Abstract| Volume 28, SUPPLEMENT 1, S34, April 2020

Comparison of three different chondrogenic differentiation protocols to obtain chondrocyte-like cells from induced pluripotent stem cells

      Purpose: Numerous studies have shown the potential of induced pluripotent stem cells (iPSc) for both treating cartilage lesions and studying molecular mechanisms of cartilage diseases, such as osteoarthritis. Nevertheless, efficient protocols to chondrogenically differentiate the iPSc are still required. There is no general agreement concerning the best approach to obtain chondrocytes from iPSc. For this reason, the purpose of this research has been to compare three different protocols to differentiate iPSc into chondrocyte-like cells.
      Methods: In this study, an iPSC-line previously established by our group from a donor without rheumatic diseases was used. Three chondrogenic differentiation protocols were tested: (1) directed differentiation protocol, (2) differentiation by means of embryoid bodies (EBs) formation and sequential addition of growth factors, and (3) differentiation through a mesenchymal stromal cell (MSC)-like state followed by micromass formation and culture in chondrogenic medium. Additionally, in order to evaluate whether MSC-like cells were obtained in the intermediate state of the differentiation protocol number 3, cells were morphological, phenotypical and functionally characterised. Finally, chondrogenic differentiation was evaluated by histological techniques in order to detect cartilage-associated markers, such as collagen and proteoglycans.
      Results: MSC-like cells were obtained from the iPSC-line during the differentiation protocol number 3, as demonstrated by their fibroblast-like morphology, the expression of specific surface markers (CD29, CD73, CD90, CD105) and their capacity to differentiate into the adipogenic, osteogenic and chondrogenic lineages. The histological evaluation of the chondrogenesis after performing each one of the protocols (figure 1) revealed that the formation of EBs followed by the sequential addition of growth factors (protocol 2) allowed the highest levels of extracellular matrix components production within the micromasses, as seen by Masson’s Trocromic and Safranin O staining. On the other hand, micromasses obtained after protocol 3 showed presence of collagen fibres but less levels of proteoglycans inside the matrix. Finally, regarding protocol number 1, iPSc formed spontaneous micromasses by day 21 of the differentiation, but neither collagen nor proteoglycans were detected histologically in those micromasses after finishing the directed differentiation protocol.
      Conclusions: Among the three protocols tested in this research, only the differentiation protocol based on the formation of EBs and the sequential addition of growth factors enables the best differentiation of the iPSc into chondrogenic-like cells. Our results should contribute to elucidate the best approach to obtain chondrocytes from iPSc, a crucial step in translating the use of these pluripotent cells to clinical applications.