Introduction: Transplantation of genetically modified human bone marrow-derived mesenchymal stem cells\r\n(hMSCs) with an accurate potential for chondrogenic differentiation may be a powerful means to enhance the\r\nhealing of articular cartilage lesions in patients. Here, we evaluated the benefits of delivering SOX9 (a key regulator\r\nof chondrocyte differentiation and cartilage formation) via safe, maintained, replication-defective recombinant\r\nadeno-associated virus (rAAV) vector on the capability of hMSCs to commit to an adequate chondrocyte\r\nphenotype compared with other mesenchymal lineages.\r\nMethods: The rAAV-FLAG-hSOX9 vector was provided to both undifferentiated and lineage-induced MSCs freshly\r\nisolated from patients to determine the effects of the candidate construct on the viability, biosynthetic activities,\r\nand ability of the cells to enter chondrogenic, osteogenic, and adipogenic differentiation programs compared with\r\ncontrol treatments (rAAV-lacZ or absence of vector administration).\r\nResults: Marked, prolonged expression of the transcription factor was noted in undifferentiated and\r\nchondrogenically differentiated cells transduced with rAAV-FLAG-hSOX9, leading to increased synthesis of major\r\nextracellular matrix components compared with control treatments, but without effect on proliferative activities.\r\nChondrogenic differentiation (SOX9, type II collagen, proteoglycan expression) was successfully achieved in all types\r\nof cells but strongly enhanced when the SOX9 vector was provided. Remarkably, rAAV-FLAG-hSOX9 delivery\r\nreduced the levels of markers of hypertrophy, terminal and osteogenic/adipogenic differentiation in hMSCs (type I\r\nand type X collagen, alkaline phosphatise (ALP), matrix metalloproteinase 13 (MMP13), and osteopontin (OP) with\r\ndiminished expression of the osteoblast-related transcription factor runt-related transcription factor 2 (RUNX2);\r\nlipoprotein lipase (LPL), peroxisome proliferator-activated receptor gamma 2 (PPARG2)), as well as their ability to\r\nundergo proper osteo-/adipogenic differentiation. These effects were accompanied with decreased levels of bcatenin\r\n(a mediator of the Wnt signaling pathway for osteoblast lineage differentiation) and enhanced parathyroid\r\nhormone-related protein (PTHrP) expression (an inhibitor of hypertrophic maturation, calcification, and bone\r\nformation) via SOX9 treatment\r\nConclusions: This study shows the potential benefits of rAAV-mediated SOX9 gene transfer to propagate hMSCs\r\nwith an advantageous chondrocyte differentiation potential for future, indirect therapeutic approaches that aim at\r\nrestoring articular cartilage defects in the human population.
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