Fig. 1

Effect of AA treatment in MSC priming during expansion on subsequent chondrogenic differentiation. (A) Schematic diagram of the experiment setup. MSCs frozen at P1 were recovered and sub-cultured until passage 3. Passage 3 MSCs were seeded and allowed to adhere overnight before the media was changed to expansion media containing 0 (Untreated), 0.05, 0.2 or 1.0 mM AA. MSCs were harvested at Passage 3 on Day 7 for subsequent analysis. (B) Histology of chondrogenic pellets after 3 weeks of chondrogenic differentiation following 1 passage of 0 (Untreated), 0.05, 0.2 and 1.0 mM of AA treatment. Formation of glycosaminoglycan (sGAG) indicated by Safranin O staining. 40x magnification; scale bar: 500 μm. Images are representative of 5 replicates per donor. (C) Quantification of sGAG in digested pellets normalized to total DNA per pellet. (D, E, F) Trilineage differentiation capacity of MSCs following 1 passage of 0 (Untreated) or 1.0mM AA (AA) treatment. (D) Chondrogenic differentiation is indicated by the ratio of total sGAG to total DNA in chondrogenic pellet; (E) osteogenic differentiation is represented by the amount of Alizarin red stain for calcium deposits, and (F) adipogenic differentiation is represented by the amount of oil red stain for oil droplets. (G) Comparison between chondrogenic, osteogenic and adipogenic differentiation efficiency in relative to Untreated following 1 passage of 0 (Untreated) or 1.0 mM AA (AA) treatment. Experiments were performed in 3–4 technical replicates. Data are presented as mean ± standard deviation. * P < 0.05 and ** P < 0.01 compared to Untreated