Isolation, culture and osteogenic differentiation of mesenchymal stem cells from human umbilical cord: Ultrastructure of cell membrane observed using atomic force microscope
10.3969/j.issn.1673-8225.2010.01.008
- VernacularTitle:人脐带间充质干细胞分离培养及成骨分化:原子力显微镜观察细胞膜表面的超微结构变化
- Author:
Guodong SUN
;
Shixian WU
;
Zhizhong LI
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2010;14(1):33-37
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: Mesenchymal stem cells (MSCs) are commonly observed under the scanning electron microscope, transmission electron microscope and inverted microscope. However, above-mentioned observation methods have disadvantages on observing ultrastructure of cell membrane and cytoskeleton. OBJECTIVE: To establish a more effective and appropriate method to isolate, culture and identification of human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro and to study the ultrastructure of osteogenic differentiation with the atomic force microscope. METHODS: The hUCMSCs were isolated from human umbilical cord by digested with collagenase. After serial subcultivation in vitro, the stem cells were passaged, and osteogenic differentiation was determined by alkaline phosphatase calcium-cobalt staining and alizarin red staining. hUCMSCs immunophenotype was measured by Flow cytometry.The membrane surface ultrastructure of osteogenic differentiation was observed by Atomic Force Microscope before and after induction. RESULTS AND CONCLUSION: The isolated hUCMSCs by digested with collagenase was efficient. After seeded 24 hours, the adherent cell showed spindle shape, polygonal shape and fibroblast-cell-like shape and the size of hUCMSCs was homogeneous. Flow cytometry analysis revealed that CD29, CD44, CD105 were highly expressed on the surface of passages 3 cells, but there was negative for CD34, CD45 and HLA-DR. These cells were high positive for alkaline phosphate staining and also showed significant calcium node using alizarin red staining after 4 weeks culture induction of osteogenic differentiation. Under atomic force microscopy, undifferentiated stem cells demonstrated insignificant microtubule protrution in a parallel distribution following analysis of cytoskeleton before and after differentiation.
