Sox‐9 facilitates differentiation of adipose tissue‐derived stem cells into a chondrocyte‐like phenotype in vitro

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Abstract
The purpose of this study is to test whether ectopic expression of Sox‐9 can induce adipose tissue‐derived stem cells (ASCs) to function as real nucleus pulposus (NP) cells in vitro. Adenoviral vectors expressing Sox‐9 were reported to infect the chondroblastic and human disc cells, which resulted in increased Sox‐9 and type II collagen production. ASCs were isolated from rat inguinal adipose pad, characterized, and transduced in vitro with a retroviral vector encoding the Sox‐9 gene. Sox‐9‐engineered ASCs (ASCs/Sox‐9) were induced for the chondrocyte‐like cell differentiation by 3D cultured in alginate beads and TGF‐β3 for 2 weeks. Expression of exogenous Sox‐9 protein was detected. Type II collagen and Aggrecan gene expressions of induced ASCs/Sox‐9 were measured using real‐time PCR; proteoglycans expressions were measured by checking the glycosaminoglycan content and type II collagen production by enzyme‐linked immunosorbent assay. Isolated ASCs were CD 29+/CD44+/C‐Kit−/Lin−/CD34−/CD45−. ASCs/Sox‐9 expressed marked increase in exogenous Sox‐9 protein. After induction, type II collagen gene expression was sevenfold higher in mRNA levels, with an approximately twofold increase in protein levels of ASCs/Sox‐9 compared to ASCs. Type II collagen and proteoglycan productions were significantly increased in the ASCs/Sox‐9 compared to the ASCs. In addition, co‐culture of induced ASCs/Sox‐9 with matured NP cells resulted in enhanced increase in proteoglycan and type II collagen production. Constitutive retroviral expression of Sox‐9 could efficiently induce ASCs differentiation into chondrocyte‐like cells. This novel approach may provide a practicable system for a simple and rapid differentiation of ASCs into chondrocyte‐like cells which may be potentially used as a stem cell‐based therapeutic tool for the treatment of degenerative disc diseases. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1291–1297, 2011

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