Fig. 7. Effect of CHL2 on in vitro chondrogenesis. (A) CHL mRNA expression during chondrogenic culture. hMSCs were grown in pellet culture in the presence of TGFß3. On the indicated day, RNA was extracted from particles, treated with DNase I, and subjected to RT-PCR using primers for aggrecan, COMP, COL2, CHL1, CHL2 and GAPD. The CHL2 signal was confirmed with a ³³P-labeled XbaI-SalI fragment from pSPORThCHL2. (B) hMSCs were grown in pellet culture in the presence of TGFß3 alone (a,d), or TGFß3 with either 0.2 µg/ml mCHL2-FLAG (b), 2 µg/ml mCHL2-FLAG (c), 0.1 µg/ml noggin-Fc (e), or 1 µg/ml noggin-Fc (f). On day 21, particles were harvested and stained with Toluidine Blue. Data are representative of five independent experiments. Addition of IgG-Fc did not affect growth and maturation of cartilage-containing particles (not shown). Note that cartilage nodules, in which well-separated cells were embedded, stained more intensely with Toluidine Blue. (C) Effect of CHL2 on in vitro mineralization of EB cell-derived cartilage. FACS-purified FLK1^–PDGFR⁺ EB cells were grown in pellet micromass culture to produce hyaline cartilage particles. On day 18, the medium was changed to a hypertrophic differentiation medium in the absence (a-c) or presence of 3 µg/ml mCHL2-FLAG (d-f) or 2 µg/ml noggin-Fc (g,h). On day 24, particles were harvested, stained with von Kossa (a,d,g), and immunostained with X53 for COL10 (b,e,h) or 2B1.5 for COL2 (c,f). Data are representative of four independent experiments. The Scale bar for B and C is shown in C.