Tendon healing process is complex to be managed because of the limited regeneration capacity of tendon tissue. Mesenchymal stem cells (MSCs) have potential applications in regenerative medicine and have been considered for tendon repair and regeneration. This study aimed to to evaluate the capacity of equine adipose tissue-derived cells (eASCs) to differentiate into tenocytes in response to platelet-derived growth factor-BB (PDGF-BB) and growth differentiation factor-6 (GDF-6) in vitro. Frozen characterized eASCS of 3 mares were thawed and the cells were expanded in basic culture medium (DMEM with 10%FBS). The cells at passage 5 were treated for 14 days in different conditions including: 1) control group in basic culture medium (CM), 2) induction medium as IM (CM containing L-Prolin, and Ascorbic Acid (AA)) supplemented with PDGF-BB (20 ng/ml), 3) IM supplemented with GDF-6 (20 ng/ml), and 4) IM supplemented with PDGF-BB and GDF-6. At the end of culture period (14th day), tenogenic differentiation was evaluated. Sirius Red staining was used to assess collagen production, and H&E was used for assessing cell morphology. mRNA levels of collagen type 1 (colI), scleraxis (SCX), and Mohawk (MKX), as teneogenic markers, were analyzed by using real-time reverse-transcription polymerase chain reaction (qPCR). H&E staining revealed a stretching and spindle shape (tenocyte-like) of cells in all treated groups compared to unchanged from of cells in control groups. Also, Sirius red staining data showed a significant increase in collagen production in all treated groups compared with the control group. MKX expression was significantly increased in PDGF-BB and mixed groups and COLI expression was significantly increased only in PDGF-BB group. . In conclusion, our results showed that PDGF-BB and GDF-6 could induce tenogenic differentiation in eASCs in combination together. It is hoped that these in vitro findings could be useful for cell therapy in equine regenerative medicine.

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