的特定区域。本补编要求拨款以支付费用。 这种仪器的性能。我们的长期目标，一旦试点计划确认我们的有效性 膝盖的实验平台，是将其应用扩展到广泛的骨骼组织和更多 复杂的细胞靶点作为一种新的研究资源来询问主要的遗传和 影响骨骼健康的治疗挑战。

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.