Methods
The rAAV-FLAG-hSOX9 vector was provided to both undifferentiated and lineage-induced MSCs freshly isolated from patients to determine the effects of the candidate construct on the viability, biosynthetic activities, and ability of the cells to enter chondrogenic, osteogenic, and adipogenic differentiation programs compared with control treatments (rAAV-lacZ or absence of vector administration).
Results
Marked, prolonged expression of the transcription factor was noted in undifferentiated and chondrogenically differentiated cells transduced with rAAV-FLAG-hSOX9, leading to increased synthesis of major extracellular matrix components compared with control treatments, but without effect on proliferative activities. Chondrogenic differentiation (SOX9, type II collagen, proteoglycan expression) was successfully achieved in all types of cells but strongly enhanced when the SOX9 vector was provided. Remarkably, rAAV-FLAG-hSOX9 delivery reduced the levels of markers of hypertrophy, terminal and osteogenic/adipogenic differentiation in hMSCs (type I and type X collagen, alkaline phosphatase (ALP), matrix metalloproteinase 13 (MMP13), and osteopontin (OP) with diminished expression of the osteoblast-related transcription factor runt-related transcription factor 2 (RUNX2); lipoprotein lipase (LPL), peroxisome proliferator-activated receptor gamma 2 (PPARG2)), as well as their ability to undergo proper osteo-/adipogenic differentiation. These effects were accompanied with decreased levels of β-catenin (a mediator of the Wnt signaling pathway for osteoblast lineage differentiation) and enhanced parathyroid hormone-related protein (PTHrP) expression (an inhibitor of hypertrophic maturation, calcification, and bone formation) via SOX9 treatment.