Abstract
BackgroundCartilage does not have the capability to regenerate itself. Therefore, stem cell transplantation is a promising therapeutic approach for impaired cartilage. For stem cell transplantation, in vitro enrichment is required; however, stem cells not only become senescent but also lose their differentiation potency during this process. In addition, cytokines are normally used for chondrogenic differentiation induction of stem cells, which is highly expensive and needs an additional step to culture. In this study, we introduced a novel method to induce chondrogenic differentiation of adipose-derived stem cells (ASCs), which are more readily available than bone marrow-derived mesenchymal stem cells(bMSCs), using centrifugal gravity (CG).MethodsASCs were stimulated by loading different degrees of CG (0, 300, 600, 1200, 2400, and 3600 g) to induce chondrogenic differentiation. The expression of chondrogenic differentiation-related genes was examined by RT-PCR, real-time PCR, and western blot analyses. The chondrogenic differentiation of ASCs stimulated with CG was evaluated by comparing the expression of positive markers [aggrecan (ACAN) and collagen type II alpha 1 (COL2A1)] and negative markers (COL1 and COL10) with that in ASCs stimulated with transforming growth factor (TGF)-β1 using micromass culture, immunofluorescence, and staining (Alcian Blue and Safranin O).ResultsExpression of SOX9 and SOX5 was upregulated by CG (2400 g for 30 min). Increased expression of ACAN and COL2A1 (positive markers) was detected in monolayer-cultured ASCs after CG stimulation, whereas that of COL10 (a negative marker) was not. Expression of bone morphogenetic protein (BMP) 4, an upstream stimulator of SOX9, was upregulated by CG, which was inhibited by Dorsomorphin (an inhibitor of BMP4). Increased expression of proteoglycan, a major component of cartilage, was confirmed in the micromass culture of ASCs stimulated with CG by Alcian Blue and Safranin O staining.ConclusionsChondrogenic differentiation of ASCs can be induced by optimized CG (2400 g for 30 min). Expression of SOX9 is upregulated by CG via increased expression of BMP4. CG has a similar ability to induce SOX9 expression as TGF-β1.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0445-6) contains supplementary material, which is available to authorized users.
Highlights
Cartilage does not have the capability to regenerate itself
To determine what degree of centrifugal gravity (CG) force is suitable to induce SOX9 upregulation, adiposederived stem cell (ASC) were stimulated with different degrees of CG (0, 300, 600, 1200, 2400, and 3600 g) for 15 min
Expression of SOX9 mRNA and protein was measured by real-time PCR and western blotting, respectively. *p < 0.05, **p < 0.01, and ***p < 0.001 for ASCs stimulated with CG versus the control or ASCs treated with 10 ng/mL transforming growth factor (TGF)-β1
Summary
Cartilage does not have the capability to regenerate itself. stem cell transplantation is a promising therapeutic approach for impaired cartilage. BMSCs are used, but they are hardly sufficiently collected and can unexpectedly differentiate to any lineage other than chondrocyte during in vitro cultivation [17, 18]. For these reasons, we wanted to trigger chondrogenic differentiation of ASCs, relatively abundant stem cells, before transplantation. We wanted to trigger chondrogenic differentiation of ASCs, relatively abundant stem cells, before transplantation This may reduce unexpected lineage differentiation of stem cells. Considering these concerns, the period from cell isolation to transplantation should be shortened to increase the success rate of cartilage regeneration
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