Molecular control of chondrocyte hypertrophy: an evolutionary approach

软骨细胞肥大的分子控制：一种进化方法

• 批准号: 10606678
• 负责人: Michael Palmer
• 金额: $ 6.95万
• 依托单位: MARINE BIOLOGICAL LABORATORY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10606678
• 关键词: ATAC-seq; Adult; Age; Biological; Biological Models; CRISPR/Cas technology; Cartilage; Cartilage injury; Cell Therapy; Cells; Chondrichthyes; Chondrocytes; Collagen Fiber; Collagen Type I; Collagen Type II; Degenerative polyarthritis; Development; Elderly; Embryonic Development; Engineering; Enhancers; Evolution; Fibrocartilages; Fishes; Frequencies; Friction; Gene Expression; Gene Transfer Techniques; Genes; Genetic Transcription; Genomic approach; Genomics; Growth; Health; Human; Hyaline Cartilage; Hypertrophy; Implant; In Vitro; Individual; Inferior; Injury; Joints; Knee; Laboratories; Left; Lesion; Life; Mammals; Mechanics; Mesenchymal; Mesenchymal Stem Cells; Minor; Molecular; Nature; Organism; Overweight; Pain; Pathology; Patients; Phenotype; Physiologic Ossification; Population; Prevalence; Process; Property; Quality of life; Regulator Genes; Research; Resources; Shark; Site; Skeletal Development; Skeleton; Stable Populations; Structure; Techniques; Testing; Thinness; Time; Tissue Engineering; Tissues; Transplantation; Untranslated RNA; Variant; Vertebrates; Weight-Bearing state; articular cartilage; bone; bone loss; calcification; cartilage cell; cartilage development; cartilaginous; comparative genomics; differential expression; embryo tissue; gene discovery; genome annotation; genome editing; healing; implantation; improved; in vitro Model; induced pluripotent stem cell; marine; novel; novel strategies; osteogenic; repaired; single-cell RNA sequencing; skeletal; skeletogenesis; stem cell based approach; stem cells; treatment strategy

Project Summary In mammals, cartilage is predominantly an embryonic tissue: the vast majority of cartilage is replaced by bone during the processes of hypertrophy and ossification, with cartilage persisting in relatively few places within the adult skeleton (e.g. in joints, as articular cartilage). Articular cartilage is an aneural, avascular tissue with very limited capacity for spontaneous repair, hence the prevalence in humans of cartilage pathologies like osteoarthritis. In contrast, cartilaginous fishes (sharks, skates, and rays) have undergone an evolutionary loss of bone, instead possessing a skeleton that is composed entirely of pre-hypertrophic cartilage, and that remains cartilaginous throughout life. Understanding how cartilaginous fishes arrest skeletal development prior to chondrocyte hypertrophy will shed new light on transcriptional features that could be employed for in vitro engineering of stable chondrocytes for the treatment of human cartilage injuries. Stem cell-based approaches for the treatment of articular cartilage injuries are currently hindered by the relative instability of mammalian cartilage cells (chondrocytes) in vitro and upon implantation into an injury site. In this project, the molecular development of cartilage in the little skate (Leucoraja erinacea) will be studied to discover gene expression correlates of their permanent cartilaginous skeleton. This project will begin with the use of single-cell RNA-sequencing to identify genes that are differentially expressed between developing and differentiated skate and mammalian chondrocytes (Aim 1), and that may underlie arrest prior to hypertrophy in the former. ATAC-seq and comparative genomic approaches will then be used to test for variation in non-coding regions (i.e. putative enhancers) that might account for divergent gene expression during skate and mammalian skeletogeneis (Aim 2). Finally, skate-inspired molecular manipulations (CRISPR/Cas9 genome editing and/or transgenesis) will be incorporated in an in vitro model of mammalian skeletogenesis, in order to achieve a permanent and stable chondrocyte cell state from mammalian mesenchymal progenitors (Aim 3). This project will capitalize on the unique properties of the skate skeleton, and on the biological resources, facilities, and expertise that are available at the Marine Biological Laboratory in Woods Hole. By taking an evolution-inspired tissue engineering approach, this project will contribute to the development of novel in vitro techniques for cartilage engineering and for the treatment of mammalian skeletal pathologies.

项目概要 在哺乳动物中，软骨主要是胚胎组织：绝大多数软骨被骨所取代 在肥大和骨化过程中，软骨在软骨内相对较少的地方持续存在 成人骨骼（例如在关节中，作为关节软骨）。关节软骨是一种无神经、无血管的组织，具有非常丰富的功能。 自发修复能力有限，因此人类中普遍存在软骨病变，例如 骨关节炎。相比之下，软骨鱼类（鲨鱼、鳐鱼和鳐鱼）经历了进化的丧失 骨骼，而不是拥有完全由肥大前软骨组成的骨骼，并且 终生保持软骨状态。了解软骨鱼类如何阻止骨骼发育 软骨细胞肥大将为研究可用于体外的转录特征提供新的线索 用于治疗人类软骨损伤的稳定软骨细胞工程。 基于干细胞的治疗关节软骨损伤的方法目前受到以下因素的阻碍： 哺乳动物软骨细胞（软骨细胞）在体外和植入损伤部位后的相对不稳定性。 在这个项目中，将研究小鳐鱼 (Leucoraja erinacea) 软骨的分子发育，以 发现与永久软骨骨骼相关的基因表达。该项目将从 使用单细胞RNA测序来鉴定差异表达的基因 发育和分化的鳐鱼和哺乳动物软骨细胞（目标 1），这可能是被捕的原因 肥厚之前在前。然后将使用 ATAC-seq 和比较基因组方法 测试非编码区（即推定的增强子）中可能导致不同基因的变异 鳐鱼和哺乳动物骨骼发育过程中的表达（目标 2）。最后，受滑板启发的分子 操作（CRISPR/Cas9 基因组编辑和/或转基因）将被纳入体外 哺乳动物骨骼发生模型，以实现永久稳定的软骨细胞状态 来自哺乳动物间充质祖细胞（目标 3）。 该项目将利用溜冰鞋骨架的独特特性和生物资源， 伍兹霍尔海洋生物实验室提供的设施和专业知识。通过采取 受进化启发的组织工程方法，该项目将有助于开发新型体​​外 软骨工程和哺乳动物骨骼病理治疗的技术。