380 ISOLATION AND DIFFERENTIATION OF MESENCHYMAL STEM CELLS DERIVED FROM CANINE AMNIOTIC FLUID

The dog is biologically comparable with humans with respect to stem cell kinetics, haematopoietic demand, and responsiveness to cytokines. The availability of canine mesenchymal stem cells allows for the establishment of the dog as a large animal model for testing the safety and efficacy of mesenchymal stem cells replacement therapy. Large animal models, such as the dog, are invaluable for working out the practicalities of a therapeutic regimen in a complex system and for verifying established mechanistic theories. Therefore, canine stem cells present the potential for unique and exciting biological opportunities. Recent observations also indicate that stem cells derived from second-trimester amniocentesis are pluripotent, capable of differentiating into multiple lineages, including representatives of all 3 embryonic germ layers. Compared with embryonic stem cells, amniotic fluid stem cells can be obtained without destroying embryos, thus avoiding much ethical controversy. The aim of the current study was to investigate adipogenic, osteogenic, and chondrogenic in vitro differentiation potential of canine amniotic fluid-derived mesenchymal stem cells by biological characterization. We successfully isolated and characterized canine amniotic fluid-derived mesenchymal stem cells (cAFS). Expression of stem cell-specific marker OCT3/4, SOX2, and NANOG was confirmed by RT-PCR. Flow cytometric analysis showed that cAFS were positive for CD44, CD29, and CD90 but negative for CD34. Immunocytochemical analysis also showed the expression of alkaline phosphatase, SOX2, SSEA-1, and SSEA-4. Following incubation with specific adipogenic, osteogenic, and chondrogenic agents, cAFS stained positive by Oil Red O and Alizarin Red S, respectively. In conclusion, according to the preview studies on other mammalians, cAFS is an appropriate source of pluripotent stem cells. Here, we demonstrated that cAFS has a high adipogenic, osteogenic, and chondrogenic differentiation potential in vitro. Therefore, amniotic fluid might be a suitable alternative source of stem cells. This study was financially supported by KOSEF (grant #R01-2008-000-21076-0), research fund of Chungnam National University, and the Korean MEST, through the BK21 program for creative research in animal biotechnology.