Abstract
Human mesenchymal stem cells (hMSCs) are multipotent cells capable of differentiating into a variety of mature cell types, including osteoblasts, adipocytes and chondrocytes. It has previously been shown that, when expanded in medium supplemented with fibroblast growth factor-2 (FGF-2), hMSCs show enhanced chondrogenesis (CG). Previous work concluded that the enhancement of CG could be attributed to the selection of a cell subpopulation with inherent chondrogenic potential. In this study, we show that FGF-2 pretreatment actually primed hMSCs to undergo enhanced CG by increasing basal Sox9 protein levels. Our results show that Sox9 protein levels were elevated within 30 minutes of exposure to FGF-2 and progressively increased with longer exposures. Further, we show using flow cytometry that FGF-2 increased Sox9 protein levels per cell in proliferating and non-proliferating hMSCs, strongly suggesting that FGF-2 primes hMSCs for subsequent CG by regulating Sox9. Indeed, when hMSCs were exposed to FGF-2 for 2 hours and subsequently differentiated into the chondrogenic lineage using pellet culture, phosphorylated-Sox9 (pSox9) protein levels became elevated and ultimately resulted in an enhancement of CG. However, small interfering RNA (siRNA)-mediated knockdown of Sox9 during hMSC expansion was unable to negate the prochondrogenic effects of FGF-2, suggesting that the FGF-2-mediated enhancement of hMSC CG is only partly regulated through Sox9. Our findings provide new insights into the mechanism by which FGF-2 regulates predifferentiation hMSCs to undergo enhanced CG.
Highlights
Due to the limited capacity of articular cartilage for self repair and lack of effective treatments for cartilage injuries and degenerative joint diseases like osteoarthritis [1], tissue engineering has gained considerable attention as a promising approach to cartilage repair
Expanding Human mesenchymal stem cells (hMSCs) in medium supplemented with fibroblast growth factor-2 (FGF-2) enhanced subsequent differentiation into the chondrogenic lineage compared to non-FGF-2-supplemented controls, as evidenced by the upregulation of cartilage-specific gene expression (Fig. 1A) and GAG accumulation (Fig. 1B)
Both hMSCs expanded for 2 passages in 5%- (Fig. 1A; Lane 2) and 1% fetal bovine serum (FBS)+FGF-2 (Fig. 1A; Lane 4) showed upregulated cartilage-specific gene expression compared to cells expanded in 10% FBS (Fig. 1A; Lane 1), the current gold standard for hMSC expansion, upon chondrogenic induction at each time point
Summary
Due to the limited capacity of articular cartilage for self repair and lack of effective treatments for cartilage injuries and degenerative joint diseases like osteoarthritis [1], tissue engineering has gained considerable attention as a promising approach to cartilage repair. For tissue engineering strategies to be successful, an appropriate cell source must be instructed to proliferate and differentiate by specific bioactive cues, such as growth factors and extracellular matrix molecules. Human mesenchymal stem cells (hMSCs) provide an attractive cell source for cartilage tissue engineering applications, as they are readily expandable [2] and capable of differentiating into chondrocytes [3]. HMSCs represent a heterogeneous population of cells with varying chondrogenic potential [4,5], limiting their use in cartilage tissue engineering applications. Improvements to our current expansion conditions will be necessary to purify those hMSCs with greatest chondrogenic potential and/or enhance their ability to undergo chondrogenesis (CG) if cartilage tissue engineering using hMSCs is to be of clinical relevance
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