FRI0516 CHONDROGENIC EFFECT OF KARTOGENIN ON AN IMMORTALIZED CELL LINE DERIVED FROM MESENCHYMAL STROMAL CELLS ISOLATED FROM HUMAN BONE MARROW
Authors: Carolina Guiance-Varela, Cristina Rodríguez-Pereira, Elena Fernandez-Burguera, Tamara Hermida Gómez, Noa Goyanes, Francisco J. Blanco, joana magalhães
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background</h3> Mesenchymal stromal cells (MSCs) currently tested in regenerative medicine approaches for osteoarthritis (OA) present limitations in obtaining stable hyaline-like cartilage phenotype. Kartogenin (KGN) has been recently described to promote both reparative and protective effects hampering the hypertrophic effects of chondrogenesis.<sup>1</sup> <h3>Objectives</h3> The aim of this study is to evaluate the chondrogenic effect of kartogenin on an immortalized cell line (3a6), using a conventional cell pellet model and gold-standard chondroinductor transforming growth factor 3 (TGF-β3), as positive reference. <h3>Methods</h3> 3a6 cells, originally obtained from a 61-year old Asian woman bone marrow, was cultured under pellet culture system with basal (DMEM, low glucose) or commercial chondrogenic medium, supplemented with 10 ng/ml TGF-β3 or 100nM KGN for 3, 7, 14 and 21 days. Total RNA was isolated and real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was performed, using custom-made primers for runt-related transcription factor 1 (RUNX1), proteoglycan 4 (PRG4), cartilage intermediate layer protein (CILP), collagens type-II (COL2A1), -X (COL10A1) and -I (COL1A1), using RPL13A as housekeeping. Proteoglycans and zonal markers protein synthesis was evaluated by safranin-O (SO) staining and lubricin/PRG4 and CILP immunohistochemical analysis (both 1:500), respectively. The experiment was performed in triplicate and differences judged using one-way ANOVA with Bonferroni's corrections, considering p&lt;0.05 significantly different. <h3>Results</h3> The molecular analysis of KGN- and TGF-β3-chondrogenic induced 3a6 pellets is depicted in figure 1A. For zonal markers expression, both conditions showed a significant downregulation in PRG4 with time, concomitant with an upregulation of CILP, for TGF-β3 but not for KGN-induced cells. However, PRG4 and CILP were found on the protein level (Figure 1B) for both conditions. The studied zonal markers were found heterogeneously distributed for KGN-induced pellets (Figure 1C). This difference is also supported by proteoglycans SO staining, with an earlier formation of a more mature tissue for KGN- than TGF-β3-induced pellets. No expression was found for the main hyaline-like cartilage collagen (COL2A1). RUNX1 was practically unaltered in both conditions. For hypertrophic markers, RUNX2 was upregulated at 14 days in TGF-β3- whilst, in KGN-induced cells its expression seems to be delayed, although no significant differences were found. In both conditions, COL10A1 was very low, until 21 days. COL1A1 presented a significant upregulation at 14 days for TGF-β3- whilst in KGN-induced cells expression was non-significant. <h3>Conclusion</h3> This is the first study to report the chondrogenic effect of kartogenin on 3a6 immortalized human bone marrow MSCs line. On the molecular level, no significant differences were found between KGN- and TGF-β3 chondroinduction, although transition into a hypertrophic phenotype seems to be delayed. On the protein level, zonal markers and proteoglycan synthesis were found improved by kartogenin after 14 days. <h3>Reference:</h3> [1] Johnson K, et al. Science2012, 11;336(6082):717-21. <h3>Disclosure of Interests</h3> carolina guiance-varela: None declared, Cristina Rodríguez-Pereira: None declared, Elena Fernandez-Burguera: None declared, Tamara Hermida Gómez: None declared, Noa Goyanes: None declared, Francisco J. Blanco Consultant for: AbbVie, Bioiberica, BMS, GSK, Grünenthal, Janssen, Lilly, Pfizer, Regeneron, Roche, Sanofi, TRB Chemedica, and UCB, joana magalhães: None declared