Purpose: The synovium, a specialised connective tissue, encapsulates synovial joints providing a barrier between the joint space and surrounding tissues. The healthy synovium consists of two layers, the sub-lining, composed of fibroblasts and other cell types, and the synovial lining that contains a specialised tissue-resident fibroblast known as the fibroblast-like synoviocyte (FLS). The FLS and sub-lining fibroblasts play a critical role in joint health and osteoarthritis. In the healthy joint, FLS produce essential joint lubricants such as hyaluronic acid and lubricin. In contrast, in osteoarthritis, synovial fibroblasts exhibit enhanced expression of inflammatory cytokines, chemokines, matrix metalloproteinases and other catabolic enzymes leading to cartilage breakdown. In the adult knee, FLS and sub-lining fibroblasts are derived, in part, from embryonic Gdf5-expressing joint progenitor cells, a subset of Pdgfrα-expressing fibroblasts, present in the joint interzone. These cells give rise to synovial joints during development forming joint tissues such as the synovium, cartilage, menisci and ligaments. Despite their critical roles, understanding of synovial fibroblast ontogeny, phenotypic diversity, molecular regulation of fate determination and renewal from adult stem/progenitor cells is limited.
Methods: Synovial fibroblasts were isolated from adult transgenic Gdf5-Cre;Tom;Pdgfrα-H2BGFP mouse knees, either healthy or 6 days after injury to the articular cartilage. Cells were sorted, by fluorescence-activated cell sorting, based on their ontogeny, and processed for single-cell RNA-sequencing using the 10x Genomics Chromium controller. Transcriptomic analysis was performed using the Seurat R package. RNA velocity was analysed using scVelo to infer differentiation pathways, and gene regulatory networks (regulons) were analysed to determine molecular regulation of fate determination using SCENIC. Cell cycle analysis was performed computationally to identify proliferating synovial fibroblast populations and confirmed by Ki67 immunofluorescence staining.
Results: In healthy joints, sub-lining fibroblasts were found to be of mixed ontogeny with partly overlapping phenotypes, while FLS descended from the Gdf5-expressing cells of the embryonic joint interzone. After cartilage injury, we identified an actively cycling, facultative Gdf5-lineage stem cell population that supplied new chondrocytes, immunoregulatory fibroblasts and FLS, the latter via transit-amplifying progenitors. Furthermore, we detected the appearance of injury-induced FLS and chondrocyte sub-populations that are not of Gdf5-lineage, demonstrating lineage plasticity during repair. Finally, we reveal the molecular regulation of the synovial fibroblast phenotype, with both mouse and human FLS exhibiting Sox5 and Creb5 regulon activity regardless of ontogeny or injury.
Conclusions: Our findings elucidate the functional hierarchies and differentiation trajectories of ontogenetically defined stromal cell populations in the knee, from adult stem cells to FLS and immunoregulatory fibroblasts, and provide novel insight into the molecular regulation that governs cell fate in the adult joint. These data advance our knowledge of the cell populations that maintain and repair the synovial joint in adult life.
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