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

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.

Cells within the intervertebral disc (IVD) play fundamental roles in tissue formation and homeostasis, as well as in the processes that lead to IVD degeneration and associated back pain. It is therefore vital to understand the cellular composition of the IVD and how cell phenotypes change during key stages of development, ageing and disease. We have previously generated phenotypic profiles of human foetal and adult IVD cells and identified sub-populations with phenotypic and functional differences. We now aim to build on these studies by utilising cutting-edge technologies to phenotype IVD cells at single cell resolution during key stages of development, skeletal maturation and degeneration to generate new insights into how the phenotype and function of sub-populations changes over time, how these changes may drive pathology and what strategies may be used to halt or reverse these changes. Specifically, we will focus on notochordal (NC) cells, which play vital roles in formation of the nucleus pulposus region of the IVD during development, and the loss of which are thought to lead to degeneration.
Hence the aims of the current proposal are to: (i) use single cell transcriptomic profiling on cells isolated from foetal spine, paediatric non-degenerate IVD and adult degenerate IVD to define (a) NC cell and sclerotomal cell populations within the developing IVD, (b) the phenotype and developmental origins of cell sub-populations which exist within the paediatric, non-degenerate IVD, and (c) how phenotype changes with degeneration; (ii) validate identified phenotypic marker profiles; (iii) isolate cell sub-populations to investigate responses to microenvironmental factors mimicking healthy and degenerate IVD; and (iv) conduct functional studies on the identified genes within the sub-populations of cells which express them. Together these findings will provide detailed information on phenotype and function of IVD cell sub-populations at key maturation and disease stages.