Effects of in vitro suspension culture in soft agar medium on differentiation of embryonic hepatic stem cells
10.3760/cma.j.issn.1007-8118.2010.07.015
- VernacularTitle:体外琼脂克隆培养对胎肝干细胞分化的影响
- Author:
Nan YOU
;
Kaishan TAO
;
Ren LI
;
Zhi SONG
;
Ming ZHANG
;
Zhiquan GAO
;
Kefeng DOU
- Publication Type:Journal Article
- Keywords:
Hepatic stem cell;
Soft agar colon cultures Differentiation
- From:
Chinese Journal of Hepatobiliary Surgery
2010;16(7):531-534
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop an ideal cultural method to amplify embryonic hepatic stem cells and inhibit their differentiation in vitro. Methods Suspension of ED 14 Fischer (F) 344 rat em-bryonic hepatic stem cells was prepared by collagenase digestion and mechanical disaggregation. Then cells were divided into two groups randomly. The cells in group 1 were seeded into type I collagen-coated plates by adherent culture while those in group 2 were seeded into soft agar medium by suspen-sion culture. After culture for 2 weeks, the morphology and ultrastructure of cells in both groups were observed and compared by inverted microscope and transmission electron microscope, respectivley.The expression of CD90. 1 and CD49F, the two specific stem cell surface markers, was tested by flow cytometry to manifest the establishment of embryonic hepatic stem cells. Alkaline phosphatase stai-ning was used to detect stem cell differentiation. Result Embryonic hepatic stem cells in group 2 were characterized by higher nucleus-cytoplasm ratio and less cell organelles, higher expression of CD90. 1 and CD49F, and stronger positive reaction for alkaline phosphatase staining compared with those in group 1. Moreover, the cells in group 1 showed significant differentiation features. Conclusion Em-bryonic hepatic stem cells cultured suspendedly in soft agar medium experience less differentiation than those adherently cultured in serum-added culture medium, and can proliferate and form clone ball with a specific stem cell feature.
