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.

项目摘要 在哺乳动物中，软骨主要是胚胎组织：绝大多数软骨被骨骼取代。 在肥大和骨化的过程中，软骨在关节内相对较少的地方持续存在 成人骨骼(如关节中的关节软骨)。关节软骨是一种神经性的、无血管的组织。 自发性修复能力有限，因此在人类中普遍存在软骨病理，如 骨性关节炎。相比之下，软骨鱼(鲨鱼、溜冰鱼和银鱼)经历了进化上的丧失。 骨骼，而不是拥有一个完全由肥大前期软骨组成的骨骼，而且 终生保持软骨状。了解软骨鱼是如何阻止骨骼发育的 软骨细胞肥大将为体外转录特性提供新的线索 用于治疗人软骨损伤的稳定软骨细胞工程学。 以干细胞为基础的治疗关节软骨损伤的方法目前受到 哺乳动物软骨细胞(软骨细胞)在体外和植入损伤部位时的相对不稳定性。 在本课题中，我们将对小白鲨软骨的分子发育进行研究。 发现它们永久软骨骨架的基因表达相关性。这个项目将从 使用单细胞RNA测序来鉴定差异表达的基因 发育和分化的滑冰和哺乳动物软骨细胞(目标1)，这可能是阻止的基础 前者先于肥大。然后将使用ATAC-SEQ和比较基因组学方法来 检测可能导致差异基因的非编码区(即假定的增强子)的变异 在滑冰和哺乳动物骨骼发育过程中的表达(目标2)。最后，受滑冰启发的分子 操作(CRISPR/Cas9基因组编辑和/或转基因)将被合并到体外 哺乳动物骨骼发育模型，以达到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)。 这个项目将利用溜冰鞋骨骼的独特特性，以及生物资源， 位于伍兹霍尔的海洋生物实验室提供的设施和专业知识。通过参加一次 受进化启发的组织工程学方法，该项目将有助于体外新型组织工程的开发 软骨工程技术和哺乳动物骨骼病变的治疗。