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

总体摘要： HuBMAP计划的目标是了解单个细胞在3D环境中的独特背景作用。 在转录活性、细胞信号传导和细胞反应的最基本水平上，组织的结构。 迄今为止，由于技术问题，受试组织不包括矿化骨骼系统， 排除了HuBMAP计划的要求。我们已经通过一个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 杂交方法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， 膝关节的骨小梁和软骨结构。协调核心将获得三种组织类型 从身份不明的人那里每个样本将定向到其源组织，通过µCT成像以捕获 它的矿物结构，并处理成组织学堆栈，以创建组织的3D表示。使用 在组织学堆栈中，矿化组织计划将进行原位杂交，以确定 多种细胞类型和seqFISH杂交以捕获所鉴定的细胞类型的细胞转录组。 这些技术上要求很高的步骤将需要既定的HuBMAP调查人员的指导， 康州大学教师谁是高分辨率共聚焦显微镜的专家。数据分析核心将翻译 将这些技术生成的图像文件转化为靶组织和转录组的3D细胞图谱 每种细胞类型的组成。从这些数据文件中，我们的背景分子作图程序TOPAS将 检查转录和信号传导途径，以估算相邻细胞如何协调其活动 对机械负荷和调节骨骼健康的全身因素作出反应。工作流程和 我们为骨骼系统开发的分析平台将与HIVE的要求保持一致 包括一项外联举措。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团体。第二，一旦我们的骨架数据从中央数据库中获得， 蜂巢源，我们将开发虚拟研讨会，告知骨骼生物学社区这一宝贵的 资源以及如何利用它来解开影响骨骼的罕见疾病。HuBMAP将是一个 每一个以组织为中心的团体都需要整合的变革性技术。我们的角色是确保 骨骼生物学社区被包含在这个新的实验平台中，并且它被用来解决 影响骨骼健康的主要遗传和治疗挑战。

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.