Defining the single cell transcriptomic landscape of intervertebral disc cells in development and disease to inform novel therapeutic interventions

在胎儿发育和儿童时期，间盘(IVD)在脊柱中分隔椎体，包含一种独特的细胞类型(称为脊索细胞)来维持组织。然而，随着年龄的增长，这些细胞会丢失，这会导致椎间盘组织的改变和丢失(退化)，最终导致一些人的背部疼痛。到目前为止，还不清楚这些“脊索细胞”是消失了，取而代之的是来自邻近组织的其他细胞，还是它们变成了在成人IVD中发现的细胞。根据我们的开创性研究的最新证据，我们认为成人IVD中至少有一些细胞来自‘脊索细胞’，但由于我们目前还不完全了解‘脊索细胞’的生物学，所以很难确切地知道当一个人在童年时期或后来在衰老和退化期间成长时，它们会发生什么。该项目旨在解决这种缺乏理解的问题，方法是从不同年龄和退变程度的IVD组织中收集细胞，并表征这些细胞表达的所有独特基因，以确定它们独特的“特征”。我们可以在单个细胞水平上定义这一特征，这意味着我们将能够检测在发育、成熟和退化的重要阶段存在的细胞类型是否存在差异。具体地说，在这项研究中，我们建议从：(I)胎儿人类脊柱(在选择性终止手术后捐赠)；(Ii)接受脊柱畸形手术但其IVD未患病的儿童；以及(Iii)正在接受背痛手术的IVD变性患者，然后在单细胞水平上定义他们的基因‘签名’。然后，我们将详细比较这些基因‘签名’，以帮助了解成人IVD中是否有来自‘脊索细胞’的细胞和/或是否有来自周围组织的细胞。重要的是，我们还将观察基因“签名”在IVD退变过程中的变化，以帮助我们了解IVD内细胞群体的变化或它们的功能是否会影响退变。在确定了可用于区分IVD内不同细胞类型的独特基因后，我们将在组织薄片中寻找它们在细胞中的表达。这将通过对组织中同一细胞中的多个基因进行染色，并使用显微镜来查看IVD内的哪些细胞表达这些基因。这将有助于我们确定成人IVD内的细胞起源于哪里。然后，我们将使用尖端技术来研究识别出的基因的功能，方法是选择表达这些基因的细胞群体，并对细胞进行改造，使其或多或少地表达每个基因。当我们操纵这些细胞中的基因表达水平时，我们将在一个模拟健康和退化的IVD的环境中培养这些细胞。这将帮助我们了解每个基因的重要性，以及每个基因与IVD发育、成熟和退化阶段的关系。我们将向国际社会免费提供在这里产生的所有数据。这将使我们和其他人能够更详细地研究IVD内的细胞，了解它们在组织中的功能，细胞的变化可能如何导致IVD退化和背痛，并开发旨在未来再生IVD组织的新疗法。

During foetal development and childhood the intervertebral disc (IVD), which separates the vertebral bodies in the spine, contains a unique cell type (called notochordal cells) that maintains the tissue. However, with ageing these cells are lost, which leads to alterations in and loss of disc tissue (degeneration) and ultimately back pain in some individuals. As yet, it remains unclear whether these 'notochordal cells' disappear and are replaced by other cells which move in from adjacent tissues, or whether they turn into the cells found in the adult IVD. Based on recent evidence from our seminal studies we believe that at least some of the cells in the adult IVD originate from 'notochordal cells', but as we don't currently fully understand the biology of 'notochordal cells' it is difficult to know definitively what happens to them as a person grows during childhood, or later during ageing and degeneration. This project aims to address this lack of understanding by collecting cells from IVD tissue across a wide range of ages and degeneration scores and characterising all the unique genes expressed by these cells to define their unique 'signature'. We can define this signature at a single cell level, meaning we will be able to detect whether there are differences in the types of cells present at important stages of development, maturation and degeneration. Specifically, in this study we propose to take IVD cells from: (i) foetal human spines (donated following elective terminations); (ii) children undergoing surgery for spinal deformities, but whose IVD are not diseased; and (iii) adults with IVD degeneration who are undergoing surgery for back pain, then define their gene 'signature' at the single cell level. We will then compare these gene 'signatures' in detail to help understand whether there are cells in the adult IVD that come from 'notochordal cells' and/or whether there are cells that come from the surrounding tissue. Importantly we will also look to see how the gene 'signatures' change during IVD degeneration to help us to understand whether a change in the populations of cells within the IVD, or their function, may influence degeneration. Having identified unique genes that can be used to distinguish different cell types within the IVD we will look for their expression in cells within thin slices of tissue. This will be done by staining the tissue for multiple genes in the same cell and using a microscope to see which cells within the IVD express these genes. This will help us to confirm where cells within the adult IVD originate from. We will then use cutting edge technology to investigate the function of the identified genes by selecting the cell populations that express them, and engineering the cells to express more or less of each gene. While we manipulate the gene expression levels in these cells, we will culture the cells in an environment which mimics the healthy and degenerate IVD. This will help us to understand the importance of each gene and how each gene relates to stages of IVD development, maturation and degeneration. We will make all the data generated here freely available to the international community. This will allow ourselves and others to study the cells within the IVD in more detail, to understand their functions within the tissue, how changes in the cells might cause IVD degeneration and back pain, and to develop new treatments aimed at regenerating IVD tissue in the future.