Generation of implant-type tissue-engineered cartilage from human embryonic stem cell-derived chondrocytes

Autologous cartilage transplantation has been applied clinically but its efficacy is limited by donor site morbidity, inter-donor variability, and high manufacturing costs. Primary chondrocytes also tend to dedifferentiate during expansion, which further limits their redifferentiation potential. Cartilage is weakly immunogenic and is thus an ideal tissue source for allogeneic transplantation. Allogeneic transplantation has drawn much interest due to the immune-privileged nature of cartilage and the recent success of allogeneic chondrocyte therapies. Meanwhile, human embryonic stem cells (hESCs) offer a scalable source of chondrocytes with high redifferentiation capacity. Building on the xeno-free automated chondrogenic differentiation of SEES2 hESCs in previous research, this study aims to establish an implant-type regenerative cartilage platform using hESCs for potential allogeneic transplantation.

hESCs (SEES2) were differentiated into cartilage tissue using an embryoid body-based protocol and cultured on collagen-coated dishes for 60 days. Cartilage-derived chondrocytes were isolated and expanded in a monolayer. Passage 3 (P3) cells were used for pellet culture, maintained for 21 days, and analyzed using histology and biochemical assays. Gene expression was assessed by RNA sequencing. Additionally, P3 chondrocytes were seeded onto bioresorbable PGA nonwoven fabric scaffolds, implanted subcutaneously into athymic nude mice for 4 weeks, and subjected to histological and biochemical analyses.

Human embryonic stem cell-derived chondrocytes (hESC-Chs) were successfully isolated and expanded over 10¹⁴-fold cumulatively. They expressed chondrogenic markers that were comparable to those of human auricular chondrocytes (hACs) and lacked pluripotency-associated genes. In pellet culture, hESC-Chs produced cartilage matrix, which was less robust than that produced by hACs. RNA sequencing showed overall similarity between groups, and extracellular matrix-related genes such as CHI3L1 and HAS3 were upregulated in hACs. In scaffold-based constructs, hESC-Chs exhibited inferior matrix production at low density (5.0 × 10⁷ cells/cm³) but showed improved glycosaminoglycan and type II collagen deposition at high density (2.0 × 10⁸ cells/cm³), reaching levels equivalent to those of hACs under high-density conditions. Notably, PD-L1 expression was elevated in hESC-Chs, indicating their potential immunomodulatory properties.

We successfully established a reproducible method for isolating and expanding chondrocytes from hESC-derived cartilage tissue. Under optimized culture conditions, these cells demonstrated the capacity for redifferentiation and in vivo cartilage regeneration, highlighting their potential as a stable and scalable allogeneic cell source for cartilage repair.