Characterization of mesenchymal stem cells isolated from mouse bone marrow

Abstract

Bone marrow represents the main source of stem/progenitor cells in adult individuals. Due to their high plasticity, these cells are being highly investigated in tissue engineering for restoring the damaged organs. The purpose of this study was to isolate and characterize mesenchymal stem cells (MSC) from mouse bone marrow for their subsequent use in differentiation and transplantation studies. Bone marrow extracted from the medullar channel of the posterior limb bones of adult mice (6-8-week old) was resuspended to obtain a single cell suspension and seeded on tissue culture dishes in DMEM supplemented with 10% fetal calf serum. A homogeneous culture of fibroblast-like cells was generated through repeated passages over a two month period. In agreement with the criteria for defining MSC established by the International Society for Cellular Transplant, these cells expressed specific markers (Sca-1, Stro-1 and CD105) while were devoid of hematopoietic markers (c-Kit, CD14 or CD45), as demonstrated by RT-PCR, ICC and FACS analyses. The pluripotency was established by the ability of these cells to differentiate into osteoblasts, chondrocytes and adipocytes, when cultured in specific differentiation media. Our results revealed that these cells were able to generate adipocytes (induced expression of adipsin and intracellular accumulation of lipid droplets), osteoblasts (induced expression of osteocalcin and extracellular calcium deposits shown by von Kossa staining), and chondrocytes (induced expression of aggrecan and acid mucopolisaccarides stained with Alcian Blue). By concluding, mesenchymal stem cells with multipotent abilities can be successfully derived from mouse bone marrow and can be maintained in culture without losing their potential. These cells represent a valuable source of stem cells for the progress of the regenerative medicine.