Abstract：In order to isolate the bone marrow mesenchymai stem cells (BMSCs) of dog (Canis lupus) and induce the BMSCs to differentiate into osteoblastic cells, the BMSCs were isolated in vitro by whole bone marrow adherent method. After proliferation into full confluency, the BMSCs were subcultured. During proliferation, cell growth curve assay was performed in different passages. Pluripotent markers (SRY-related high-monility-group (HMG)-box protein-2 (SOX2), octamer-binding transcription factor-4 (OCT4) and NANOG) and osteoblast specific markers (osteocalcin (OCN) and osteonectin (ONN)) were defected by qRT-PCR in undifferentiated cells and cells induced differentiation for 10 and 21 d. Alkaline phosphatase (ALP) and alizarin red were defected by staining in undifferentiated cells and cells induced differentiation for different days. The results showed that the growth curve of undifferentiated cells in p5 and p10 were similar. However, the proliferation rate of cells in p20 was significantly slower than that of cells in p5 and p10. Before differentiation, the pluripotent genes such as OCT4 and SOX2, but not NANOG expressed in the BMSCs. The low expression levels of OCT4 and SOX2 could also be detected after induced differentiation for 10 days. However, after induced differentiation for 21 days, there was no expression of OCT4 and SOX2 in differentiated cells. For osteoblast specific markers, the expression levels of OCN and ONN increased along with induced differentiation. Alkaline phosphatase activity of differentiated cells significantly increased after induced for 7 days. Calcified nodules could be observed and stained red after induced for 21 days, and the number of calcified nodules increased along with induced differentiation. These results indicated that BMSCs of dog could be isolated and induced differentiation into mature osteoblasts successfully in vitro, which offer a new approach to the clinical treatment of severe fractures and osteodystrophic arthropathy of the pet dogs.
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