292 MULTILINEAGE POTENTIAL OF CANINE MESENCHYMAL STEM CELLS DERIVED FROM BONE MARROW

Mesenchymal stem cells (MSCs) isolated from bone marrow have unique self-renewal capability for multilineage differentiation and show promise in the field of medicine for cell therapies and tissue engineering. In the present study, we examined the maintenance of cell populations and the differentiation ability of canine MSCs into mesodermal lineages, oesteoblasts and adipocytes, under different culture conditions. MSCs isolated from bone marrow by Ficoll gradient treatment were cultured in (1) ADMEM supplemented with 10% fetal bovine serum (FBS) (basic medium), (2) basic medium supplemented with 10 ng mL–1 basic fibroblast growth factor (bFGF), (3) basic medium supplemented with 1000 IU mL–1 human leukemia inhibitory factor (hLIF), and (4) basic medium supplemented with 10 ng mL–1 bFGF and 1000 IU LIF. In Experiment 1, under a phasecontrast microscope (40×), morphological changes of MSC populations, including the appearance of more spherical shapes and extending processes arranged into network-like structures, were observed in all media with cytokines added, but not in basic medium. In Experiment 2, MSCs cultured under different conditions at 3–5 passages were induced into osteogenic and adipogenic lineages, and their characteristics were evaluated based on the formation of mineral nodules by alkaline phosphatase-positive cells, von Kossa and alizarin red staining of osteoblasts, and oil red O staining of lipid vacuoles following the protocol described earlier (Jin et al. 2007 Int. J. Dev. Biol. 51, 85–90). Only MSCs cultured in basic medium were successfully differentiated into osteoblasts and adipocytes. In conclusion, this study indicates that supplementation of culture media with cytokines might have a negative effect on the pluripotency maintenance of canine MSCs. Further studies are required to evaluate more characteristics of gene expression in canine MSCs.