Isolation, Cultivation, Biological Features Identification And Labelling Of The Canine Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMSCs) are another kind of adult stem cellsaddition to the hematopoietic stem cells that presented in bone marrow. BMSCs have beenattracting much attention by the medical researchers since being discovered in mid-20thcentury, and a series of biological characteristics of BMSCs have been revealed. First of all,as the adult stem cells, BMSCs can differentiate into various phenotypes of each germ layersunder appropriate induction conditions and have abilities to differentiate into more lineagesthan hematopoietic stem cells. Second, compared with embryonic stem cells, BMSCs are easyto obtain and culture, and no limit from the ethical and moral. In addition, BMSCs, withmagic immune suppression, immune conditioning, homing and neoplastic tropism, have greatprospects in the field of tissue engineering and gene therapy. Although many encouragingresults for the study of BMSCs have been achieved, but many basic problems have not beensolved. An effective isolation and purification method of BMSCs has not yet been found.There has been no universally accepted identification method for the isolated BMSCs. In vivotransplantation studies, an efficient and reliable method for marking BMSCs remains to bestudied. To solve these problems, the method of whole bone marrow culture was used to isolatcanine BMSCs in this study. The surface antigens were detected by flow cytometry. Thedifferentiation potentials were identified by in vitro induction towards osteoblasts andmyocardial-like cells. The pEGFP-C3, adenoviral vectors, lentiviral vectors carrying EGFPgene were respectively transfected into canine BMSCs. Transfection efficiency was calculated,and proliferation abilities of BMSCs after transfection were detected. Testing results were asfollows:1. Canine BMSCs with uniform morphology and, vigorous condition were successfullyisolated by the method of whole bone marrow culture.2. The flow cytometry analysis showed that the CD44was positive, CD34and D45werenegative.3. Calcified nodules were observed after osteogenic differentiation and alizarin redstaining. Myofilament-like structures were appeared30days after myocardiomyocyte-likecells differentiation and HE staining. 4. Transfection efficiency was separately calculated and comparatived at72h afterBMSCs transfected by Plasmid pEGFP-C3, adenovirus and lentivirus. Lentiviral transfectionefficiency was much higher than the others, EGFP-positive cells attained more than90%when MOI=100. Lentiviral was an ideal vector to transfect and mark BMSCs.5. Proliferation ability of canine BMSCs after transfection was detected. Plasmid almosthas no effect on cell proliferation. Canine BMSCs proliferation was not significantly affectedby adenovirus when the MOI≤100,while the lentivirus was not significant when MOI≤200.In summary, the whole bone marrow adherent culture method is a convenient andreliable method for the isolation of BMSCs. The isolated canine BMSCs have high purity andstrong ability of proliferation and differentiation. Canine BMSCs labeled bylentiviral-mediated EGFP is more significant efficiency than by pEGFP-C3and adenovirusunder the multiplicity of infection without affecting cell proliferation, which provides an ideallabeling method for subsequent studies of BMSCs transplantation and tissue engineering.