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

定义发育和疾病中椎间盘细胞的单细胞转录组景观，为新的治疗干预措施提供信息

• 批准号: MR/W019418/1
• 负责人: Stephen Richardson
• 金额: $ 74.03万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FW019418%2F1
• 关键词: Defining; single; cell; transcriptomic; landscape

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.

在胎儿发育和童年期间，将脊椎椎体分开的椎间盘（IVD）包含一种维持组织的独特细胞类型（称为脊索细胞）。但是，随着年龄的增长，这些细胞会丢失，这会导致盘状组织的改变和丢失（变性），并最终导致某些个体的背痛。到目前为止，尚不清楚这些“垂直细胞”是否消失，并被其他从相邻组织转移的细胞代替，或者它们是否变成成人IVD中发现的细胞。根据我们的开创性研究的最新证据，我们认为至少成人IVD中的某些细胞源于“垂直细胞”，但是由于我们目前不完全理解“脊索细胞”的生物学，因此很难确切地知道，因为他们在童年时期或后期在老化和变性期间会生长什么。该项目的目的是通过从IVD组织中收集细胞的各种年龄和变性得分来解决这种缺乏理解，并表征这些细胞表达的所有独特基因，以定义其独特的“签名”。我们可以在单个细胞水平上定义该签名，这意味着我们将能够检测到在重要阶段的发展，成熟和变性的细胞类型中是否存在差异。具体而言，在这项研究中，我们建议从以下原因获取IVD细胞：（i）胎儿人棘（选修终止后捐赠）； （ii）接受脊柱畸形手术的儿童，但IVD没有患病； （iii）正在接受背痛手术的IVD变性的成年人，然后在单细胞水平上定义其基因“签名”。然后，我们将详细比较这些基因“特征”，以帮助了解成年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，我们还将研究基因在IVD变性过程中的“特征”变化如何帮助我们了解IVD内部细胞种群的变化是否可能影响退化。确定了可用于区分IVD中不同细胞类型的独特基因，我们将在组织薄片中寻找它们在细胞中的表达。这将通过对同一细胞中多个基因的组织染色并使用显微镜查看IVD中的哪些细胞表达这些基因来完成。这将有助于我们确认成年IVD中的细胞起源于哪里。然后，我们将使用尖端技术来研究鉴定基因的功能，从而选择表达它们的细胞群体，并设计细胞以或多或少地表达每个基因的功能。当我们操纵这些细胞中的基因表达水平时，我们将在模仿健康和退化IVD的环境中培养细胞。这将有助于我们了解每个基因的重要性以及每个基因与IVD发展，成熟和变性的阶段的关系。我们将在此处免费生成的所有数据向国际社会免费使用。这将使我们自己和他人能够更详细地研究IVD中的细胞，了解它们在组织中的功能，细胞的变化如何导致IVD变性和背痛，并开发旨在将来再生IVD组织的新疗法。