Taxonomy of fibroblasts and progenitors in the synovial joint at single-cell resolution
Fraser L. Collins, Anke J. Roelofs, Rebecca A Symons, Karolina Kania, E. A. Campbell, Elaina Collie–Duguid, Anna H.K. Riemen, Susan M Clark, Cosimo De Bari
Abstract
<h3>Objectives</h3> Fibroblasts in synovium include fibroblast-like synoviocytes (FLS) in the lining and <i>Thy1</i>+ connective-tissue fibroblasts in the sublining. We aimed to investigate their developmental origin and relationship with adult progenitors. <h3>Methods</h3> To discriminate between <i>Gdf5</i>-lineage cells deriving from the embryonic joint interzone and other <i>Pdgfrα</i>-expressing fibroblasts and progenitors, adult <i>Gdf5-Cre;Tom;Pdgfrα-H2BGFP</i> mice were used and cartilage injury was induced to activate progenitors. Cells were isolated from knees, fibroblasts and progenitors were sorted by fluorescence-activated cell-sorting based on developmental origin, and analysed by single-cell RNA-sequencing. Flow cytometry and immunohistochemistry were used for validation. Clonal-lineage mapping was performed using <i>Gdf5-Cre;Confetti</i> mice. <h3>Results</h3> In steady state, <i>Thy1</i>+ sublining fibroblasts were of mixed ontogeny. In contrast, <i>Thy1-Prg4</i>+ lining fibroblasts predominantly derived from the embryonic joint interzone and included <i>Prg4</i>-expressing progenitors distinct from molecularly defined FLS. Clonal-lineage tracing revealed compartmentalisation of <i>Gdf5</i>-lineage fibroblasts between lining and sublining. Following injury, lining hyperplasia resulted from proliferation and differentiation of <i>Prg4</i>-expressing progenitors, with additional recruitment of non-<i>Gdf5</i>-lineage cells, into FLS. Consistent with this, a second population of proliferating cells, enriched near blood vessels in the sublining, supplied activated multipotent cells predicted to give rise to <i>Thy1</i>+ 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. <h3>Conclusions</h3> 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.