Dynamic heterogeneity of nucleus pulposus cells from development to degeneration

摘要 腰椎间盘退变是一系列细胞、结构和力学变化， 强烈暗示这是引起腰痛的原因。目前治疗疼痛性椎间盘退变的保守疗法 或外科手术，只专注于减轻症状。迫切需要新的治疗方法， 结构和机械功能，直接解决潜在的生物学原因。方面面临的关键挑战 开发椎间盘退变的有效治疗是需要概括结构复杂性， 细胞外基质是组成组织的特化细胞外基质（ECM），其包含多种细胞因子的细胞。 发育谱系中央髓核（NP）与椎间盘退变的发生有关。 NP细胞的发育起源直到最近还是一个争论点，但通过命运解决了 在小鼠中进行的定位研究最终证明，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 这些特征将被更小的软骨细胞样“成熟”NP细胞所取代。的消失 然而，长期以来，人们认为脊索细胞在以后的生活中会导致进行性椎间盘退变， 其潜在的分子机制尚不清楚。随着退化，NP经历了 炎症介导的纤维转化，损害椎间盘生物力学功能;然而， 启动和驱动该过程的潜在细胞事件知之甚少。单细胞RNA-Seq （scRNA-Seq）是一种新兴的工具，其允许对单个细胞进行高分辨率转录组分析， 有助于鉴定稀有亚群。结合尖端的计算工具，scRNA- Seq可以阐明细胞亚群之间的过渡状态和关系，以预测细胞 分化轨迹本提案的总体目标是使用scRNA-Seq来研究 在椎间盘发育和生长过程中出现不同的NP细胞亚群（目的1），以及 建立这些亚群在损伤和退化过程中的命运和功能（目标2）。对于这两个目标， 使用最高的簇特异性基因，不同NP细胞亚群的存在和定位将被确定。 使用免疫荧光和qPCR确认。这项研究的结果将对以下方面产生重要影响 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异性标记可允许随后鉴定和富集 人椎间盘组织中的治疗性细胞群，并且定义分化轨迹可以允许 将这些细胞重编程用于治疗应用。

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.