Methods. To discriminate between Gdf5-lineage cells deriving from the embryonic joint interzone and other Pdgfrα-expressing fibroblasts and progenitors, adult Gdf5-Cre;Tom;Pdgfrα-H2BGFP mice were used and cartilage injury was induced to activate progenitors. Cells were isolated from knees, sorted fluorescence-activated cell-sorting based on developmental origin, and analysed by single-cell RNA32 sequencing. Flow cytometry and immunohistochemistry were used for validation. Clonal-lineage mapping was performed using Gdf5-Cre;Confetti mice.
Results. In steady state, Thy1+ sub-lining fibroblasts were of mixed ontogeny, while Thy1-Prg4+ lining fibroblasts predominantly derived from the embryonic joint interzone. The latter included Prg4- expressing progenitors distinct from molecularly defined FLS. Clonal-lineage tracing revealed compartmentalisation of Gdf5-lineage fibroblasts between lining and sub-lining. Following injury, lining hyperplasia resulted from proliferation and differentiation of Prg4-expressing progenitors, with additional recruitment of non-Gdf5-lineage cells, into FLS. Consistent with this, a second population of proliferating cells, enriched near blood vessels in the sub-lining, supplied activated multipotent cells predicted to give rise to Thy1+ fibroblasts, and to feed into the FLS differentiation trajectory. Transcriptional programmes regulating fibroblast differentiation trajectories were uncovered, identifying Sox5 and Foxo1 as key FLS transcription factors in mice and humans.
Conclusions. Our findings blueprint a cell atlas of mouse synovial fibroblasts and progenitors in healthy and injured knees, and provide novel insights into the cellular and molecular principles governing the organisation and maintenance of adult synovial joints.
- Synovial fibroblasts
- fibroblast-like synoviocytes
- progenitor cells
- single-cell RNA-sequencing
- cell atlas