Objectives: Research on the use of mesenchymal stem cells (MSCs) in tissue regeneration has generally been driven by the needs of human and veterinary medicine. Adipose-derived mesenchymal stem cells (AMSCs) have potential to differentiate into various lineages such as cartilage. As first step, the capability of their chondrogenic differentiation should be confirmed in vitro. Materials & Methods: To isolate AMSCs, fat samples were collected from three mares aged 3, 6 and 9- years old. Cells were isolated by mechanical and enzymatic (Collagenase I) process and were cultured in optimized conditions. After 3 subcultures, to analyze chondrogenic differentiation, 500,000 cells were cultured under five different treatments and were differentiated with appropriate chondrogenic medium supplemented with or without TGF-beta3 and BMP-6 in 3D micropellet system for 21 days. Chondrogenic differentiation was assessed by staining with toluidine blue and aggrecan expression as a cartilage-specific gene. Results: Isolated AMSCs were plastic-adherent, fibroblast-like morphology and expressed specific markers. After the period of chondrogenic culture, histological results confirmed chondrogenic differentiation in both groups containing chondrogenic media with or without growth factors compared with control group. More lacuna formation and more adhesive matrixes was seen in growth factors supplemented groups. Moreover, the band of aggrecan fragment in PCR gels were sharp and most highlighted in group supplemented with two growth factors compared with other group. Conclusion: The results suggest that addition of growth factors to the standard chondrogenic medium improves chodnrogenic differentiation of AMSCs and increases the expression of cartilage-specific genes such as aggrecan.

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