Anticancer Activity and Mechanisms of Action of MAPK pathway inhibitors

TdTomato-tagged (for tracing) ci-chon pellets (2? 105 cells) were implanted into a full-thickness cartilage defect model validated by other groups (Eltawil et?al

TdTomato-tagged (for tracing) ci-chon pellets (2? 105 cells) were implanted into a full-thickness cartilage defect model validated by other groups (Eltawil et?al., 2009, Wang et?al., 2017). CHIR98014, Repsox, TTNPB, and celecoxib). Using single-cell transcriptomics, we revealed the inhibition of fibroblast features and activation of chondrogenesis pathways in early reprograming, and the intermediate cellular process resembling cartilage development. The implantation of chemical-induced chondrocytes at defective?articular surfaces promoted defect healing and rescued 63.4% of mechanical function loss. Our approach directly converts fibroblasts into functional cartilaginous cells, and also provides insights into potential pharmacological strategies for future cartilage regeneration. transgene driven by promotor/enhancer, we also demonstrated the poor chondrogenesis ability of untreated MEFs (Figures S1B and S1C). During stage 1 of the induction, expanded MEFs were treated with chemical cocktails under 5% O2 for 6?days. Basic chemicals in stage 1 contained valproic acid (V, histone deacetylase inhibitor), CHIR98014 (C, GSK-3 kinases inhibitor), and Repsox (R, transforming growth factor [TGF-] inhibitor), as they have been used to facilitate the direct reprogramming of other lineages (Cheng et?al., 2014, Han et?al., 2017). Stage 2 involved culturing the cocktail-treated cells in chondrogenic differentiation medium for an additional 14?days (days 6C20). At the end of the induction, we calculated the cell number in Safranin O+ clusters to quantify the fibroblast-to-chondrocyte conversion (Figure?1B), as Safranin O-fast green staining was used for chondrocyte glycosaminoglycan recognition (Oldershaw et?al., 2010). Immunostaining for chondrocyte markers SOX9 and COL2 was conducted to characterize their chondrocyte identity (Figure?1C). Using Col2-pd2EGFP reporter mice, we also demonstrated the real-time expression of chondrocyte marker Col2 (Figure?1D). The cellular morphology of MEFs changed into polygonal after chemical reprogramming (Figure?S1D). Elimination of individual components of VCR, and extension of induction time during stage 1 reduced the formation of Safranin O+ cells (Figures S1E and S1F). TGF-3 was identified as an essential component for chondrogenic medium in stage 2 (Figures S1H and S1I). Thus, these results validated the establishment of the basic model. We used VCR treatment followed by culturing in chondrogenic medium as RU-302 a basis for further optimizing our induction system. To identify additional chemical compounds capable of boosting the fibroblast-to-chondrocyte conversion, we screened a library of 48 small molecules known to facilitate reprogramming or regulate chondrogenesis (Table S1). In primary screening, each compound was added either at stage 1 or 2 (Figure?1A). We identified five compounds, treatment RU-302 with which, together with the VCR cocktail during stage 1, potentially increased the Safranin O+ efficiency (Figure?S1J). These were kartogenin (Kgn, K), olanzapine (O), dopamine HCl (D), celecoxib (c), and TTNPB (T) (Table S2). We tested RU-302 30 different combinations of these five candidates and found that the combination of TTNPB (a?retinoic acid receptor agonist) and celecoxib (a cyclooxygenase [COX] 2 inhibitor) (Figure?S1L) together with the VCR (VCRTc) led to one of the best outcomes (Figures 1E and S1K). We further validated the function of the candidate combinations by reprogramming Col2-pd2EGFP MEFs (Figures 1F and 1G). When compared with other groups, cocktail VCRTc resulted in the greatest conversion efficiency, which increased the initial efficiency (VCR group) by 4-fold (Figures 1E and 1F). Altogether, we have established a chemical reprogramming system to convert MEFs into chondrocytes using chemical cocktail VCRTc (Figure?1H). Chemical-Induced Chondrocytes Form Scaffold-free Cartilage Organoids The micro-mechanical environment provided by 3D cultures has been reported to be essential for chondrogenesis (Benoit et?al., 2008). We, therefore, applied bionic 3D culture to the generation of chemical-induced chondrocytes (ci-chons). Although VCRTc produced the most efficient lineage conversion among other groups, the (Figure?S2A), and the immunostaining images showed they were SOX9+ and COL2+ (Figure?S2C). In the 3D system, we also used suspended pellet culture for better cell collection. VCRTc-treated MEFs self-organized into dense suspended pellets and were cultured for 4?weeks. Mesenchymal condensation marker N-cadherin was expressed in early phase (day Rabbit Polyclonal to TRIM24 7C10) and SOX9 was continuously expressed during chondrogenic induction, and presented in a higher expression level in late period (days 13C20) (Figure?S2C). The pellets grew in size over time (Figure?2C) and expressed Col2 from day 20 (Figure?2A). Open in a separate window Figure?2 Chemical-Induced Chondrocytes Form Scaffold-free Cartilage Organoids (A) Representative.