Centrifugal gravity-induced BMP4 induces chondrogenic differentiation of adipose-derived stem cells via SOX9 upregulation

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.

Spontaneous adipogenic differentiation potential of adipose‑derived stem cells decreased with increasing cell passages

“…For chondrogenic or osteogenic regeneration, the spontaneous adipogenic differentiation of ADSCs should be minimized as possible. Adipogenic, chondrogenic, and osteogenic differentiation was observed in passage 3 in many studies (29)(30)(31)(32)(33)(34). The results of the present study indicated that the impact of spontaneous adipogenic differentiation reduced sufficiently after passage 3, and the cells passaged more than three times were probably suitable for chondrogenic and osteogenic regeneration.…”

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

“…Some groups use monolayer culture of hiPSCs to directly convert the phenotype into MSC-like cells . However, TGFβ3 is used more frequently by researchers than TGFβ1 7,10,18,19 . BMP2 enhances the expression of genes related to the chondrogenic matrix components in human articular chondrocytes under in vitro conditions 20 .…”

Different Chondrogenic Potential among Human Induced Pluripotent Stem Cells from Diverse Origin Primary Cells

“…Successful generation of hyaline cartilage in tissue engineering and in vitro cultivation systems can lead to tremendous therapeutic benefits in the related fields. Early in vitro chondrogenesis was done using MSCs or adipocyte-derived stem cells [ 20 , 28 ]. These two adult stem cells are also actually used in current clinics along with autologous chondrocytes for the regeneration of the damaged cartilage [ 29 – 31 ].…”

“…It is well known that the adult articular cartilage lacks natural healing ability [ 17 – 20 ]. Chondrogenesis using hiPSCs was attempted for several years with hiPSCs generated from various origin cells (i.e., adipose-derived stem cells, fibroblasts, and articular chondrocytes) [ 15 , 21 – 25 ].…”