Taxonomy of fibroblasts and progenitors in the synovial joint at single-cell resolution

Objectives Fibroblasts in synovium include fibroblast-like synoviocytes (FLS) in the lining and Thy1+ connective-tissue fibroblasts in the sublining. We aimed to investigate their developmental origin and relationship with adult progenitors. 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.


INTRODUCTION
The synovium consists of two layers, lining and sublining. The sublining is composed of Thy1+ fibroblasts, immune cells, blood vessels and nerves in a meshwork of extracellular matrix (ECM). The lining consists of type A macrophage-like synoviocytes and type B fibroblast-like synoviocytes (FLS). The FLS are specialised fibroblasts, negative for Thy1, which are unique to the synovium and critical for the maintenance of joint homeostasis through secretion of lubricating factors including lubricin (encoded by Prg4) and hyaluronic acid. 1 Here, we will use the term synovial fibroblasts to collectively refer to Thy1-Prg4+ FLS in the lining and Thy1+ fibroblasts in the sublining.
Synovial fibroblasts express platelet-derived growth factor receptor α (Pdgfrα), 2 a pan-fibroblast marker 3 also expressed by skeletal progenitors. 4 They are ontogenetically heterogeneous and derive in part from the growth differentiation factor 5 (Gdf5)-expressing cells of the embryonic joint interzone. 2 The joint-interzone cells give rise to joint tissues during development, including articular cartilage and synovium. [5][6][7] Tracing of Gdf5expressing cell progeny into adulthood, using Gdf5 regulatory sequence to control Cre expression that is active in the embryonic knee joint interzone but not in healthy, injured or osteoarthritic adult knees, [8][9][10] revealed that the Gdf5-lineage cells in adult mouse knees proliferate following cartilage injury and WHAT IS ALREADY KNOWN ABOUT THIS TOPIC ⇒ Synovial fibroblasts, consisting of lining fibroblast-like synoviocytes (FLS) and sublining connective tissue fibroblasts, play a critical role in joint health and arthritis pathology. However, their phenotypic diversity, developmental origin and relationship with adult progenitors is incompletely understood.

WHAT THIS STUDY ADDS
⇒ This study reveals the relationship between ontogeny and phenotypic diversity of synovial fibroblasts, and shows at single-cell level the cellular and molecular pathways involved in the response to injury. Findings also identify Prg4-expressing FLS progenitors in the lining and facultative progenitors in sublining that are activated by cartilage injury and give rise to FLS and Thy1+ sublining fibroblasts.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
⇒ This study provides novel insight into the hierarchical pathways and molecular regulation that govern synovial fibroblast cell fate in the adult joint.
repair cartilage. 2 More recently, we identified within the adult Gdf5-lineage cell population two progenitor cell subsets, Prg4expressing cells in synovial lining and Sox9-expressing cells in periosteum, which cooperate to form osteophytes during osteoarthritis. 11 FLS express Prg4, and whether the progenitor activity of Prg4+ cells in the lining reflects FLS plasticity or true progenitor cells exist that are distinct from FLS, remains to be determined. Furthermore, it is not known whether a common adult stem/progenitor cell lineage or distinct pools of progenitors supply the different subsets of synovial fibroblasts.
Here, we used transgenic mice allowing the separation of ontogenetically distinct Gdf5-lineage mesenchymal stromal cells from other Pdgfrα-expressing fibroblasts and progenitors, we analysed at the single-cell level the transcriptome of these lineages in healthy and injured adult knees to construct a stromal cell atlas of the joint and elucidate the relationships between fibroblasts and progenitors in synovium.

METHODS
Materials and methods are available inonline supplemental materials and tables 1-4

Developmental origin and taxonomy of adult synovial fibroblasts in steady state.
To investigate the developmental origin of adult synovial fibroblasts, we used Gdf5-Cre;Tom;Pdgfrα-H2BGFP mice to trace cells from the Gdf5-expressing embryonic joint interzone based on tdTomato (Tom) expression and to identify fibroblasts and progenitors based on Pdgfrα-promoter-driven green fluorescent protein (GFP) expression (figure 1A). Cells isolated from adult mouse knees were sorted by FACS into Tom+ Gdf5-lineage cells, which coexpressed GFP, and Tom-GFP+ cells, and analysed independently by scRNA-seq, to ensure high purity (figure 1B; online supplemental figures 1 and 2). Unsupervised clustering of integrated datasets (figure 1C) and analysis of differentially expressed genes (DEGs) (figure 1D; online supplemental figure 3; table 5) identified FLS, osteoblast-lineage cells, chondrocytelineage cells, tenocyte-lineage cells, and 6 fibroblast clusters (F1-F6) expressing the synovial sublining fibroblast markers Thy1 and Cd34 (figure 1E, F). 12 13 Gene Ontology (GO) analysis of significant cluster genes indicated functional diversity between the Thy1+ fibroblast clusters (figure 1G). The two ontogenetic lineages made variable contributions to the different fibroblast clusters, and within each cluster, Tom+ and Tom-GFP+ cells were highly transcriptomically similar (online supplemental figure 4). Strikingly, FLS were only detected in the Tom+ population, deriving from the embryonic joint interzone (figure 1C).
To identify putative developmental relationships among cell clusters, we performed unsupervised Slingshot lineage inference. 14 This predicted, for both ontogenetic cell lineages, trajectories that emerged from the F4 fibroblast cluster towards the specialised cells of the skeletal joint ( figure 1H). The transcriptome of the F4 cluster was characterised by Pi16 and Cd55 expression (figure 1I), and correlated with the transcriptome of a population of Pi16+ fibroblasts recently identified across multiple tissues that has been postulated to represent a reservoir of non-specialised, universal fibroblasts that can develop into specialised, tissue-specific fibroblasts (figure 1J). 15 Collectively, these data reveal that the adult joint contains functionally distinct fibroblast subsets of heterogeneous developmental origin, with each ontogenetic lineage comprising a universal fibroblast population predicted to give rise to specialised cells.

Identification of FLS and progenitors in synovial lining
Analysis of the scRNA-seq data showed that the FLS cluster only included Tom+ cells deriving from the embryonic joint interzone (figure 1C). We sought to confirm this in a larger cohort of mice and other synovial joints. Tom+ cells were present in synovial lining in all joints analysed (online supplemental figure 5). Flow cytometry confirmed that Thy1-Itga6+ FLS were enriched in the Tom+GFP+ population, while Thy1+Cd55+ universal fibroblasts were similarly or less abundant in the Tom+GFP+ compared with the Tom-GFP+ population (figure 2A; online supplemental figure 6). Furthermore, immunofluorescence staining on tissue sections showed that the vast majority of FLS, identified by Clic5 expression, expressed Tom (figure 2B; online supplemental figure 7). These findings show that, consistent with the scRNA-seq data, the FLS in the adult synovial joints predominantly derive from the embryonic joint interzone.
To define the spatial patterns of adult synovial fibroblasts by their derivation from individual embryonic joint interzone cells, we carried out clonal-lineage mapping using Gdf5-Cre mice crossed with Confetti multi-colour reporter mice. 16 This revealed clonal fibroblast clusters in synovium to be typically aligned longitudinally, along the proximal-distal axis, parallel to the lining ( figure 2C). This indicates that parallel clonal cell stacking underpins synovial tissue architecture and suggests that lining fibroblasts are a self-maintaining cell population throughout life.
Interestingly, we observed Tom+ cells in synovial lining that were negative for the FLS marker Clic5 (figure 2B), raising the possibility of the existence of distinct progenitors within the Gdf5-lineage synovial lining fibroblast population. Consistent with this notion, we identified in the scRNA-seq data a Tom+ subcluster of Thy1-Prg4+ cells, which were distinct from mature FLS (defined by expression of Cd44, Cd55, Hbegf, Has1, Tspan15, Itga6 and Clic5) 17 and superficial zone chondrocytes 18-21 (figure 2D and E; online supplemental figure 8, table 6). We additionally identified growth plate chondrocytes and vascular smooth muscle cells (figure 2D; online supplemental figure 8, table 6), and a putative progenitor subset within the osteoblast-lineage cluster (online supplemental figure 9). These findings further define the mouse synovial joint stromal cell atlas and identify Prg4-expressing synovial lining cells distinct from FLS.

Activation of synovial fibroblasts following joint injury
To study fibroblast activation, we used a mouse model whereby injury to articular cartilage triggers a healing response characterised by fibroblast proliferation that underpins synovial hyperplasia and chondrogenesis to repair cartilage. 2 We analysed cells 6 days after cartilage injury, a time when synovial hyperplasia peaks (figure 3A), 2 and integrated the data with steady-state data (figure 3B; online supplemental figure 10). Clusters identified by unsupervised clustering were annotated by analysing the top DEGs for each cluster and mapping cells from each steady-state cluster onto the integrated UMAP  similar to each other and to the steady-state Tom+ FLS (online supplemental figure 15). Transcriptomic comparisons between steady-state and injured-state cells within the fibroblast clusters revealed upregulation of genes involved in ECM remodelling and fibroblast migration, such as Cthrc1, Postn, Timp1, Bgn, Lum, Sparc, Lox and various collagens (online supplemental figure 16). 13 22-24 Four injury-induced fibroblast (IF) clusters were identified with no steady-state analogous cluster (figure 3B-D; online supplemental figures 11-13), which were characterised by activity of regulons associated with cell proliferation and activation (figure 3F; online supplemental tables [8][9][10][11][12]. Individual cells in these clusters coexpressed Sox9 (chondrocyte-lineage), Runx2 (osteoblast-lineage) and Scx (tenocyte-lineage) transcription factors (figure 3G; online supplemental figure 17A, B), suggestive of multilineage differentiation potential. In addition, the cluster analogous to the osteoblast-lineage cluster in steady state displayed an osteochondral phenotype after injury (online supplemental figure 17C, D), similar to the hybrid skeletal cells that form the early osteophyte in osteoarthritis. 11 Immunostaining confirmed upregulation of Sox9 and Runx2 expression after injury, especially at the joint margin where synovium and periosteum merge and chondrophyte formation is typically observed ( figure 3H).
Altogether, these data indicate that injury triggers expansion of the Prg4+ progenitor and FLS populations, which in part involves recruitment of cells that do not derive from the Gdf5expressing joint interzone, and induces activation of fibroblasts expressing genes indicative of multi-lineage differentiation potency.

Context-dependent activated fibroblast phenotypes
Next, we sought to determine the specificity of the synovial fibroblast response to cartilage injury. Recently, Buechler et al analysed single-cell transcriptomic data of fibroblasts from multiple injured or diseased mouse tissues and identified three perturbed-state fibroblast (PF) populations. 15 Two of these showed transcriptomic similarity to the injury-induced clusters in our study (online supplemental figure 18).
We then focused on a comparison with fibroblasts from joints of mice with serum transfer-induced inflammatory arthritis (STIA), by integrating our injured-state dataset with the STIA dataset published by Croft et al. 25 Unsupervised clustering revealed five perturbedstate fibroblast clusters (PF1-5) (figure 4A-C; online supplemental figures 19,20), which showed expression of lineage-specifying transcription factors (Sox9, Runx2 and Scx) in both models ( figure 4D). Strikingly, few FLS were present in STIA, while Thy1-Prg4+ cells

Animal models
extended from the progenitor cluster into the perturbed-state clusters (figure 4A-C).
PF clusters included one proliferating cluster (PF1), and four clusters predominant in either the injured state (PF2 and PF3) or STIA (PF4 and PF5) (figure 4A-C), the latter characterised by Cxcl5 expression (figure 4B). Cells in STIA-dominant PF clusters showed a more inflammatory and catabolic transcriptome compared with cells in injury-dominant PF clusters, and this remained true when injuredstate and STIA cells were analysed separately (figure 4E).
These findings indicate that different perturbed-state synovial fibroblast phenotypes exist, in varying proportions, both after injury and during immune-mediated inflammation, and suggest that adoption of multilineage potency by fibroblasts is a generic response to a perturbed state.

Identification and molecular regulation of progenitor cell differentiation trajectories
We next analysed differentiation trajectories using RNA velocity and lineage reconstruction 14 26 27 (figure 5A) and by pseudotemporally ordering the cells based on changes in gene expression using Monocle 3 27 ( figure 5B). This revealed inferred differentiation trajectories originating from cells in injury-induced fibroblast clusters IF1 and IF2, with a branchpoint towards either Thy1-Prg4+ lining fibroblasts (P and FLS) or Thy1+ sublining fibroblasts (F1-F6 and tenocytes) ( figure 5A and B). Cell cycle analysis revealed two clusters of cycling cells after injury supplying new cells feeding into the differentiation trajectories, one in IF1 and IF2 clusters, and one in the Thy1-Prg4+ progenitor cluster extending into the FLS cluster (figure 5C; online supplemental figure 21).
To confirm these findings in situ, we costained for GFP and the proliferation marker Ki67, together with the endothelial marker Cd31. Proliferating fibroblasts were detected in lining, and in sublining enriched near blood vessels (figure 5D, online supplemental figure 22A). To further determine the identity of proliferating fibroblasts in the lining, we costained for Tom and Ki67, together with the FLS marker Clic5. We observed Tom+Ki67+ proliferating cells in the lining located immediately adjacent to Clic5+ FLS (figure 5E, online supplemental figure 22B), and occasional Tom+Ki67+Clic5+ FLS (online supplemental figure 22B), supporting the notion that the Thy1-Prg4+ progenitors identified by scRNA-seq (P cluster) are located in the lining where they proliferate and give rise to new FLS after injury. Clonal-lineage tracing using the Gdf5-Cre;Confetti model indicated clonal expansion along the medial-lateral axis (figure 5F), although clones typically remained locally confined to either lining or sublining compartments . Altogether, these data indicate that synovial lining hyperplasia after injury in large part results from proliferation of Gdf5-lineage FLS progenitors in the lining, with additional

recruitment from proliferating cells in injury-induced clusters into the FLS trajectory.
To gain insight into the molecular regulation of synovial fibroblast differentiation, we identified transcription factors that significantly changed in expression across pseudotime ( figure 6A). SCENIC analysis revealed regulon activity associated with these transcription factors along their respective differentiation trajectories, suggesting they are key to driving this process For clinical relevance, we extended our analysis to published data from knee synovial tissues of osteoarthritis patients, 13 28 29 which similarly showed that THY1-PRG4+CLIC5+ FLS exhibited SOX5, FOXO1 and CREB5 regulon activity (figure 6F, online supplemental figure 25). Immunohistochemistry confirmed SOX5 and FOXO1 expression in both quiescent and hyperplastic lining of human synovium (figure 6G, online supplemental figure 24B). These data indicate that molecular regulation of the FLS phenotype is conserved across species and states.

DISCUSSION
Comprehensive cell atlases from diseased joints with inflammatory or degenerative arthritis have documented heterogeneity of synovial fibroblasts, identifying perturbed-state subsets. 13 15 25 30

Animal models
We previously reported that in the adult knee synovium, the Gdf5-lineage cell population contains fibroblasts that become pathogenic in inflammatory arthritis 31 and progenitors that form cartilage after injury. 2 11 However, little was known about the fibroblasts in healthy joints, and it remained to be determined whether progenitors and fibroblasts are distinct cells, or plastic fibroblasts adopt progenitor activity. In this study, single-cell transcriptomic analysis of ontogenetically distinct stromal cell lineages from steady-state mouse knee joints led to the identification of FLS and distinct Prg4-expressing progenitors in the lining, both largely deriving from the embryonic joint interzone. Joint surface injury, employed to study repair mechanisms, 2 triggered proliferation of progenitors in the lining, and additional cells located near blood vessels in sublining predicted to supply specialised fibroblasts.
Traditionally, Prg4-expressing synovial lining fibroblasts are considered to be specialised FLS that maintain joint homeostasis through secretion of lubricating factors. 1 Here, we disentangle the identity of the lining fibroblasts and show that they comprise two distinct cell subsets, FLS and progenitors postulated to replenish FLS lost to physiological turnover. The observation that in synovium, clonal fibroblasts are arranged longitudinally parallel to the lining, supports this notion. We also show that the synovial lining hyperplasia following joint surface injury 2 32 is largely underpinned by an expansion of FLS driven by proliferating Prg4-expressing progenitors. A previous study tracing the progeny of cells expressing Prg4 showed their proliferation and expansion in synovium after cartilage injury. 32 Our data identify a population of Prg4-expressing progenitors in synovial lining that are distinct from FLS and respond to injury with proliferation to supply new FLS.
The FLS population is further expanded after injury by differentiation of cells that do not derive from the Gdf5lineage population. Although the non-Gdf5 lineage FLS are transcriptomically highly similar to their Gdf5-lineage counterparts, to which extent they are functionally equivalent remains to be determined. Similarly, while Gdf5-lineage cells are the main progenitors that form articular cartilage during development, repair cartilage after injury in adulthood, 2 and form osteophytes in osteoarthritis, 11 other cells can give rise to new chondrocytes, especially ectopically in synovium after injury. 2 Thus, while the Gdf5-lineage cells are the natural progenitors for FLS and articular chondrocytes, under conditions of stress, other cells in the joint supply FLS and chondrocytes in a compensatory mechanism.
The quiescent cells from which the injury-induced cells with multipotent phenotype originate remain to be determined. A recent study identified a population of fibroblasts that reside near blood vessels in many tissues, marked by expression of Pi16. These cells were postulated to be unspecialised reservoir cells giving rise to tissue-specific specialised fibroblasts. 15 We identified a transcriptomically similar Pi16+ fibroblast cluster in the adult mouse knee, which was predicted to give rise to specialised cells of the steady-state skeletal joint. After injury, proliferating cells feeding into differentiation trajectories were found to be enriched in a sublining perivascular niche, and we previously showed these perivascular cells to be distinct from pericytes. 33 While these cells could be progeny of the Pi16+ fibroblasts, we speculate that quiescent fibroblasts in the joint, under the stress resulting from damage, would be opportunistically recruited to function as facultative progenitors, showing a plasticity that has been reported in other tissues. 34 A comparative analysis of synovial fibroblasts in joint surface injury and various perturbed states revealed an overall similar fibroblast response. Fibroblasts with an inflammatory phenotype were detected in our injury model, although at a much lower prevalence compared with the STIA mouse model of inflammatory arthritis. Inflammation plays a crucial role in repair. 35 Likewise, activated cells with a multipotent phenotype were detected in both injury and STIA models. These data suggest that inflammatory and multipotent fibroblast transcriptional states reflect a generic response to insult, although their prevalence and level of activation would be context-dependent and likely to determine structural outcome. It was interesting to observe that while few FLS were present in the STIA dataset, Prg4-expressing progenitors were abundant and extended into the PF clusters. This suggests that under inflammatory conditions such as rheumatoid arthritis, Prg4-expressing progenitors are shifted towards pathogenic fibroblasts.
Our data define the molecular identity of FLS distinct from Prg4+ progenitors, and reveal the transcriptional programmes underpinning synovial fibroblast differentiation. Notably, both mouse and human FLS are characterised by Sox5, Foxo1 and Creb5 regulon activity. This suggests that the identified gene regulatory networks are crucial for the FLS phenotype and that their disruption could result in dysregulation of FLS formation or function. The transcription factors Foxo1 and Sox5 have been linked to skeletal cell survival and fate. 36 37 Creb5 was shown to be required for the induction of Prg4 expression in articular chondrocytes. 19 Since the synovial lining shares many properties with the superficial zone of the articular cartilage, including production of lubricin (encoded by Prg4), it is likely that the Creb5 regulon has similar functions in FLS and superficial zone chondrocytes.
In summary, our analysis at single-cell resolution of stromal cells isolated from steady-state and injured mouse knees provides novel insights into the ontogeny and taxonomy of fibroblast and progenitor populations in synovium and defines differentiation trajectories and their molecular regulation. This study critically advances our knowledge of the cell populations that maintain the synovial joint in adult life.
Provenance and peer review Not commissioned; externally peer reviewed.
Data 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.
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