Isolation, Culture, In-Vitro Differentiation and Characterization of Canine Adult Mesenchymal Stem Cells

Abstract

Mesenchymal stem cells are multipotent stem cells capable of differentiating into various cell types, including osteocytes, chondrocytes, adipocytes, myocytes, and tenocytes. They are being highly investigated in tissue engineering and regenerative studies. This study was carried out to isolate, identify and examine the differentiation potential of canine mesenchymal stem cells (MSCs). Bone marrow aspirates were collected from the wing of ilium (iliac crest) from the clinical fracture cases of dogs and processed by centrifugation over Histopaque-1077. The putative mesenchymal stem cells were cultured in Dulbecco’s modified eagle’s medium-low glucose supplemented with 10 % fetal calf serum. Initially very few cells adhered to the flask surface, subsequently the cells started to grow and divide gradually which were then subcultured at 60–70 % confluency. MSCs at third to fifth passage were examined for their stemness properties by immunostaining where positive expression of surface markers CD44+, CD105+ and Stro-1 and negative expression of hematopoeitic markers CD34+ and CD45+ were detected which revealed intact stemness properties. Thereafter, Canine MSCs were subjected to differentiation studies by growing them in osteogenic, chondrogenic and adipogenic differentiation media for 21 days. The differentiated cells were characterized by reverse transcriptase-polymerase chain reaction (RT-PCR) for detection of the cell specific marker gene expressions, cytochemistry and also by immunocytochemistry using antibodies against specific markers of these cells. RT-PCR could detect osteopontin, SOX9 and Lpl gene expression in differentiated cMSCs. On cytochemistry, osteogenesis was confirmed by Von Kossa, chondrogenesis by Alcian blue and adipogenesis by Oil Red O stain. Anti-Collagen type I, anti-SOX9 and PPAR-γ antibodies could detect the osteogenic, chondrogenic and adipogenic differentiation of the cMSC, respectively. In conclusion, canine MSCs were successfully isolated and cultured in the laboratory. The isolated cells showed osteogenic, chondrogenic and adipogenic differentiation potential as revealed by cytochemistry, immunocytochemistry and RT-PCR assays.