High resolution 3D mapping of cellular heterogeneity within multiple types of mineralized tissues

总体摘要： HuBMAP计划的目标是了解3D中各个单元格的独特上下文角色 处于转录活动、细胞信号和细胞反应的最基本水平的组织结构。 到目前为止，由于技术问题，主题组织还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 能够进行包括先进和重复原位方法的多模式组织学 杂交。我们将为三种非常不同的矿化骨骼组织开发这项技术：牙齿， 膝关节的松质骨和软骨结构。协调核心将获得三种组织类型 来自未确认身份的人类来源。每个样本将定位到其源组织，由µCT成像以捕获 它的矿物结构，并加工成组织堆叠，以创建组织的3D表示。vbl.使用 组织学堆叠，矿化组织计划将进行原位杂交，以确定 多种细胞类型和seqFISH杂交捕获细胞转录组的鉴定细胞类型。 这些技术要求很高的步骤将需要老牌HuBMAP调查人员以及 康涅狄格州大学教授，是高分辨率共聚焦显微镜方面的专家。数据分析核心将转换 将这些技术生成的图像文件转换为目标组织和转录组的3D细胞图 每种细胞类型的组成。从这些数据文件中，我们的上下文分子作图程序Topas将 检查转录和信号通路以推测相邻细胞如何协调它们的活动 对机械负荷和调节骨骼健康的系统因素作出反应。工作流程和 我们为骨骼系统开发的分析平台将与蜂箱的要求保持一致 包括一项外展倡议。首先，我们将提供机会将组织学技术转移到 主要的学术骨骼研究小组。第二，一旦我们的骨骼数据从中央获得 蜂巢来源，我们将开发虚拟工作坊，向骨骼生物学社区通报这一宝贵的信息 资源，以及如何利用它来解开影响骨骼的罕见疾病。HuBMAP将成为 每个以组织为中心的组织都需要整合的变革性技术。我们的角色是确保 在这个新的实验平台中加入了骨骼生物学社区，并利用它来解决 影响骨骼健康的重大遗传和治疗挑战。

Overall Abstract: The goal of the HuBMAP program is to appreciate the unique contextual role of individual cells within the 3D structure of a tissue at its most basic level of transcriptional activity, cellular signaling and cellular response. To date the subject 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. We will develop this technology for three very different mineralized skeletal tissues: tooth, trabecular bone and cartilage structures of the knee. The Coordination Core will acquire the three tissue types from de-identified human sources. Each sample will be oriented to its source tissue, 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 Program will perform both in situ hybridization to identify the multiple cell types and seqFISH hybridization to capture the cell transcriptome of the identified cell types. These technically demanding steps will require direction from established HuBMAP investigators as well as UConn faculty who are experts in high resolution confocal microscopy. The Data Analysis Core will translate the image files generated by these techniques into 3D cellular maps of the target tissue and transcriptome composition of each cell type. From those data files, our contextual molecular mapping program, TOPAS, will examine the transcriptional and signaling pathways to impute how neighboring cells coordinate their activities to respond to mechanical loading and systemic factors that regulate skeletal health. The workflow and analytical platforms that we developed for the skeletal system will be aligned with the requirement of the HIVE including an outreach initiative. First, we will provide opportunities to transfer the histological technology to major academic skeletal research groups. Second, once our skeletal data becomes available from the central HIVE source, we will develop virtual workshops to inform the skeletal biology community of this valuable resource and how it can be utilized to unravel rare diseases affecting the skeleton. HuBMAP will be a transformational technology that every tissue centric group needs to incorporate. Our role is to ensure that the skeletal biology community is included in this new experimental platform, and that it is employed to solve the major genetic and therapeutic challenges affecting skeletal health.