Background/Purpose: Osteoarthritis (OA) is characterized by progressive breakdown of articular cartilage. Regeneration of cartilage has been an attractive approach to OA therapy. Sox9 is a transcription factor belonging to the Sox (Sry-type HMG box) gene family and has been identified as a “master regulator” of the chondrocyte phenotype. We investigated whether a super positively charged cell penetrating Sox9 fusion protein can induce bone marrow-derived mesenchymal stem cells (MSC) to differentiate into chondrocytes for potential use to promote cartilage regeneration in situ. 

Methods: A bioactive supercharged Sox9 (scSox9) was generated by fusing Sox9 with a super positively charged green fluorescence protein (GFP) using molecular engineering technology. Human bone marrow-derived MSC at passage 5 were cultured with scSox9 in monolayer or in cell aggregate for differentiation into chondrocytes. Chondrogenesis was verified by toluidine blue staining for aggrecan and by RT-PCR and immunohistochemistry for collagen type I, II and X production.

Results: scSox9 could readily enter HHF cells, a human skin fibroblasts-derived cell line and  human bone marrow- derived MSC. After one hour incubation with scSox9, both HHF cells and MSC showed intracellular expression of green fluorescence indicating entry of scSox9 into these cells. scSox9 was able to induce MSC proliferation and differentiation with no requirement of additional growth factors. The number of scSox9 treated MSC was increased two folds after 72 hours in culture compared with scGFP treated MSC. As early as 48 hours, scSox9 treated MSC started changing morphology to chondrocyte-like cells and the changed morphology maintained for 21 days in culture when observation was ended. These morphologically changed cells stained positive for toluidine blue, which suggests aggrecan production. Furthermore, scSox9 induced collagen type II expression and down- regulated collagen type I and type X production. Most importantly, one time addition of scSox9 at the initiation of chondrogenesis was sufficient to induce MSC chondrogenic differentiation and maintain the chondrocyte phenotype.

Conclusion: These in vitro data demonstrated that scSox9 is able to enter bone marrow-derived MSC and induce chondrogenesis and maintain chondrocyte phenotype. Further investigation is warranted for the potential therapeutic use in cartilage repair by promoting regeneration of hyaline cartilage.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/supercharged-sox9-protein-induces-chondrogenic-differentiation-of-bone-marrow-derived-mesenchymal-stem-cells/