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

单细胞分辨率下人类膝关节和骨盆的分子结构

• 批准号: 10659650
• 负责人: Terence D Capellini
• 金额: $ 76.1万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10659650
• 关键词: 3-Dimensional; ATAC-seq; Aging; Anatomy; Architecture; Articular ligaments; Behavior; Biological; Biological Assay; Biology; Bone structure; Cartilage; Cell Separation; Cell physiology; Cells; Cellular biology; Chondrocytes; Chromatin; Communities; Complex; Connective Tissue; Data Set; Development; Developmental Process; Disease; Distal; Elements; Ensure; Epigenetic Process; Epiphysial cartilage; Event; Extracellular Matrix; Femur; Fibroblasts; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Processes; Genetic Transcription; Genomic Segment; Genomics; Grant; Growth; Hip Joint; Hip region structure; Histologic; Human; Immunohistochemistry; In Situ Hybridization; In Vitro; Individual; Investigation; Joint Capsule; Joints; Knee; Knee joint; Knowledge; Ligaments; Limb structure; Link; Location; Locomotion; Mediating; Medical; Meniscus structure of joint; Methods; Molecular; Molecular Profiling; Morphology; Multiomic Data; Musculoskeletal; Nucleic Acid Regulatory Sequences; Orthopedics; Osteogenesis; Pattern; Pelvis; Pharmacologic Substance; Phenotype; Population; Process; Proliferating; Property; Protocols documentation; Regenerative Medicine; Regulatory Element; Reporter; Research; Research Personnel; Resolution; Resources; Role; Sampling; Shapes; Skeleton; Specimen; Structure; Synovial Membrane; Technology; Therapeutic; Tissue-Specific Gene Expression; Tissues; Weight-Bearing state; acetabulum; cartilage cell; developmental disease; femur head; functional genomics; ilium; in utero; in vivo; innovation; insight; ischium; multiple omics; novel; population based; programs; regenerative therapy; skeletal; skeletal disorder; skeletal maturation; soft tissue; spatial relationship; tibia; tool; transcription factor; transcriptome sequencing; transcriptomics

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.

项目摘要/摘要 在人类两足动物的基石上是骨盆和膝盖，两个后肢骨骼结构 我们对他们在人类的各自发展知之甚少。确实，这些结构具有复杂 3-D形态的初始模式在软骨阶段出现，当 细胞分化和增殖建立各种组织类型以及之间的空间关系 不同的结构成分（例如，膝盖的股股骨con和近端胫骨平台之间， 或在骨盆的ilium，耻骨，坐骨和髋臼亚域之间）。但是，对于发展人类的骨骼 结构，我们对这些细胞事件及其与组织形态的关系一无所知 功能。此外，虽然可以设想这些事件是由多效性或常见的骨骼介导的 生长的基因集和伴随的调节设备，该工具套件如何用于开发人类 构建每个结构，仍然是一个谜。随着生物医学朝着关节组织的再生疗法的发展 以及机械投资对骨骼的发育障碍，获得一个至关重要的 更好地了解骨骼和关节的细胞如何掌握其功能角色，这都是及时的 至关重要的是在单细胞和空间分辨率上建立这一点。迄今为止，大型功能基因组学 财团，例如编码或路线图表观基因组学项目，并未关注 骨骼由于后勤问题而引起的骨骼元素提取软骨细胞而引起的骨骼问题 硬细胞外基质。但是，赠款调查人员允许他们的最新进展 分离和研究从发展人类骨骼的单个软骨细胞。该提议的重点， 因此，是为了更深入地研究子宫内的膝盖，骨盆和后肢如何 单个细胞的水平以及了解其生物学和行为的变化如何推动相对 每个后肢结构的发展。这将通过两个主要目标来完成，一个专注于使用 空间转录组学以检查表达动力学（AIM 1），另一个关于使用 单细胞（SC）多组学方法（AIM 2），由SCRNA测序（检测基因）和 在同一细胞上进行SCATAC-SECER-STERESCONS（检测调节区域）。通过简单分析基因 来自许多细胞的单个细胞水平的表达和调节元件可用性 人类结构以及空间上将实现必要的分辨率来定义小但重要 控制解剖位置特异性软骨细胞生物学及其如何链接到遗传程序的细微差别 后肢形态。赠款团队制定的这些协议的使用将有助于确保 向肌肉骨骼生物学社区提供了非凡的资源，以及这些关键信息 开发新型药物和基于再生医学的疗法所需的需要公开。