## 1 Introduction  
The intervertebral disc (IVD) provides for motion and flexibility in the spine [1]. Degeneration of the IVD is characterized by a loss of cellularity, changes in composition, and loss of hydration that is manifested as changes in disc height and MRI signal intensity. These features of IVD degeneration in the lumbar and cervical spine can contribute to mal-alignment of the adjoining vertebral bodies and affect the integrity and health of the IVD and adjacent neural structures, leaving the IVD vulnerable to progressive damage during the loading conditions of daily living [2; 3; 4]. The cell-mediated changes affecting IVD growth, extracellular matrix synthesis, and metabolism can affect IVD proteolytic degradation, and are of great interest for identifying the relevant cellular and molecular targets to support IVD repair and regeneration. The cell density of the nucleus pulposus (NP) region is at the lowest in the IVD, and are the principal contributors to the loss of cellularity with age. NP cells have long been considered to share features with chondrocytes in the adult IVD, due to their roundedness and high expression of multiple chondrocyte markers including SOX9, type II collagen,and aggrecan upon bulk mRNA transcriptional profiling [5; 6; 7; 8; 9]. NP cells are derived from the embryonic notochord and express transcriptionally unique markers that reflect their notochord origins, including CD24, cytokeratins, and the brachyury and FOX transcription factors [10; 11; 12; 13]. Cells of the annulus fibrosus (AF) are derived from mesenchyme and express some overlapping and some differing molecular markers from adjacent NP cells. Indeed, there have been numerous studies designed to identify the unique cellular phenotypes of NP and AF cells, as well as non-resident cells involved in degeneration and repair, using tools of bulk RNA transcription or RNA-sequencing, proteomic profiling, flow cytometry, and protein assays [5; 14; 15; 16; 17; 18]. The improved understanding of cell-specific molecular changes is crucial for identifying important protein targets that drive progress degeneration and molecular targets for stem cell-mediated IVD repair. In recent years, single-cell RNA sequencing (scRNA-seq) of IVD cells, both from anatomically distinct regions and for comparisons with pathology, has been used to characterize the phenotype and abundance of distinct cell populations in the IVD in human and animal tissue sources [5; 15; 17; 19; 20; 21]. When cells are isolated rapidly and immediately prepared for RNA-sequencing, this technique has the potential to reveal the endogenous RNA expression of resident cells and thus identify differences in cell sub-populations in the native IVD [5; 15; 17; 19; 20; 21; 22]. Results of sc-RNA-seq from rat and bovine IVD tissues, and from non-degenerate human IVD tissues, rely on mapping cells with similar transcriptomic profiles into \"clusters\" in order to name and number discrete cell populations in the non-degenerate IVD. While the number of clusters is highly variable in the absence of a consensus-based approach, it is most commonly observed that the majority of IVD cells map to a few clusters based on their expression of common genes (50-99% of isolated cells). Studies have used this approach to identify small but meaningful stem cell or progenitor cell populations in the IVD [15; 20], or to identify relationships between identified clusters and primary cell functions of matrix regulation, stress responses, cell cycle, and more [22]. Only a few studies have used sc-RNA-seq to better understand the progression of IVD degeneration at the cellular level.