Analyses in osteogenically and adipogenically differentiated cultures of human mesenchymal stem cells (hMSCs). Cells in monolayer cultures were transduced with rAAV-lacZ or rAAV-FLAG-hSOX9 (100 μl each vector) or left untreated and induced toward osteogenic or adipogenic differentiation, as described in Materials and methods. Cultures were processed on day 21 for (a) ALP staining (osteogenesis; magnification ×4) and Oil Red O staining (adipogenesis; magnification ×10) and (b) and (c) gene-expression analysis with real-time RT-PCR amplification, as described in Figure 3. The genes analyzed included ALP, COL1A1, OP, and RUNX2 for osteogenically differentiated cultures (b) and LPL and PPARG2 for adipogenically differentiated cultures (c), with GAPDH serving as a housekeeping gene and internal control in both cases. Ct values were obtained for each target and GAPDH as a control for normalization, and fold inductions (relative to untreated cultures) were measured by using the 2-ΔΔCt method. Statistically significant compared with (a) condition without vector treatment or (b) rAAV-lacZ.
Venkatesan et al. Stem Cell Research & Therapy 2012 3:22 doi:10.1186/scrt113