Induced pluripotent stem cells (iPSCs) can generate multiple tissues with close to normal phenotype. This is valuable for hyaline cartilage which tends to hypertrophy when derived from adult stem cells. Horses commonly suffer from joint injuries and regenerative strategies are increasingly needed. Moreover, evidence generated in horses is highly transferable to human medicine based on their similar joint biomechanics. However, the lack of robust methodologies to generate and differentiate animal iPSCs hinders veterinary applications. We aimed to optimise the generation of equine iPSCs (eiPSCs) and to explore different strategies to derive cartilage from these cells. We compared the reprogramming potential of equine cells of three developmental stages (embryonic, perinatal, adult) using different vectors and culture conditions. Generated eiPSCs were characterised by morphology, pluripotent markers expression and three-germ layer differentiation. These were induced to differentiate into chondroprogenitors (iCHO) and mesenchymal stem cells (iMSCs) by adapting human protocols. The chondrogenic potential of these was compared to bone marrow (BM)-MSCs. Only embryonic-derived cells produced fully pluripotent eiPSCs, confirmed by marker expression and three-germ layer differentiation, while maintaining transgene expression (putative lines). iCHO induction was achieved as indicated by adoption of a chondrocyte morphology, downregulation of pluripotent genes and increased SOX-9. However, complete differentiation was not achieved based on histology of chondrogeneic pellets. iMSCs had higher chondrogeneic potential than iCHO, but not all the lines assessed generated good quality pellets. Overall, BM-MSCs were superior to eqiPSCs in generating cartilage. We propose two potential explanations for limited eqiPSCs chondrogenesis: 1) transgene expression retained pluripotency and hindered differentiation; 2) equine pluripotent profile differs from human so differentiation protocols need further adjustment and/other differentiation pathways should be explored. Future comparative transcriptomic/epigenomic studies can reveal equine-specific requirements for differentiation. Nevertheless, despite some observed limitations, putative iPSCs are critical to enhance our understanding of pluripotency of animal cells. iPSCs hold the key for transforming veterinary regenerative medicine, but further studies are needed.

Poster presented at the International Society of Cell and Gene Therapy (ISCT) Europe 2024 Regional Meeting, celebrated in Ghotenburg (Sweden) on 4-6 Septembre 2024. Abstract is provided in its acceptance form. Poster is provided in its presentation form.