| Bone marrow stromal stem cells ( BMSCs) are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues , including bone, cartilage, and fat et al. Under controlled in vitro conditions , these cells could be induced to differentiate exclusively into the adipocyt-ic, chondrocytic, or osteocytic lineages. Using BMSCs to fabricate engineered tissue is a foreground technology, which offer an ideal therapy for reconstruction of imperfect tissue caused by various reasons.The purpose of this study were to observe the growth characteris of cultured murine marrow stromal stem cells (BMSCs ) ;to investigate the differentiate potential of MSCs under the induced condition; to evaluate the differentiated state of BMSCs on the basis of the Hoechst 33342 stained degree of cells. Based the experiment, we expect to understand the biological characters of BMSCs further more, and offer reliable bases before they are applied to clinic.Methods1. A population of homogeneous murine marrow stem cells were isolated and cultured from bone marrow by their abilities to proliferate in culture with an attached well-spread morphology. And the shapes and structures of the cells were observed by phase contrast microscope.2. Immunohistochemistry was used to examine the expression of the suface markers: CD34 CD 44 CD54 and CD106.3. The osteogenic differentiation were induced under the influence of 10-8M dexamethasone, 10 mmol/L p-glycerol phosphate, and 50ug/ml ascorbate and in the presence of 15% v/v FBS. Two weeks later, examine the expression of alkaline phosphatase and type I collagen.4. Adipogenic differentiation were induced in the expanded marrow stem cell cultures by treatment with 0. 5 mmol/L l-methyl-3-isobutylxanthine, 1 umol/L dexamethasone, and 1 ug/ml insulin. Observe the changes of induced cells. Two weeks later, Oil red-0 stain show adipogenic differentiation.5. Drop the liquid including Hoechst 33342 into foster which creeped original and passed BMSCs. Incubate 30 min avoided the light. Remove the dye-stuff. Rinse 3 times with PBS. Observe the stained degree of cells by Olympus-BX51 fluorescence phase contrast microscope.Results1. Mor phologic observation of BMSCs: 24h after the initial seeding, BMSCs attached the surface of foster. Purification of BMSCs was achieved by removal of the nonadherent cells during subsequent changes of medium. There were distinct formation of clonies 4 ~5 days later, and the shape, the size and the density of the clonies were different; and BMSCs reached confluence 6 ~ 8 days later.At the fourth and fifth day of initial culture, we observed some lipocytes by phase contrast microscope. Oil red-0 stain show positive.2. The result of the surface markers expression of BMSCs : the cells were negative for reactivity to antigen CD34 which is common on cells of the hemato-poietic lineages; and positive forCD44,CD54 and CD106.3. Osteogenic Differentiation of BMSCs : after 3-4 days , the shape of the cells change from slightly shuttle into triangle and polygon; at the eighth day, the isolated BMSCs fonned aggregates or nodules. The expression of alkaline phosphatase and type I collegon were positive.4. Adipogenic Differentiation of BMSCs: after 5 days, the shape of cells change from polygon to ellipse ; at 7 ~9 days, there were lipid vacuoles within cells. After 14 days, there were apparent and accumulation of lipid-rich vacuoles within cells with an eccentric deviation of the nucleus. The Oil red-0 stained result of the induced cells was positive.5. The result of Hoechst 33342 staining: most nucleolus of original cells were not stained. But the nucleolus of the fifth generation were stained generally , and the stained degree of cells are different.Conclusion1. Bone marrow marrow stromal stem cells( BMSCs) could be isolated from bone marrow, and proliferated in vitro.2. Bone marrow marrow stromal stem cell... |
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