Generation of a senescent synovial organoid model using rat synovial fibroblasts treated with oxidative stress.
- 1Center for Stem Cell and Regenerative Medicine, Institute of Science Tokyo, 1-5-45 Yushima, Bunkyo-Ku, Tokyo, 113-8510, Japan.
- 2Center for Stem Cell and Regenerative Medicine, Institute of Science Tokyo, 1-5-45 Yushima, Bunkyo-Ku, Tokyo, 113-8510, Japan. [email protected].
Abstract
Background
Osteoarthritis (OA) is a common age-related joint disease characterized by cartilage loss and synovitis. Cellular senescence contributes to chronic inflammation through senescence-associated secretory phenotypes (SASPs). Recent evidence indicates that senescent synovial fibroblasts (SFs) contribute to OA pathogenesis. However, the in vitro modeling of the microenvironment formed by senescent SFs via SASPs remains limited. Here, we developed a three-dimensional (3D) senescent synovial organoid model using senescent rat SFs.Methods
We created the senescent SFs by treating normal rat SFs in monolayer culture with hydrogen peroxide (H₂O₂). Senescence induction was confirmed by senescence-associated beta-galactosidase (SA-β-gal) staining, γH2AX expression, and transcriptomic analysis. Synovial organoids were then generated by embedding normal or senescent SFs in Matrigel. Transcriptomic and protein-protein interaction analyses were performed and the expression of key regulatory factors was evaluated by immunostaining. Transcriptomic profiles of the organoids were further compared with bulk RNA sequencing data from rat OA synovium and single-cell RNA sequencing data from human OA synovium. Rat chondrocytes and RAW264.7 cells were co-cultured with control or senescent organoids.Results
H₂O₂ treatment significantly increased SA-β-gal positivity and the expression of γH2AX and senescence-associated genes in monolayer-cultured SFs. Senescent organoids had thickened lining layers and distinct transcriptomic profiles compared to normal organoids. Enrichment analyses identified the upregulation of genes associated with extracellular matrix (ECM) organization, growth factor responses, and PI3K-Akt/MAPK signaling. Senescent organoids had a unique secretory phenotype characterized by elevated expression of ECM components, ECM-degrading enzymes, and proinflammatory factors. The expression of senescence-associated genes was greater in senescent organoids than in control organoids or H₂O₂-treated monolayer SFs. Protein-protein interaction analysis of senescent organoids identified central hub genes, including Il6, Tgfb1, Mmp13, and Ngf, with upregulation also confirmed at the protein level. Comparison with transcriptomic data from rat and human OA synovium revealed transcriptional similarities between senescent organoids and OA SFs in both species. Co-culture assays showed that senescent organoids decreased Sox9 expression and increased Col10a1 expression in chondrocytes, whereas they increased Cd80 expression and decreased Cd206 expression in RAW264.7 cells.Conclusions
Senescent organoids can faithfully recapitulate key features of the senescent SF microenvironment, offering an ideal platform for cellular senescence-based OA research.
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BibTeX
@article{lee2026generation,
title = {{Generation of a senescent synovial organoid model using rat synovial fibroblasts treated with oxidative stress.}},
author = {Sunghyun Lee and Kentaro Endo and Ichiro Sekiya},
journal = {Inflammation and regeneration},
year = {2026},
month = aug,
doi = {10.1186/s41232-026-00436-2},
url = {https://europepmc.org/article/MED/42968193},
note = {CC BY},
}
RIS
TY - JOUR
TI - Generation of a senescent synovial organoid model using rat synovial fibroblasts treated with oxidative stress.
AU - Sunghyun Lee
AU - Kentaro Endo
AU - Ichiro Sekiya
PY - 2026
DA - 2026/08/05
JO - Inflammation and regeneration
DO - 10.1186/s41232-026-00436-2
UR - https://europepmc.org/article/MED/42968193
AB - Background Osteoarthritis (OA) is a common age-related joint disease characterized by cartilage loss and synovitis. Cellular senescence contributes to chronic inflammation through senescence-associated secretory phenotypes (SASPs). Recent evidence indicates that senescent synovial fibroblasts (SFs) contribute to OA pathogenesis. However, the in vitro modeling of the microenvironment formed by senescent SFs via SASPs remains limited. Here, we developed a three-dimensional (3D) senescent synovial organoid model using senescent rat SFs.Methods We created the senescent SFs by treating normal rat SFs in monolayer culture with hydrogen peroxide (H₂O₂). Senescence induction was confirmed by senescence-associated beta-galactosidase (SA-β-gal) staining, γH2AX expression, and transcriptomic analysis. Synovial organoids were then generated by embedding normal or senescent SFs in Matrigel. Transcriptomic and protein-protein interaction analyses were performed and the expression of key regulatory factors was evaluated by immunostaining. Transcriptomic profiles of the organoids were further compared with bulk RNA sequencing data from rat OA synovium and single-cell RNA sequencing data from human OA synovium. Rat chondrocytes and RAW264.7 cells were co-cultured with control or senescent organoids.Results H₂O₂ treatment significantly increased SA-β-gal positivity and the expression of γH2AX and senescence-associated genes in monolayer-cultured SFs. Senescent organoids had thickened lining layers and distinct transcriptomic profiles compared to normal organoids. Enrichment analyses identified the upregulation of genes associated with extracellular matrix (ECM) organization, growth factor responses, and PI3K-Akt/MAPK signaling. Senescent organoids had a unique secretory phenotype characterized by elevated expression of ECM components, ECM-degrading enzymes, and proinflammatory factors. The expression of senescence-associated genes was greater in senescent organoids than in control organoids or H₂O₂-treated monolayer SFs. Protein-protein interaction analysis of senescent organoids identified central hub genes, including Il6, Tgfb1, Mmp13, and Ngf, with upregulation also confirmed at the protein level. Comparison with transcriptomic data from rat and human OA synovium revealed transcriptional similarities between senescent organoids and OA SFs in both species. Co-culture assays showed that senescent organoids decreased Sox9 expression and increased Col10a1 expression in chondrocytes, whereas they increased Cd80 expression and decreased Cd206 expression in RAW264.7 cells.Conclusions Senescent organoids can faithfully recapitulate key features of the senescent SF microenvironment, offering an ideal platform for cellular senescence-based OA research.
SN - 1880-9693
SN - 1880-8190
ER -