Single-Cell RNA-Seq Reveals Transcriptomic Heterogeneity and Post-Traumatic Osteoarthritis-Associated Early Molecular Changes in Mouse Articular Chondrocytes

Abstract

Articular cartilage is a connective tissue lining the surfaces of synovial joints. When the cartilage severely wears down, it leads to osteoarthritis (OA), a debilitating disease that affects millions of people globally. The articular cartilage is composed of a dense extracellular matrix (ECM) with a sparse distribution of chondrocytes with varying morphology and potentially different functions. Elucidating the molecular and functional profiles of various chondrocyte subtypes and understanding the interplay between these chondrocyte subtypes and other cell types in the joint will greatly expand our understanding of joint biology and OA pathology. Although recent advances in high-throughput OMICS technologies have enabled molecular-level characterization of tissues and organs at an unprecedented resolution, thorough molecular profiling of articular chondrocytes has not yet been undertaken, which may be in part due to the technical difficulties in isolating chondrocytes from dense cartilage ECM. In this study, we profiled articular cartilage from healthy and injured mouse knee joints at a single-cell resolution and identified nine chondrocyte subtypes with distinct molecular profiles and injury-induced early molecular changes in these chondrocytes. We also compared mouse chondrocyte subpopulations to human chondrocytes and evaluated the extent of molecular similarity between mice and humans. This work expands our view of chondrocyte heterogeneity and rapid molecular changes in chondrocyte populations in response to joint trauma and highlights potential mechanisms that trigger cartilage degeneration.

Keywords: PTOA; cartilage; chondrocyte heterogeneity; gene expression; knee injury; osteoarthritis; scRNA-seq.

Figure 1

Single-cell analysis of 10-week-old BL6 mouse knee joints. (A) Graphical representation of the experimental workflow. Cartilage from mouse knee joints was dissected, dissociated into single cells, and subjected to immune and blood cell depletion. Viable cells from the remaining fraction were sequenced. (B) Cell clusters from scRNA-seq analysis visualized by Uniform Manifold Approximation and Projection (UMAP). Colors indicate clusters of various cell types. (C) Feature plot showing the expression of chondrocyte marker Acan. (D) Dot plot showing the expression of selected markers of various cell types. Dot size represents the % of cells expressing a specific marker, while the intensity of color indicates the average expression level for that gene, in that cluster. (E) Violin plot showing the expression of key transcription factors enriched in chondrocytes.

Figure 2

Identification of potential OA targets enriched in chondrocytes. (A) Heatmap showing potential OA targets enriched in chondrocyte clusters compared to other connective-tissue forming cell types in the joint. (B) Violin plot showing the expression of selected OA targets that are expressed in all chondrocyte subtypes and OA targets with a restricted expression pattern. (C) Dot plot showing the expression of selected markers of various chondrocyte clusters. Dot size represents the fraction of cells expressing a specific marker and color intensity indicates the average expression level in that cluster.

Figure 3

Characterization of chondrocyte subtypes. (A) Feature plots showing the expression of key chondrocyte markers. Blue: high expression, grey: low expression. (B) UMAP plots of various chondrocyte subtypes in mouse knee joints. Colors indicate clusters of various cell types with distinct gene expression profiles. (C) Heatmap showing the scaled expression of top genes differentially expressed in each cluster. (D) Violin plots showing the expression of selected markers of various chondrocyte subtypes. (E) Monocle pseudotime trajectory colored based on chondrocyte clusters in (A). (F) Expression of chondrocyte subtype markers on a pseudotime scale (colored based on clusters in (A)).

Figure 4

Functional characterization of chondrocyte subtypes. (A) Enriched ontologies for each chondrocyte subtype. Circle size indicates the number of genes associated with each category. (B) Circos plot showing overlap between genes enriched (>1.25-fold) in each cluster compared to all other clusters. The purple curves link identical genes. (C) Feature plots showing the expression of Matn3, Cytl1, Spp1, and Chil1. (D) Protein-level expression of Matn3 (red) and Cytl1 (green). (E) Protein-level expression of Chil1/CHI3L1 (red). (F) Protein-level expression of Spp1 (red). DAPI (blue); C: cartilage; B: bone; M: meniscus.

Figure 5

Comparison of human and mouse articular chondrocytes. (A) UMAP plots of various chondrocyte subtypes in human osteoarthritic knee joints. Colors indicate clusters of various cell types with distinct gene expression profiles. (B) Feature plots showing the expression of key chondrocyte markers in human chondrocytes. Blue: high expression, grey: low expression. (C) Violin plot showing the expression of Cytl1 and Col10a1 in human chondrocyte subtypes. (D) Dot plot showing the expression of selected markers of various clusters. Dot size represents the fraction of cells expressing a specific marker in a particular cluster and intensity of color indicates the average expression level in that cluster. (E) Feature plots showing the expression Jun and Fos in mouse chondrocytes.

Figure 6

Injury-induced changes in chondrocytes. (A) Histological assessment of uninjured and injured joints at 3 days (3DPI), 7 days (7DPI), and 6 weeks (6WPI) post-injury using Safranin-O and Fast Green staining. Severe cartilage degeneration was observed at 6WPI and minor proteoglycan loss was observed at 1WPI (yellow arrow). (B) UMAP plots of various chondrocyte subtypes identified in uninjured, 3DPI, and 7DPI joints. Colors indicate clusters of various cell types with distinct gene expression profiles. (C) UMAP plots of various chondrocyte subtypes identified in uninjured, 3DPI, and 7DPI joints. Colors indicate cells from each experimental group. (D) Feature plots showing the expression of chondrocyte markers Col2a1 and Acan; high expression (blue), low expression (grey). (E) Violin plot showing the expression of chondrocyte subtype markers. (F) Sequenced cells per chondrocyte cluster.

Figure 7

Injury-induced early molecular changes in articular chondrocytes. (A) Heatmaps showing key differentially expressed genes in each chondrocyte cluster. U: uninjured; 7D: 7 days post-injury. (B) Immunohistochemistry analysis showing protein-level expression of Cytl1 (20×, Mmp3 (40×), and Inhba (20×) in uninjured and 3DPI joints. B: bone; C: cartilage, M: meniscus.