INTRODUCTION: Harnessing essential developmental cues represents a promising strategy for reversing or treating Intervertebral disc degeneration (IVDD). The notochord of the developing embryo, serving both as a structural and signalling entity, is also the direct provider of notochordal cells that reside within the developing nucleus pulposus (NP). These notochordal cells (NCs) exert regenerative effects and hold promise for new therapeutic approaches1. As the disc matures, the notochordal cells are replaced by smaller nested NP cells (NPCs). The objective of this study was to obtain insights into gene regulatory networks orchestrating this cellular transition and identify approaches to reversing this, using the dog as a model.
METHODS: We characterized the different cell populations within the NP by transcriptomic analysis of single cells isolated from enzyme-digested tissues from healthy IVDs (stillborn and cadaveric healthy adult dogs from unrelated experiments), and degenerated NP tissue collected as waste material during standard-of-care surgery from dog patients with their owners’ consent. We used SORT-seq; a single-cell RNA sequencing (scRNA-seq) method based on flow cytometry sorting into barcoded plates2. To understand the lack of regenerative capacity as the disc ages, it is not only essential to know which genes are expressed, but also which genes are actively silenced. To address this, we utilized T-ChIC (combined sortChIC)3 and VASA-seq4.)to cluster cells on the histone H3 lysine 27 trimethyl (H3K27me3) modification and assess how chromatin states change during IVDD-associated cell fate decisions.
RESULTS: The scRNA-seq contained 2597 cells derived from discs of stillborn juvenile (n=3), healthy (n=3), and degenerate (n=3) donor spines. 505 healthy adult and 584 patient cells were processed for the H3K27me3 histone modification. UMAP-based clustering of the scRNA-seq identified 13 clusters, including 3 clusters of juvenile vacuolated NCs that mature into 4 clusters of mature vacuolated NCs. Trajectory analysis revealed that as the disc ages and degenerates, a specific subcluster of mature NCs in the adult healthy discs differentiates into the chondrogenic NPCs. IVDD was associated with fibrotic cells (IVDD-Fs). The classical IVD markers TBXT, KRT18, THY1, and CDH2 were dominantly expressed in the juvenile and mature vacuolated NCs. The chondrogenic NPCs formed a distinct cluster with increased expression of COL2A1 and ACAN, readily identifiable from the chondrogenic inner cells of the annulus fibrosis. Further, the IVDD-Fs formed a distinct population of fibrotic cells identifiable by the expression of CD4 and CD45, suggesting their haematopoietic origin. H3K27me3 profiling revealed distinct and cluster-dependent heterochromatin-repressed states suggesting distinct chromatin regulation during the cellular transitions. We confirm that H3K27me3 enrichment occurs at key genes that regulate NC cell phenotypes such as TBXT, consistent with the silencing observed with ageing and degeneration.
DISCUSSION: Using a multi-omics approach, combined with trajectory analysis, we model the denominators of the NC fate and cellular transition and highlight the essential instructions (essential transcription factors and histone modification). We demonstrate how a higher understanding of how cell states are maintained in health and disease can change the game of existing regenerative treatment strategies. (iPSpine: Horizon2020 (No.825925)