Molecular architecture of the human knee joint and pelvis at single cell resolution

项目摘要/摘要 人类两足运动的基石是骨盆和膝盖，两个后肢骨骼结构用于 我们对它们各自在人类体内的发育情况知之甚少。事实上，这些结构具有复杂性 3-D形态，当协调时，其初始图案出现在软骨形成的原生期 细胞分化和增殖建立了各种组织类型和它们之间的空间关系 不同的结构部件(例如，在膝盖的远端股骨髁和胫骨近端平台之间， 或骨盆的髂骨、耻骨、坐骨和髋臼亚域之间)。然而，对于发育人类骨骼来说 结构，我们对这些细胞事件及其与组织形态的关系知之甚少 功能。此外，虽然人们可以想象这些事件是由多效性或共同的‘骨骼’所介导的 Growth‘基因集和伴随的调节装置，该工具包如何用于发育人类以 建造每一座建筑，仍然是一个谜。随着生物医学走向关节组织的再生疗法 和骨骼发育障碍的机制研究，至关重要的是获得一个 更好地了解骨骼和关节的细胞如何获得其功能角色，这两个都是及时的 这对在单细胞和空间分辨率下建立这一点至关重要。到目前为止，基于大型功能基因组学 Encode或路线图表观基因组学项目等财团并未将重点放在 从发育中的骨骼元素中提取软骨细胞时由于后勤问题而导致的骨骼 硬的细胞外基质。然而，最近拨款调查人员在这方面的进展使他们能够 从发育中的人骨骼中分离和研究单个软骨细胞。这项提案的重点是， 因此，更深入地研究人的膝盖、骨盆和后肢在子宫中的一般形态，在 单个细胞的水平，以及了解它们的生物学和行为变化如何推动各自的 每个后肢结构的发育。这将通过两个主要目标来实现，一个集中在使用上 空间转录切割学用于组织学研究表达动力学(目标1)，以及另一个关于使用 单细胞(Sc)多组学方法(目标2)，包括单链RNA测序(检测基因)和 SCATAC-在同一细胞上进行测序(以检测调控区域)。通过同时分析基因 这些发育中的许多细胞在单个细胞水平上的表达和调控元件的可用性 人类结构，在空间上，将达到必要的分辨率，以确定微小但重要的 支配解剖部位特定软骨细胞生物学的遗传程序的细微差别以及它如何与 后肢形态。使用由赠款团队开发的这些协议将有助于确保 为肌肉骨骼生物界提供了非凡的资源，而这一关键信息 需要开发新的药物和以再生医学为基础的疗法是公开的。

Project Summary/Abstract At the cornerstone of human bipedal locomotion are the pelvis and knee, two hind limb skeletal structures for which we know little about their respective development in humans. Indeed, these structures have complex 3-D morphologies whose initial patterns arise during the chondrogenic anlagen stage, when coordinated cellular differentiation and proliferation establish various tissue types and the spatial relationships between different structural components (e.g., between the knee’s distal femoral condyles and proximal tibial platform, or between the pelvis’ ilium, pubis, ischium, and acetabular subdomains). Yet, for developing human skeletal structures, we understand little about these cellular events and their relationships to tissue morphology and function. Moreover, while one can envision that these events are mediated by a pleiotropic or common ‘skeletal growth’ gene set and accompanied regulatory apparatus, how this tool kit is used in developing humans to build each structure, remains a mystery. As biomedicine move towards regenerative therapies for joint tissues and mechanistic investigations into developmental disorders of the skeleton, it is crucially important to gain a better understanding of how cells of the skeleton and joints acquire their functional roles, and it is both timely and critical to establish this at single cell and spatial resolutions. To date, large functional genomics-based consortia, such as an ENCODE or the ROADMAP EPIGENOMICS PROJECT, have not focused on the skeleton due to logistical issues in extracting cartilage cells from developing skeletal elements composed of hard extracellular matrix. However, recent advances on this front by the grant investigators have allowed them to isolate and study individual cartilage cells from developing human skeletons. The focus of this proposal, therefore, is to more deeply investigate how the human knee, pelvis, and hind limb in general form in utero, at the level of individual cells and in understanding how changes in their biology and behavior drive the respective development of each hind limb structure. This will be accomplished via two main aims, one focused on the use of spatial transcriptomics to examine expression dynamics histologically (Aim 1), and another on the use of a single cell (sc) multiomics approach (Aim 2), consisting of scRNA-sequencing (to detect genes) and scATAC-sequencing (to detect regulatory regions) on the same cell. By simultaneously profiling gene expression and regulatory element availability at the individual cell level from many cells of these developing human structures, and spatially, the necessary resolution will be achieved to define small but important nuances in the genetic programs that govern anatomical-site-specific cartilage cell biology and how it links to hind limb morphology. Use of these protocols developed by the grant’s Team will help ensure that an extraordinary resource is provided to the musculoskeletal biology community, and that crucial information needed to develop novel pharmaceutical and regenerative medicine-based therapeutics is made public.