Stratification of nucleus pulposus cell sub-populations during intervertebral disc development, ageing and degeneration

在英国，背痛是导致残疾的主要原因，而椎间盘退变（IVD）已被确定为高比例患者的原因。由于目前治疗慢性背痛的方法长期疗效不佳，迫切需要先进的基于细胞的再生疗法。然而，开发这种疗法的一个重要步骤是详细了解天然细胞生物学，以及它如何随着发育、衰老和退化而变化。IVD的中央髓核（NP）区域是由胚胎脊索在胎儿发育过程中断裂形成的，而周围的韧带纤维环（AF）区域是由硬膜/中胚层形成的。出生时，NP由形态不同的大液泡脊索细胞（NCs）填充，但这些细胞在骨骼成熟过程中逐渐消失，最终无法检测到，取而代之的是一群较小的圆形细胞，即NP细胞。细胞群的这种变化与基质内退行性变化的开始相关，这表明NC细胞的丢失可能是退行性变化和最终背痛的原因。然而，目前尚不清楚成人IVD中是否存在nc或其他祖细胞，以及NP的细胞组成如何随着发育、衰老和变性而变化。阐明这些变化将允许鉴定合成代谢细胞亚群，这可能对IVD稳态至关重要，因此可能在再生治疗中具有潜力。该实验室最近对人类胎儿NCs和成人NP细胞进行了表型分析，发现了NC和NP群体的潜在标记，并有初步证据表明细胞亚群存在于成人IVD中。他们还发现了成年NP细胞在变性过程中表型的变化，包括细胞衰老和采用分解代谢表型的证据。本研究的总体目标是：(i)使用一组新的表型标记来鉴定和分离人类IVD在胎儿发育、青少年骨骼成熟和衰老/退化的不同阶段的细胞亚群；（ii）比较分离亚群体的表型和功能，以确定是否存在具有再生潜力的群体。这些目标将通过一系列最先进的技术来实现，包括CyTOF大规模细胞术、RNAseq转录组分析和体外功能人类细胞研究，以阐明人类NP细胞在发育、骨骼成熟、衰老和退化过程中的表型和行为。

Back pain is a leading cause of disability in the UK and intervertebral disc (IVD) degeneration has been identified as the cause in a high proportion of patients. As current therapies for chronic back pain demonstrate poor long-term efficacy, there is an urgent clinical need for advanced cell-based regenerative therapies. However, an essential step in development of such therapies is a detailed understanding of native cell biology and how this changes with development, ageing and degeneration. The central nucleus pulposus (NP) region of the IVD is formed from the embryonic notochord as it segments during foetal development, while the surrounding ligamentous annulus fibrosus (AF) region is formed from the sclerotome/mesoderm. At birth, the NP is populated by morphologically distinct, large vacuolated notochordal cells (NCs), but these cells gradually disappear during skeletal maturation eventually becoming undetectable and replaced by a population of smaller round cells - i.e. the NP cells. This change in cell population correlates with initiation of degenerate changes within the matrix suggesting that loss of NC cells may be responsible for degeneration and ultimately back pain. However, it is currently unclear whether there is persistence of NCs or other progenitor cells in the adult human IVD, or how the cellular composition of the NP changes with development, ageing and degeneration. Elucidation of such changes will allow identification of anabolic cell sub-populations which may be critical to IVD homeostasis and as such may have potential in regenerative therapies.The lab has recently undertaken phenotypic profiling of both human foetal NCs and adult NP cells, which have identified potential markers of NC and NP populations and have preliminary evidence that subpopulations of cells exist with the adult IVD. They have also identified changes in phenotype of adult NP cells during degeneration, including evidence of cellular senescence and adoption of a catabolic phenotype. The overall aims of this study are: (i) to use a panel of novel phenotypic markers to identify and isolate cell sub-populations from the human IVD during different stages of foetal development, adolescent skeletal maturation, and ageing/degeneration; and (ii) to compare the phenotype and function of isolated sub-populations to establish whether a population exists which has regenerative potential. These aims will be achieved through a range of state-of-the-art techniques, including CyTOF mass cytometry, RNAseq transcriptomic profiling and functional in vitro human cell studies to elucidate the phenotype and behaviour of human NP cells across development, skeletal maturation, ageing and degeneration.