Differentiation of Human Induced Pluripotent Stem Cells Toward Implantable Chondroprogenitor Cells

Background. Post-traumatic chondral and osteochondral lesions can be treated with autologous chondrocyte implantation (ACI), but the high cost of autologous cell expansion under strict Good Manufacturing Practice (GMP) regulations limits patient access. Stem cell-based advanced therapy medicinal products (ATMPs) offer more cost-effective alternatives, with human induced pluripotent stem cells (iPSC) showing great promise due to their expandability, low immunogenicity, commercialization potential, and fewer ethical concerns. Aim. To develop a protocol to direct iPSC through a mesenchymal stage into chondroprogenitors (iCHOp), resembling autologous chondroprogenitor cells used in ACI. Methods. The derived chondroprogenitor cells were expanded in monolayer and in 3-dimensional (3D) cultures and subsequently analyzed using transcriptomic profiling via RNA sequencing and reverse transcription quantitative polymerase chain reaction and compared with ACI chondrocytes. Results. Transcriptomic profiling confirmed successful differentiation, with iCHOp showing 83% similarity to ACI chondrocytes. Further 3D culture maturation led to upregulation of chondrogenesis-related genes and activation of cartilage-specific pathways. Histological analysis confirmed extracellular matrix production, including proteoglycans, collagen, and versican. Furthermore, the protocol's reproducibility was demonstrated using 3 distinct iPSC lines, successfully expanded in both serum-containing and defined serum-free media. Conclusion. Our optimized approach yields iCHOp with phenotypes closely matching ACI chondrocytes, offering a solid foundation for further development and potential clinical applications in cartilage repair.