Student Catholic University of Korea Catholic university, Republic of Korea
Abstract: Cartilage regeneration is crucial for treating joint disorders, yet conventional differentiation methods rely on TGF-β3 and BMP-2, which are costly and require complex protocols. Small-molecule alternatives have been explored to simplify the differentiation process while maintaining efficiency. Retinoic acid (RA) and its derivatives, including Retinoic acid analogs like TTNPB, play an essential role in limb bud formation and subsequent chondrogenesis during skeletal development. Previous studies have demonstrated that TTNPB can efficiently induce chondrogenesis in human pluripotent stem cells using a 2D differentiation approach (Kawata et al., 2019). However, to further evaluate its efficacy in a physiologically relevant environment, this study investigates whether TTNPB, a retinoic acid analog that activates retinoic acid receptors (RARs), can support chondrogenic differentiation of induced pluripotent stem cells (iPSCs) in a 3D pellet culture system, which more closely mimics native cartilage tissue formation. iPSCs were differentiated into embryoid bodies (EBs) and further into EB outgrowth cell cells (EBOGCs) following our established protocols. At the final stage, cells underwent chondrogenic pellet culture. As a control, differentiation was carried out using conventional BMP-2 and TGF-β3 supplementation, while the experimental group was treated with TTNPB. Chondrogenic differentiation was assessed by analyzing key cartilage-specific markers, including COL2A1, ACAN, and SOX9. Immunofluorescence (IFA) staining was performed to visualize the expression and localization of these markers at the protein level. Additionally, Alcian blue and Safranin O staining were used to evaluate glycosaminoglycan deposition, a critical indicator of cartilage formation. Based on previously confirmed results, we believe that TTNPB has the potential to simplify the chondrogenic differentiation process while maintaining comparable efficacy to conventional growth factors.
Funding Source: This work was supported by the National Research Foundation of Korea (NRF) and ministries including Science and ICT, Trade, Industry and Energy, Health & Welfare, and Food and Drug Safety (RS-2023-KH142779, RS-2019-NR040058).
