Enhancing the hyaline cartilage regenerative potential of induced pluripotent stem cell-derived chondrocyte cell sheets

Takahashi, T.; Uchiyama, R.; Toyoda, E.; Maehara, M.; Takahashi, Y.; Wasai, S.; Oomura, H.; Ogawa, M.; Yamashita, A.; Watanabe, M.; Tsumaki, N.; Sato, M.

doi:10.64898/2026.09.25.754531

bioRxiv · 10.64898/2026.09.25.754531

Enhancing the hyaline cartilage regenerative potential of induced pluripotent stem cell-derived chondrocyte cell sheets

Takahashi, T.·Uchiyama, R.·Toyoda, E.·Maehara, M.·Takahashi, Y.·Wasai, S.·Oomura, H.·Ogawa, M.·Yamashita, A.·Watanabe, M.·Tsumaki, N.·Sato, M.

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Abstract

Introduction Autologous and allogeneic chondrocyte cell sheet transplantation has shown safety and potential efficacy for cartilage defects associated with knee osteoarthritis. Although polydactyly-derived chondrocyte (PDC) cell sheets have been used clinically for allogeneic transplantation, a scalable and reliable alternative cell source is needed. This study aimed to establish a proof-of-concept protocol for generating chondrocyte cell sheets from clinical-grade human induced pluripotent stem cells (iPS cells). Methods Cartilaginous tissue was generated from human iPS cells, and isolated chondrocytes were used to fabricate iPS cell-derived chondrocyte sheets. In Phase 1, sheets were produced in high-serum medium under normoxic, hypoxic, or hypoxic/high-seeding-density conditions and compared with PDC sheets using a three-dimensional in vitro model and xenogeneic orthotopic transplantation in an immunodeficient rat model. In Phase 2, sheets were produced in low-serum medium under hypoxic/high-seeding-density conditions and evaluated for cartilage-related gene expression, secreted factors, cell surface marker expression, and in vivo cartilage repair. Results In Phase 1, hypoxic/high-seeding-density culture enhanced in vitro chondrogenic properties. However, after transplantation, iPS cell-derived chondrocyte sheets formed fibrotic repair tissue, whereas PDC sheets generated hyaline-like cartilage. In Phase 2, low-serum culture increased expression of cartilage-related genes, including collagen type II alpha 1 (COL2A1) and SRY-box transcription factor 9 (SOX9), enhanced secretion of transforming growth factor beta 1 and melanoma inhibitory activity, and upregulated CD9, CD26, CD56, CD201, CD227, and GD2. Low-serum iPS cell-derived chondrocyte sheets regenerated hyaline cartilage at 4 weeks, and the repair tissue was maintained at 12 weeks without bone formation. Conclusions A robust fabrication protocol enabled human iPS cell-derived chondrocyte sheets to regenerate hyaline-like cartilage in vivo. These findings support their potential as a scalable cell source for clinical cartilage repair.

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Takahashi, T., Uchiyama, R., Toyoda, E., Maehara, M., Takahashi, Y., Wasai, S., Oomura, H., Ogawa, M., Yamashita, A., Watanabe, M., Tsumaki, N., Sato, M.. 2026-09-28. Enhancing the hyaline cartilage regenerative potential of induced pluripotent stem cell-derived chondrocyte cell sheets. https://doi.org/10.64898/2026.09.25.754531

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