Profiling Human Chondrocytes and Synoviocytes Using Single Cell RNA Sequencing Identifies Cell Diversity in the Pathogenesis of Osteoarthritis in the Joint Organ

Purpose: The goal of our stduy was to use single-cell transcriptomic analysis to systematically identify the molecular census of cell types and cell states within osteoarthritic (OA) synovium and matched cartilage from human knee OA joints. We hypothesized that single cell sequencing would elucidate the inflammatory pathways and cell origin of inflammatory mediators involved in the pathophysiology of OA. Methods: We use droplet-based single-cell RNA sequencing (scRNAseq) (10x Genomics) to profile the transcriptomes of 3 OA patients undergoing knee arthroplasty surgery. A total of 14,613, 11,579 and 10,640 individual cells were profiled from intact cartilage, damaged cartilage and synovium, respectively. We utilized principal component analysis (PCA) and t-distributed statistical neighbor embedding (t-SNE) for dimensionality reduction, and K-nearest-neighbor (KNN) algorithm for cluster identification and visualization using the R package Seurat. Ingenuity pathways analysis was used to identify upstream regulators of the genes most representative (differentially expressed) of the four different classes of chondrocytes revealed by PCA; the tissue of origin of these upstream regulators was evaluated using Seurat software (Figure 1). Results: We identified 12 synovial cell clusters with distinct gene expression profiles in synovia consistent with intimal synovial fibroblasts (ISF), sub-intimal synovial fibroblasts (SSF), and activated macrophages (A-Mϕ). We also identified 4 distinct chondrocyte subpopulations from OA cartilage consistent with being representative of different classes of chondrocytes in OA, including homeostatic chondrocytes (HomC), extracellular matrix chondrocytes (EMC), hypertrophic chondrocytes (HTC) and fibrocartilage chondrocytes (FC). We predicted potential upstream regulators in chondrocytes during OA progression and revealed potential molecular networks between cartilage and synovium. Growth factors, such as TGFB1 and IGF1, were expressed by OA chondrocytes consistent with upregulation of anabolic processes to maintain cartilage homeostasis. However, several pro-inflammatory cytokines, including IL1A, IL1B, IL6 and TNF, were specifically highly expressed in A-Mϕ, but not expressed in chondrocytes and other immune cells in synovia. Conclusions: Our high resolution single cell expression data provide insights into the spectrum of cellular heterogeneity within human OA cartilage and synovium. Moreover, based on these data, many of the signals regulating chondrocyte transcription in OA originate in the synovium not the cartilage. These results could inform diagnostic and therapeutic options for human OA.