Influence of chondrocytes on the chondrogenic differentiation of adipose stem cells

Abstract

In this study, whether or not chondrogenic differentiation of adipose-derived stem cells (ASCs) could be enhanced by soluble factors from or coculture with chondrocytes was determined. In vitro pellet cultures were carried out in five ways using ASCs or chondrocytes in passage 3 as follows: #1, 2.5 × 10⁵ ASCs were cultured in Dulbecco's modified Eagle's medium/F-12 supplemented with 1% ITS, 10⁻⁷ M dexamethasone, 50 μM ascorbate-2-phosphate, 50 μM L-proline, and 1 mM sodium pyruvate; #2, 2.5 × 10⁵ chondrocytes were cultured in the same medium as #1: #3, 1.25 × 10⁵ ASCs and 1.25 × 10⁵ chondrocytes were mixed and cocultured in the same medium as #1; #4, 2.5 × 10⁵ ASCs were cultured in a medium that was a 1:1 mixture of the same fresh medium as #1 and conditioned medium from chondrocyte culture (#2); #5, 2.5 × 10⁵ ASCs were cultured in the same medium as #1 and 5 ng/mL of transforming growth factor-β₂ and 100 ng/mL of BMP-7. After 3 weeks, the glycosaminoglycan level that normalized to the DNA amount was significantly increased by 25% in ASCs treated with condition medium from chondrocyte cultures (p = 0.028) and by 37% in ASC-chondrocyte cocultures (p = 0.042). The glycosaminoglycan level was 37% greater in chondrocytes (p = 0.046) and 50% greater in ASCs cultured under growth factor cocktails than the control ASCs. The gene expression of SOX-9 significantly increased by >10-fold (p < 0.05) in ASCs treated with the conditioned medium from chondrocyte cultures and ASC-chondrocyte cocultures compared with the control ASCs; whereas COL2A1 significantly increased ~100-fold (p < 0.05) in either condition. COL10A1 gene expression increased by treating either with conditioned medium or with coculture (p < 0.05), but COL1A1 gene expression did not significantly change in either condition. Western blotting of SOX-9 and immunochemistry for types II, I, and X collagen largely parallel the results from gene expression studies. It is concluded that the signals from chondrocytes, in the form of soluble factors or by direct interaction, effectively promote chondrogenic differentiation of ASCs during in vitro pellet culture. This work may present a simple and innovative method for generating cartilaginous tissue from ASCs and shed a new light in cartilage tissue engineering from ASCs.