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, 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 Gdf5-Cre;Confetti mice.
Results In steady state, Thy1+ sublining fibroblasts were of mixed ontogeny. In contrast, Thy1-Prg4+ lining fibroblasts predominantly derived from the embryonic joint interzone and included Prg4-expressing progenitors distinct from molecularly defined FLS. Clonal-lineage tracing revealed compartmentalisation of Gdf5-lineage fibroblasts between lining and sublining. 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 sublining, 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.
The authors thank all members of the Arthritis and Regenerative Medicine Laboratory at the University of Aberdeen, with special thanks to Alison Richmond, Iain Cunningham and Megan Robertson for technical assistance. The authors are also grateful to Animal Facility staff for care of our animals, the NHS Grampian Biorepository for facilitating the collection of human tissue samples, and staff in the Centre for Genome Enabled Biology and Medicine, the Microscopy and Histology Facility, and the Iain Fraser Cytometry Centre, for their expert support. Part of this work has been previously presented at OARSI 2022 World Congress: F.L. Collins, A.J. Roelofs, K. Kania, E. Campbell, E.S. Collie-Duguid, C. De Bari. Defining the Hierarchy of Fibroblasts and Their Stem Cells in the Adult Synovial Joint At Single Cell Resolution. Osteoarthr Cartil 2022;30:S40. doi:10.1016/j.joca.2022.02.041
This work was supported by funding from Versus Arthritis (grants 20775, 21156, 21800), Medical Research Council (grant MR/L020211/1), and Tenovus Scotland (grant G18.11).
© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.
Data Availability StatementData availability statement
Data are available in a public, open access repository. Data are available on reasonable request. Single-cell RNA sequencing data that support the findings of this study have been deposited in Gene Expression Omnibus (GEO) with the accession code GSE214500. All data relevant to the study are included in the article or uploaded as online supplemental information.
Additional supplemental material is published online only. To view, please visit the journal online (http://dx.doi.org/10.1136/ard-2021-221682).
- Synovial fibroblasts
- fibroblast-like synoviocytes
- progenitor cells
- single-cell RNA-sequencing
- cell atlas