Biological and cellular features of mesenchymal stem cells from canine bone marrow

Objective To study and establish the method for isolation,culture,proliferation and identification of canine bone marrow mesenchymal stem cells (MSCs).MethodsTen ml canine bone marrow aspirate,taken from the iliac crest of normal canines,were diluted 1∶1 with Dullbecco's Modified Eagle Medium (DMEM).The washed cells were resuspended in DMEM to a final volume of 10 ml and layered over an equal volume of 1.063 g/ml Percoll gradient solution.After centrifugation at 600 g for 30 minutes,the mononuclear cells(MNCs)were recovered from the gradient interface and washed with PBS.Percoll fractionated MNCs were suspended in DMEM containing 1 g/L of glucose supplemented with 10% fetal bovine serum,100 U/ml penicillin,100 μg/ml streptomycin,and 25 μg/ml amphotericin B.All cells were plated in 10 ml of medium in a culture dishes.The cultures were maintained at 37℃ in air of 5% CO 2,with an initial medium change at 24 hours after initial plating and then medium change every 3 or 4 days.The MSCs were identified by special cellular surface antigens and pluripotent committing differentiation potential.Results The methodology for isolation and expansion of MSCs was established.The mononuclear cells from canine bone marrow were separated on Percoll gradient sedimentation solution at 600 g for 30 minutes in small percentage (about 0.01～0.001%).The MSCs were set very good in culture of LG-DMEM supplemented with 10% selected fetal bovine serum.MSCs attached and grew as fibroblastic cells at 24 hours after initial plating,about 10 -6 out of mononuclear cells.The hematopoietic stem cells that did not attach to the dish were washed from the culturewith each medium change.MSCs grew rapidly,developed into visible symmetric colonies at about 3 days and reached confluence at 7 to 10 days.While the cells were permitted to proliferate to confluence,MSCs were spindle-shaped morphology,small and arranged like pectinate.As many as(1.1～1.5)×10 7 cells were generated by passage 3 from a 10 ml marrow aspirate.The MSCs did not differentiate spontaneously during culture expansion and maintained a normal mesenchymal stem cells biological features.These expanded attached mesenchymal stem cells were positive for SH2,and negative for CD45 surface antigen.The mesenchymal stem cells have shown the ability to give rise to a varity of differentiated cell types such as cardiomyocytes,adipocytes and osteocytes.Conclusions The mesenchymal stem cells from canine bone marrow could be generated,cultured and expanded in vitro.These cells displayed a stable phenotype,biological and cellular features.The cultivation and selective differentiation of MSCs should provide a new cell source for stem cells transplantation,and the potential of new therapeutic approaches for the restoration of damaged or diseased tissue.