## 3 Results  
### Cell Populations Identified in IVD Tissues from 2 Weeks Post-Surgery  
Samples in control IVD tissues 2 weeks post-surgery. Unsupervised K-means clustering was used for the CON samples to group the cells into clusters with equal variance. Results show that five clusters separated all cells into discrete populations. There were identified four named clusters: intervertebral disc cells, endothelial cells, myeloid, and lymphoid cells (Figure 2a); cluster 5 was annotated as \"other cells\" due to the lack of common identification markers. Identification of cell populations within each cluster was based on known gene markers that were pre-selected as described in methods (Figure 2b). The identified clusters had a distribution of cell counts that was very similar across samples for the four CON samples derived from four separate rats (Figure 2c). Each sample showed a similar cell distribution profile where intervertebral disc cells contributed to most of the cluster (~87.7%), followed by endothelial cells (~7.3%), myeloid (~1.0%) and lymphoid cells (~0.2%). Figure 2: (**a**) UMAP plot representation of five cell types within CON group at two weeks after surgery. (**b**) Expression levels of marker genes within intervertebral disc cells, endothelial cells, myeloid, and lymphoid cells. (**c**) Pie chart showing the distribution of each cell analyzed for the four rat samples in the CON group. (**d**) The expression level of marker genes for three subclusters of intervertebral disc cells: nucleus pulposus, inner annulus fibrosus, and outer annulus fibrosus. The selected marker genes were chosen based on _a priori_ knowledge for identifying the discrete cell clusters (Figure 2b). There was some commonality in extracellular matrix genes across clusters, primarily for _Col2a1_ and _Compy_; this observation is consistent with the prevalent expression of these extracellular matrix proteins in different tissues and suggests that relying on non-extracellular matrix genes as differentiating markers may be preferable. Markers selected as lymphoid and myeloid markers proved to be effective in separating cells into clusters with more separation. As more than 85% of all cells mapped to the \"intervertebral disc cells\" cluster, we sought to sub-cluster this population to identify the known cell populations. The IVD cell cluster was partitioned into three distinctively adjacent subclusters that were classified as nucleus pulposous (NP), inner annulus fibrosus (IAF), and outer annulus fibrosus (OAF) based on relative expression levels of classical IVD cell markers (Figure 2d). Subcluster 1 was identified to be NP cells based on a relatively higher expression for _Acan_ and _Col2a1_ while also expressing lower _Col1a1_ compared to other IVD cells [8; 29; 30; 31; 32]. Additionally, these cells highly express _Cd24_, a classical marker for NP cells [13; 33; 34; 35], previously identified markers of the chondrocyte lineage including S100B, and a novel marker for bovine NP tissues, PDIA4 [19; 36; 37]. Cells mapping to the IAF cluster were similar in the pattern of expression to that of NP cells, differing with modestly increased _Col1a1_ expression and decreased presence of _Cd24_. Finally, OAF cells were identified based on the expression of a number of previously identified markers for this cell type including _lgfbp5_, _Igfbp6_, _Lum_, _Myoc_, and _Fibin_ (Figure 2d) [8; 19; 29; 31; 32; 38]. Samples in degenerated IVD tissues. Samples harvested from the intervertebral discs of the LDP rats at 2 weeks post-surgery underwent unsupervised K-means clustering as applied to the CON group. Five clusters provided for optimal separation of the LDP cells based on known gene markers (Figure 3a,b). The IVD cell cluster contributed to the greatest proportion of the cells (\(\sim\)82.4%), followed by endothelial (\(\sim\)6.4%), myeloid (1.4%), and lymphoid clusters (\(\sim\)8.2%). The myeloid and lymphoid clusters exhibited the most notable difference 2 weeks after LDP (Figure 3c).