Document Type : Original Article
STEM CELLS DEPARTMENT, ROYAN INSTITUTE, TEHRAN, IRAN
Introduction: An investigation of bone, cartilage and fat differentiation potentials of canine bone marrow-derived mesenchymal stem cells.
Materials and methods: Bone marrow aspirates were obtained from 10 canine’s iliac crest under general anesthesia. Each sample was separately loaded on lymphodex and centrifuged at 1200 rpm for ten minutes to obtain mononuclear cell fraction. 5×104 cell/cm2 of mononuclear cells were cultured in 150 cm2-flask in a DMEM medium containing 15% fetal bovine serum. Fibroblastic cells were then purified and expanded through several subcultures. Passaged-3 cells were differentiated into osteogenic, chondrogenic and adipogenic lineages for a period of 3 weeks. The differentiated cells were evaluated by histochemistry and RT-PCR analysis.
Results: Passaged-3 fibroblastic cells cultured in osteogenic medium were stained red with Alizarin red method, indicating that the matrix was mineralized. The cell also produced a large amount of bone specific markers including collagen I and osteopontine. In the adipogenic cultures, lipid droplets were stained red by oil red method. Furthermore, RT-PCR analysis indicated that these cells produced a large amount of adiopocyte specific genes including lipoprotein lipase and PPARG2. Sections from chondrogenic cultures turned into purple upon safranin O staining that was indicative of significant amount of glycosaminoglycans in the cultures. In addition, RT-PCR analysis revealed that the mRNA of Decorin and collagen II as cartilage specific genes were produced in these cells but aggreacan was not expressed.
Conclusion: Canine bone marrow fibroblastic cells could easily be differentiated into bone and fat but in chondrogenic differentiation, while glycoseaminoglycan-rich matrix containing collagen II and Decorin was produced, the agreacan gene was not expressed