Single cell RNA sequencing reveals a shift in cell function and maturation of endogenous and infiltrating cell types in response to acute intervertebral disc injury

Abstract

Intervertebral disc (IVD) degeneration contributes to disabling back pain. Degeneration can be initiated by injury and progressively leads to irreversible cell loss and loss of IVD function. Attempts to restore IVD function through cell replacement therapies have had limited success due to knowledge gaps in critical cell populations and molecular crosstalk after injury. Here, we used single cell RNA sequencing to identify the transcriptional changes of endogenous and infiltrating IVD cell populations, as well as the potential of resident mesenchymal stem cells (MSCs) for tissue repair. Control and Injured (needle puncture) tail IVDs were extracted from 12 week old female C57BL/6 mice 7 days post injury and clustering analyses, gene ontology, and pseudotime trajectory analyses were used to determine transcriptomic divergences in the cells of the injured IVD, while immunofluorescence was utilized to determine mesenchymal stem cell (MSC) localization. Clustering analysis revealed 11 distinct cell populations that were IVD tissue specific, immune, or vascular cells. Differential gene expression analysis determined that Outer Annulus Fibrosus, Neutrophils, Saa2-High MSCs, Macrophages, and Krt18+Nucleus Pulposus (NP) cells were the major drivers of transcriptomic differences between Control and Injured cells. Gene ontology of DEGs suggested that the most upregulated biological pathways were angiogenesis and T cell related while wound healing and ECM regulation categories were downregulated. Pseudotime trajectory analyses revealed that cells were driven towards increased cell differentiation due to IVD injury in all IVD tissue clusters except for Krt18+NP which remained in a less mature cell state. Saa2-High and Grem1-High MSCs populations drifted towards more IVD differentiated cells profiles with injury and localized distinctly within the IVD. This study strengthens the understanding of heterogeneous IVD cell populations response to injury and identifies targetable MSC populations for future IVD repair studies.Lay SummaryThe intervertebral disc (IVD) is a spinal joint that accumulates damage with age but has limited tissue repair capabilities. IVD damage progresses into degeneration, and IVD degeneration is a leading cause of lower back pain. There are no effective therapies to treat IVD degeneration, but understanding the cell populations that change and respond to injury will uncover targets to restore IVD function. Mesenchymal stem cells (MSCs) are cells within the IVD that can potentially replenish the cells lost after IVD damage. To identify the cell populations of the IVD and how they change with injury, we performed single cell RNA sequencing of IVD tissue 7 days post injury and analyzed the differences in gene regulation. We identified diverse cells populations such as IVD specific tissues, immune cells, vascular cells, and MSCs. We discovered the presence ofSaa2andGrem1expressing MSCs that become less stem cell-like and express higher levels of IVD gene markers after injury. We also determined thatSaa2andGrem1have slightly different expression patterns in IVD tissues, and this expression becomes reduced after injury. These MSCs could be used in future stem cell therapies to prevent IVD degeneration.Abstract FigureGraphical AbstractIsolation of mouse coccygeal intervertebral discs for scRNASeq analyses.