Fig. 2
cAMP regulators control TGFβ-induced osteogenic differentiation of hMSCs. Osteogenic differentiation was measured by ALP activity and corrected for the level of Neutral Red uptake as a measure for the number of cells present in the well. a Osteogenic differentiation of hMSCs in osteogenic differentiation medium supplemented with or without 125 ng/ml BMP2, 2 ng/ml TGFβ1, 10 μg/ml insulin, 500 μM IBMX, or 10−7 M rosiglitazone. ALP activity is significantly higher (p < 0.001) in medium with BMP2 and TGFβ in the presence of IBMX than in the absence of IBMX. b Osteogenic differentiation of hMSCs in adipogenic differentiation medium supplemented with or without 125 ng/ml BMP2 and 2 ng/ml TGFβ1, and following omission of 10 μg/ml insulin, 500 μM IBMX, or 10−7 M rosiglitazone. ALP activity is significantly higher (p < 0.01) in medium with all supplements than in the absence of IBMX. c Effect of PGE2, added at the indicated nanomolar concentrations, on osteogenic differentiation of hMSCs in osteogenic differentiation medium. A comparison is made with the effects of BMP2 (125 ng/ml), TGFβ (2 ng/ml), and IBMX (500 μM). Enhancement of ALP activity is significant (p < 0.05) at concentrations of 10 nM PGE2 and above. d Effect of BMP2 (125 ng/ml) and TGFβ (2 ng/ml) on total Ca2+ deposition (μg) in a six-well plate well (10 cm2) by hMSCs, cultured for 13 days in either osteogenic or adipogenic differentiation medium. Ca2+ deposition is significantly enhanced by BMP2 alone in osteogenic differentiation medium (p < 0.01) and by BMP2 + TGFβ in adipogenic differentiation medium (p < 0.05). ALP alkaline phosphatase, BMP bone morphogenetic protein, IBMX 3-isobutyl-1-methylxanthine, PGE2 prostaglandin E2, TGFβ transforming growth factor beta