Isolation of rabbit bone marrow mesenchymal stem cells and differentiontion into bone, cartilage, and adipose cell lineages

By far rabbit marrow-derived mesenchymal stem cells (MSCs) have been used in several experimental cell therapies especially in reconstruction of bone and cartilage defects. While in vitro differentiation potentials of these cells have rarely been investigated, it is the subject of the present study. In this experimental study, rabbit MSCs were extracted and culture-expanded through several passages using 0.5 ml bone marrow sample aspirated from the animal’s tibia. The passaged-3 cells were then cultivated in the osteogenic, adipogenic and chondrogenic conditions for 21 days after which differentiations were evaluated by specific staining as well as RT-PCR analysis for related gene expression. The culture-expanded cells appeared to be fibroblastic in morphology. In osteoinductive cultures, the cells formed nodules which were positively stained red following alizarin red staining. In adipogenic cultures, the cells developed some lipid droplets which were positively stained red with oil red. The sections prepared from the chondroinductive cultures exhibited metachromatic property with toluidine blue staining. According to RT-PCR analysis, the bone-related genes including osteocalcin, osteopontin and collagen I, the adipocyte-specific genes such as LPL, PPAR-alpha and PPAR-gamma, and also the cartilage-specific genes including aggreacan, collagen II and X were expressed in the osteogenic, adipogenic and chondrogenic cultures respectively. Taken together, MSCs isolated from rabbit’s bone marrow are tripotent cells with the ability to differentiate into osteocytic, adipocytic and chondrocytic cell lineages.