Dynamic heterogeneity of nucleus pulposus cells from development to degeneration

髓核细胞从发育到退化的动态异质性

• 批准号: 10401258
• 负责人: Neil Malhotra
• 金额: $ 17.7万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-05 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10401258
• 关键词: Acute; Address; Aging; Automobile Driving; Behavior; Biological; Biomechanics; Cell Differentiation process; Cell Therapy; Cells; Characteristics; Chondrocytes; Chronic; Data; Development; Embryo; Event; Exhibits; Extracellular Matrix; Genes; Goals; Growth; Growth and Development function; Heterogeneity; Histologic; Human; Immunofluorescence Immunologic; Individual; Inflammation; Inflammatory; Injury; Intervertebral disc structure; Life; Low Back Pain; Maps; Mechanics; Mediating; Modeling; Molecular; Mus; Natural regeneration; Nature; Needles; Operative Surgical Procedures; Pain; Population; Process; Property; Proteoglycan; Puncture procedure; Research; Resolution; Role; Severities; Signal Transduction; Structure; Therapeutic; Time; Tissues; base; computerized tools; disc regeneration; effective therapy; in vivo; intervertebral disk degeneration; notochord; novel; novel therapeutics; nucleus pulposus; postnatal; progenitor; reduce symptoms; repaired; response to injury; single-cell RNA sequencing; stem cells; tool; transcriptome; transcriptome sequencing; transcriptomics; validation studies

Abstract Lumbar intervertebral disc degeneration is a cascade of cellular, structural and mechanical changes that is strongly implicated as a cause of low back pain. Current therapies for painful disc degeneration, conservative or surgical, focus only on alleviating symptoms. There is a critical need for new therapies that restore disc structure and mechanical function by directly addressing the underlying biological causes. A key challenge to developing effective treatments for disc degeneration is the need to recapitulate the structural complexity and specialized extracellular matrix (ECM) of the component tissues, which comprise cells of multiple developmental lineages. The central nucleus pulposus (NP) is implicated in the initiation of disc degeneration. The developmental origin of NP cells was until recently a point of contention, but was resolved though fate mapping studies in mice that demonstrated conclusively that these cells in their entirety are descendants of embryonic notochordal cells. During growth and aging, NP cells progressively lose their notochordal characteristics to be replaced by smaller, chondrocyte-like “mature” NP cells. The disappearance of notochordal cells has long been considered to contribute to progressive disc degeneration later in life, however the underlying molecular mechanisms are not understood. With degeneration, the NP undergoes an inflammation-mediated, fibrous transformation that compromises disc biomechanical function; however the underlying cellular events that initiate and drive this process are poorly understood. Single cell RNA-Seq (scRNA-Seq) is an emerging tool that permits high resolution transcriptome analysis of individual cells, facilitating identification of rare subpopulations. In combination with cutting-edge computational tools, scRNA- Seq can elucidate transitional states and relationships between cell subpopulations to predict cell differentiation trajectories. The overall objective of this proposal is to use scRNA-Seq to investigate the emergence of distinct NP cell subpopulations during intervertebral disc development and growth (Aim 1), and establish the fate and function of these subpopulations during injury and degeneration (Aim 2). For both Aims, using the top cluster-specific genes, the presence and localization of distinct NP cell subpopulations will be confirmed using immunofluorescence and qPCR. The results of this study will have important implications for the development of novel cell-based therapies for disc regeneration. For example, establishing NP progenitor- specific markers through cluster-specific gene analysis may allow subsequent identification and enrichment of therapeutic cell populations in human disc tissue, and defining differentiation trajectories may allow reprogramming of such cells for therapeutic application.

抽象的 腰椎间盘退变是一系列细胞、结构和机械变化， 目前治疗疼痛性椎间盘退变的保守疗法与腰痛密切相关。 或手术，仅关注减轻症状 迫切需要恢复椎间盘的新疗法。 通过直接解决潜在的生物学原因来实现结构和机械功能是一个关键挑战。 开发针对椎间盘退变的有效治疗方法需要概括其结构复杂性和 组成组织的专门细胞外基质（ECM），由多种细胞组成 发育谱系。中央髓核（NP）与椎间盘退变的起始有关。 直到最近，NP 细胞的发育起源仍然是一个争论点，但最终通过命运得到了解决 对小鼠进行的绘图研究最终证明这些细胞整体上是 胚胎脊索细胞在生长和衰老过程中，NP 细胞逐渐失去其脊索。 特征被更小的软骨细胞样“成熟”NP 细胞取代。 长期以来，脊索细胞一直被认为会导致生命后期进行性椎间盘退变，然而 潜在的分子机制尚不明确，随着退化，NP 会发生变化。 炎症介导的纤维转化会损害椎间盘的生物力学功能； 对启动和驱动这一过程的潜在细胞事件知之甚少。 (scRNA-Seq) 是一种新兴工具，可以对单个细胞进行高分辨率转录组分析， 与尖端计算工具 scRNA 相结合，促进稀有亚群的识别。 Seq可以阐明细胞亚群之间的过渡状态和关系以预测细胞 该提案的总体目标是使用 scRNA-Seq 来研究分化轨迹。 在椎间盘发育和生长过程中出现不同的 NP 细胞亚群（目标 1），以及 确定这些亚群在损伤和退化过程中的命运和功能（目标 2）。 使用顶部簇特异性基因，不同 NP 细胞亚群的存在和定位将被 通过免疫荧光和 qPCR 证实，这项研究的结果将具有重要意义。 开发新的基于细胞的椎间盘再生疗法，例如，建立 NP 祖细胞。 通过簇特异性基因分析的特定标记可以允许随后的鉴定和富集 人类椎间盘组织中的治疗细胞群，并定义分化轨迹可能允许 对此类细胞进行重新编程以用于治疗应用。