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TGFβ activation and inhibition of Wnt and MAPK signaling pathways promote the specification of joint lineage cells in vitro and ex vivo

ABSTRACT: The developmental processes of synovial joint initiation and subsequent differentiation of progenitor cells towards anatomically and functionally distinct joint tissues are not well understood. Here we quantified the induction of cells expressing growth and differentiation factor 5 (Gdf5), an early marker of joint formation, and Prg4, a proteoglycan found in the superficial zone of articular cartilage and other joint tissues, utilizing a dual reporter mESC line and directed differentiation methodologies. We found TGF signaling to be necessary and sufficient for the induction of Gdf5-RFP and Prg4-GFP. Inhibition of either canonical Wnt signaling or MAPK signaling significantly increased the induction of Gdf5-RFP and chondrogenesis, while activation of either pathway prohibited this. Activation of MAPK significantly promoted the emergence of Prg4-GFP-expressing cells and elevated levels of tendon-ligament fibroblast genes such as Tnmd. We used single cell transcriptomics to determine the cellular composition of these cultures and determined that Gdf5+ cells are chondrogenic in both in vitro cultures and in mouse E12.5 and E14.5 limb bud cells. We validated the joint cell- promoting role of Wnt and MAPK inhibition in both systems. In summary, we used a novel ESC model to elucidate the roles of TGF, Wnt and MAPK signaling in the specification and differentiation of joint lineage cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE274060 | GEO | 2025/09/22

REPOSITORIES: GEO

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Project description:Objectives. Fibroblasts in synovium include fibroblast-like synoviocytes (FLS) in the lining and Thy1+ connective-tissue fibroblasts in the sub-lining. 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, 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+ 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 data 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.

Project description:Objective: Joint formation begins with the establishment of an interzone within the cartilaginous anlagen of the future skeleton and both Gdf5 and Erg are proposed as regulators of chondrocyte differentiation during and post interzone formation. The aim of this study was to examine the relationship between Gdf5 and Erg expression and downstream effects on chondrocyte gene expression. Design: Erg expression was identified in mouse knee joints at E13.5. Expression and microarray analyses were performed using micromass cultures of murine C3H10T1/2 mesenchymal cells undergoing induced chondrogenesis in the presence of absence of Gdf5 and Erg. Results: At E13.5, Erg expression was found to surround epiphyseal chondrocytes and span the interzone up to the intermediate zone. Erg splice forms were expressed in micromass cultures, and their expression profile was altered by the addition of recombinant Gdf5 depending on the stage of differentiation. Overexpression of Erg-010 resulted in a downregulation of Col2a1 and Col10a1. Microarray analysis following Erg-010 overexpression identified two potential downstream targets, Ube2b and Osr2, which were also differentially regulated by Gdf5. Conclusion: Erg regulation by Gdf5 in mesenchymal cells in vitro is dependent on the stage of chondrogenesis, and its expression in vivo demarcates chondrocytes that are not destined to be consumed by endochondral ossification. Functionally, Erg expression causes downregulation of Col2a1 and Col10a1 expression and this effect is potentially mediated by Osr2, which is a known regulator of chondrocyte differentiation. The identification of Ube2b as a putative downstream target of Erg-010 suggests that it may contribute to the regulation of the ubiquitination pathway and thereby BMP2 signaling, which is essential for normal knee joint development. RNA from overexpression experiments was extracted as described above. Total RNA quality was confirmed using a Bioanalyzer 2100 (Agilent). Labelled targets were generated from total RNA and hybridized to GeneChip Mouse Genome 430 2.0 arrays (Affymatrix) (Genomics Centre, University of Manchester). The raw fluorescence intensity values were normalized using a Robust Multi-array Average approach using dChip (V2005)

Project description:Background: Human pluripotent stem cells (hPSCs) give rise to chondrogenic activity in culture. Such activity was found in mesodermal and neural crest like progeny of hPSCs, and readily expanded as SOX9+ msenchymal cells in a medium containing FGF2. However, once expanded, the type of chondrocytes they perfer to form is growth plate-ike transient chondrocytes that are destined to form bone. However, we have recently developed a method to generate GDF5+ mesenchymal cells from such SOX9+ cells which ehibit activity to preferentially form articular-like permanent chondrocytes. The aim of the present prospective follow-up study was to reveal the cell-type and developmental origin of the GDF5+ cells as well as the SOX9+ cells. Methods: Paraxial mesoderm was generated from human PSCs, purified by FACS, and expanded to generate SOX9+ chondroprogenitors. Then, the SOX9+ cells were further differentiated into joint progenitor-like GDF5+ chondroprogenitors. These two chondroprogenitors were subjcted to bulk RNA-seq. Results: Based on the transcriptomic data, we examined differentially expressed genes, cell-type deconvolution analysis, and cell-type correlation analysis. The results suggested that GDF5+ cells might contain a teno/ligamento-genic potential (e.g., SCX+ cells), while SOX9+ cells were related to (neural crest-derived) ectomesenchymal chondroprogenitors. The followup biological analyses indicated as follows. The fast-growing SOX9+ cells formed cartilage that expressed chondrocyte hypertrophy markers and was readily mineralized after ectopic transplantation. In contrast, the slowly-growing GDF5+ cells were derived from SOX9+ cells. They formed cartilage that tended to express low to no chondrocyte hypertrophy markers, while expressing PRG4, a marker of embryonic articular chondrocytes, and to remain largely unmineralized in vivo. Conclusions: Our results imply that GDF5+ cells specified to generate permanent-like cartilage are likely to emerge in association with the commitment of SOX9+ cells to the tendon/ligament lineage.

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TGFβ activation and inhibition of Wnt and MAPK signaling pathways promote the specification of joint lineage cells in vitro and ex vivo

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