In vitro differentiation and identification of adult bone marrow mesenchymal stem cells into isletsecreting cells
- VernacularTitle:成人骨髓间充质干细胞体外诱导分化为胰岛素分泌样细胞及其鉴定
- Author:
Nailong YANG
;
Fen YANG
;
Lili XU
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2007;0(38):-
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: There is no ideal method about adult bone marrow mesenchymal stem cells (BMSCs) differentiate into islet-secreting cells and appreciation in vitro at present. Transgene requests strict conditions and complex program. The induction using chemicals is a present-used method. OBJECTIVE: To investigate the culture conditions for inducing adult BMSCs into islet-like cells in vitro. DESIGN, TIME AND SETTING: The cytology in vitro experiment was performed at the Central Laboratory of Affiliated Hospital of Medical College of Qingdao University from January to October 2007. MATERIALS: Bone marrow was collected from diabetic patients undergoing autologous stem cell transplantation at Department of Endocrinology, Affiliated Hospital of Medical College of Qingdao University after signing the informed consent. Epidermal growth factor and basic fibroblast growth factor were obtained from Peprotech Asia. B27 adjunct was purchased from Gibco. Nicotinamide, 2-mercaptoethanol, glutamine and dithizone were bought from Sigma, USA. METHODS: The adult BMSCs were isolated by Percoll from adult bone marrow aspirates and cultured in LG-DMEM, were suspended by Trypsin and passaged for subsequent passages. At the third to fifth passages, BMSCs were incubated at a density of 1?108 L-1 and were induced differentiation into islet-like cells through three developmental stages. In the first stage, BMSCs were incubated in HG-DMEM supplemented with 2-mercaptoethanol, glutamine for 2 days. In the second stage, BMSCs were incubated in HG-DMEM supplemented with epidermal growth factor, basic fibroblast growth factor, B27 and glutamine for 6 days. In the third stage, BMSCs were incubated in HG-DMEM supplemented with nicotinamide and 2-mercaptoethanol for 6 days. BMSCS in the control group were only incubated in the HG-DMEM. MAIN OUTCOME MEASURES: Morphological changes in BMSCs were analyzed under a phase contrast microscopy. Duodenal Homeobox 1 (PDX-1) expression was detected during the second and differentiation stage by immunofluorescence assay. Islet ?-like cell clusters from the 3rd stage were identified by positive dithizone staining. The insulin secretions under the stimulation of high or low glucose were detected by electrochemiluminescence immunoassay. RESULTS: The undifferentiated BMSCs exhibited adherent long spindle-shaped cells. After induction, cells gradually became round and formed clusters. Cells expressed the PDX-1 gene at 8 days and formed islet-like cell clusters that exhibited positive dithizone staining at 14 days. After high and low glucose treatment, no insulin was detected in the control group; insulin content significantly increased at 14 days (t=3.638-9.387, P
