Differentiation of bone marrow mesenchymal stem cells into epithelial cells induced by fetal intestinal connective tissue
- VernacularTitle:胎儿肠壁结缔组织诱导骨髓间充质干细胞分化为上皮细胞
- Author:
Weiwei WANG
;
Bei WANG
;
Jianhua ZHANG
;
Rong JIANG
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2008;12(25):4952-4956
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: Mesenchymal stem cells have ability of multi-directional differentiation, and can be induced to differentiate into epithelial cells in vitro. The differentiation of epithelial cells in fetal primitive gut is induced by mesochymal cells of intestines. The report of bone marrow mesenchymal stem cells (BMSCs)differentiate into epithelial cells induced by intestinal connective tissue has not been identified. OBJECTIVE: To observe the possibility of BMSCs differentiate into epithelial cells induced by fetal intestinal connective tissue. DESIGN: Culture in "vitro and comparative observation. SETTING: The experiment was carried out in the Department of Histology and Embryology, Chongqing Medical University from July 2004 to July 2006. MATERIALS: Epidermal growth factor (EGF, Sigma); CK, CK20 (Zhongshan Bio-Tech, Co.,Ltd). Bone marrow of limbs was collected from 6 aborted fetus samples aged 4-5 months. Adding Ficoll to centrifugalize, BMSCs were isolated, cultured and proliferated. The intestinal segment about 15 mmx5 mm was obtained sterilely from fetal duodenal papilla to colon, then muscular tunic and adventitia were peeled. Enzymatic digestion was used to remove the epithelial cells on the mucosa surface. The lump of intestinal connective tissue was cut into 15 minx5 nun. Fetus samples were provided by Department of Gynaecology and Obstetrics in Clinical College, all the parturients agreed to the offer, and the experiment was approved by the hospital ethical committee. METHODS: The experiment was assigned into 4 groups. In groups A and B, the DAPI labelled BMSCs (3x104) at the third generation were transplanted on the submucosa of intestinal connective tissue;.In groups C and D, the DAPI labelled BMSCs were only cultured on the cover glass; In groups B and D, EGF in final concentration of 10 ng/mL was added. MAIN OUTCOME MEASURES: After cultured for 12 days, the morphous and distribution of DAPI labelled BMSCs were observed under fluorescence microscope; the cell morphous on surface of the same intestinal connective tissue was observed with hematoxylin-eosin stain. Expressions of CK and CK20 as well as whether the intestinal connective tissue express EGF were all detected by immnnohistochemical stain. Production of basal lamina between the DAPI labelled cells and connective tissue was assayed with periodic acid-Schiff reaction.RESULTS: On the surface of the tissue lump in groups A and B, the DAPI labelled BMSCs could be seen under fluorescenc microscope, in the same section stained by hematoxylin-eosin, there were some epithelioid cells. During culture, the medium were changed several times, the marked cells that were not eluted, grew on the surface of connective tissue.Immunohistochemistry revealed, in groups A and B, cells on the surface of the tissue lump both expressed CK and CK20. Immunofluorescence stain shown that some of cells on the tissue lump had the DAPI marked nncelus (sapphirine fluorescence) and CK (red fluorescence) in cytoplasm simultaneously. It indicated that the 3rd generation of DAPI labelled BMSCs had differentiated into epithelial cells. Cells in group C were negative for CK and CK20, but those in group D were positive, which indicated that EGF could induce BMSCs differentiate into epithelial cells. There were glycosidoprotein with basal membrane-like structures appeared between the cells and connective tissues in periodic acid-schiff stained section. The uncultured connective tissue expressed EGF. CONCLUSION: Both EGF and fetal intestinal connective tissues can induce BMSCs differentiate to epithelial cells in vitro; the EGF presented in the intestinal connective tissues may be one of factors in this induction process.
