Figure 4

Expression analyses in chondrogenically induced cultures of human bone marrow-derived mesenchymal stem cells transduced with rAAV-hIGF-I. Human bone marrow-derived mesenchymal stem cell aggregates were transduced with rAAV-hIGF-I or rAAV-lacZ as described in Figure 2 and processed on day 21 for gene expression analysis by real-time reverse transcription-polymerase chain reaction amplification after total cellular RNA extraction and cDNA synthesis, as described in Methods. The genes analyzed included the transcription factor SOX9, type II, type I, and type X collagen (COL2A1, COL1A1, COL10A1), matrix metalloproteinase 13 (MMP13), the transcription factor RUNX2, alkaline phosphatase (ALP), and β-catenin, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as a housekeeping gene and internal control (primers are listed in Methods). Threshold cycle (Ct) values were obtained for each target and GAPDH as a control for normalization, and fold inductions (relative to lacZ-treated aggregates) were measured using the 2^–ΔΔCt method. *Statistically significant compared with rAAV-lacZ. hIGF-I, human insulin-like growth factor I; rAAV, recombinant adeno-associated virus.