Mesenchymal stem cells and endothelial progenitor cells obtained from rabbit bone marrow with differential adhesion methods and their biological characteristics / 中国实验血液学杂志

ABSTRACT

This study was aimed to evaluate an effective and stable method for obtaining mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC) from the rabbit bone marrow and to investigate the biological characteristics of MSC and EPC. Mononuclear cells were obtained from rabbit bone marrow using density gradient method, and were differentially adhered to the cell culture plate enclosed with fibronectin. Then, MSC and EPC were amplified with EGB-2MV medium. Trypan blue method was used to test the passage survival rate. Growth curve, MTT and DNA cycle were used to evaluate the proliferation ability of MSC and EPC. MSC were identified with induced differentiation into the osteoblasts and adipocytes, and their immune phenotype was detected by flow cytometry (FCM). EPC were characterized by the special digestion of Dil-ac-LDL, FITC-UEA-I, and the conjunction with CD133, VEGFR2/KDR and CD34, their purity was also calculated. The results indicated that the colony was obviously formed when the mononuclear cells were cultured for 24 hours and, 80% of the cells became long spindle and integrated at d 8. Cells, which were adhered for twice, were cultured with EGM-2MV medium, began to extend at d 3, and became strip-shaped and integrated for about 80% at d 8. Passage survival rates were more than 90% for both cells, and after passage 2 the growth curve was like "S". Optical density was changed obviously when the cells were cultured for 3 - 5 d, but there were no significant difference of cell cycles between MSC and EPC, which G0-G1 was (93.32 ± 1.65)% and (93.05 ± 1.95)% respectively. Positive rates were (99.7 ± 1.12)%, (99.1 ± 2.33)%, (4.8 ± 0.38)%, (6.8 ± 0.49)% and (0.4 ± 0.08)% for CD90, CD44, CD14, CD45 and CD79a respectively. MSC were identified by induced differentiation into osteoblasts and adipocytes. Positive rates of the EPC, which were adhered for twice and passaged 2, were (82.1 ± 3.4)% for fluorescent staining of Dil-ac-LDL and FITC-UEA-I, and (74.2 ± 3.2)%, (64.7 ± 4.3)% and (43.5 ± 1.5)% for CD133, VEGFR2/KDR and CD34 respectively. It is concluded that high-purity MSC can be obtained with density gradient and differential adhesion method, and high-proliferation EPC can be cultured with EGM-2MV medium in cell plates enclosed with fibronectin, so they may become the optimal seed cells for tissue engineering study.