Single-cell RNA-sequencing analysis reveals cellular states and intercellular communication of human lumbar facet-joint degeneration

Figure 1 Single-cell RNA sequencing analysis of human lumbar facet joint cartilage. A: Schematic workflow of the experimental strategy to isolate cells from human lumbar facet joint cartilage for subsequent single-cell RNA sequencing; B: Uniform Manifold Approximation and Projection showing the single-cell transcriptomic landscape of human lumbar facet joint cartilage. Fifteen clusters were visualized; C: Heatmap of the top marker genes of each cluster (defined in B); D: Distribution of cell clusters (defined in B) in human FJ1, 2 and 3 (degeneration grade I, II and III) cartilage samples. LFJ: Lumbar facet joint; DC: Dendritic cell; HSC: Hematopoietic stem cell; pDC: Plasmacytoid dendritic cell.

Figure 2 Identification of lumbar facet joint cartilage chondrocyte subpopulations and their marker-gene expression in different degeneration grades of human lumbar facet joint cartilage. A: Uniform Manifold Approximation and Projection plot showing the single-cell transcriptomic landscape of human lumbar facet joint chondrocytes. Eight subpopulations were identified; B: Heatmap of the top marker genes of each cluster (defined in A); C: Violin plot of representative genes of each chondrocyte population (defined in A); D: Immunohistochemistry staining of representative markers of the indicated cell clusters in different degenerated lumbar facet joint cartilage samples. Scale bar 100 μm. UMAP: Uniform Manifold Approximation and Projection; ProCs: Proliferative chondrocytes; PreHTCs: Prehypertrophic chondrocytes; HTCs: Hypertrophic chondrocytes; FCs: Fibrocartilage chondrocytes; ECs: Effector chondrocytes.

Figure 3 Distribution and trajectories of lumbar facet joint cartilage chondrocyte subpopulations during the degeneration process of human lumbar facet joint. A: Gene ontology functions of eight lumbar facet joint (LFJ) cartilage chondrocyte subpopulations; B: Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of eight LFJ cartilage chondrocyte subpopulations; C: Distribution of chondrocyte subclusters in mild (FJ1) and severe (FJ2-3) degenerated LFJs; D: RNA velocity-based pseudotime analysis inferred from Uniform Manifold Approximation and Projection embedding of LFJ chondrocyte subclusters. ProCs: Proliferative chondrocytes; PreHTCs: Prehypertrophic chondrocytes; HTCs: Hypertrophic chondrocytes; FCs: Fibrocartilage chondrocytes; ECs: Effector chondrocytes.

Figure 4 Identification of macrophage subpopulations and their marker-gene expression in different degeneration grades of the human lumbar facet joint. A: Uniform Manifold Approximation and Projection plot showing the single-cell transcriptomic landscape of macrophages. Five subclusters were identified. Numbers 1-5 represent Mφ-F13A1, Mφ-TCN2, Mφ-COL27A1, Mφ-CD52, and Mφ-IL3RA, respectively; B: Dotplot of top ten marker genes for five macrophage subclusters (defined in A); C: Violin plot of representative genes of macrophage subclusters (defined in A); D: Representative immunohistochemistry staining of CD68 of macrophage subclusters in different degenerated lumbar facet joint cartilage samples. Scale bar 100 μm; E: Gene enrichment for these subclusters (defined in A) in comparisons with the classical cell type M1 and M2 macrophage. Bubble size represents the proportion of upregulated and downregulated genes of subclusters based on the gene signature on the X axis. The color of the circle represents the directional false discovery rate. Red, enrichment of upregulated genes in the specific cell subsets; blue, depletion of downregulated genes.

Figure 5 Distribution and trajectories of macrophage subpopulations during the degeneration process of human lumbar facet joint. A: Gene ontology functions of macrophage subpopulations. Number 1-5 represents Mφ-F13A1, Mφ-TCN2, Mφ-COL27A1, Mφ-CD52 and Mφ-IL3RA respectively; B: Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of macrophage subpopulations; C: Distribution of macrophage subclusters in mild and severe degenerated lumbar facet joint cartilage samples; D: Representative immunohistochemistry staining of IL3RA of macrophage subclusters in different degenerated lumbar facet joint cartilage samples. Scale bar 100 μm; E: RNA velocity-based pseudotime analysis inferred from Uniform Manifold Approximation and Projection embedding of macrophage subclusters.

Figure 6 Intercellular crosstalk networks between chondrocytes and other cell clusters in the degeneration process of human lumbar facet joint. A: Intercellular crosstalk network of the interaction number between cell clusters in severely degenerated lumbar facet joints (LFJs) compared with mildly degenerated LFJs. Dots indicate cell clusters. The thickness of the directed line indicates the relative quantity of significant ligand-receptor pairs between any two pairs of cell clusters. Red represents the interaction amounts increased. Blue represents the interaction amounts decreased; B: Heatmap of the interaction number between cell clusters. The colored bar at the top and right represent the sum of receptor and ligand signals in each cell cluster, respectively; C: Intercellular crosstalk network of the interaction strength between cell clusters in severe degenerated LFJs comparing with mild degenerated LFJs. Red represents the interaction strength increased. Blue represents the interaction strength decreased; D: Heatmap of the interaction strength between cell clusters in severe degenerated LFJs comparing with mild degenerated LFJs.

Figure 7 Overview of the signaling pathways between cell clusters in the degeneration process of human lumbar facet joint. A: Signaling pathways between cell clusters in different degenerated grades of lumbar facet joints (LFJs); B: SPP1 signaling pathways in different degenerated LFJs; C: C-X-C motif chemokine ligand and LAMININ signaling pathways in different degenerated LFJs. CXCL: C-X-C motif chemokine ligand.