Effects of in vitro suspension culture in soft agar medium on differentiation of embryonic hepatic stem cells / 中华肝胆外科杂志

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.