Effects of NR2F2 expression during chondrogenesis of hMSCs in alginate beads

Osteoarthritis, which is a leading cause of pain and disability in the United States, has a non-curative treatment of physical therapy, anti-inflammatory medication, and finally total joint replacement. This treatment, specifically joint replacement, has an average lifetime of 15 years and is therefore inappropriate for young patients with chondral lesions caused by osteoarthritis or traumatic injury. Emerging curative techniques, such as osteochondral plugs and autologous chondrocyte injection have many downsides including: donor site morbidity, improper integration, and difficulty expanding cells ex vivo. A potential solution lies in the use of autologous mesenchymal stem cells (hMSC), which have well known proliferation protocols and ease of harvest, to produce large amounts of autologous chondrocytes. A hurdle with this solution however is the difficulty in producing chondrocytes that will not undergo hypertrophy, leading them to differentiate to osteocytes and increasing the chance for ectopic bone formation if implanted in vivo. In order to prevent this, there is a need to improve techniques for chondrogenic differentiation. Previous work has show that the upregulation of NR2F2 in hMSCs results in expression of SOX9, COL2a1, and ACAN; all of these markers are known chondrogenic markers. Furthermore there was a resultant down expression of COL10a1 and MMP13, both of which are hypertrophic markers. This study aims to use the TetOn promoter system to regulate the over expression of NR2F2. In this way, cells are not burdened with overexpression until after expansion. The chondrogenic gene expression 3D culture conditions and in both hMSC and chondrogenic media, was assessed via qPCR. Furthermore, a GAG content assay and histology was performed on the 3D alginate bead cultures. The future directions of this study will involve in vivo implantation of 3D constructs, as well as 3D co-culture, and intra-construct spatial control of NR2F2 expression.