Optimization of induction and differentiation condition of hepatocyteª²like cells from human fetal marrow mesenchymal stem cells in vitro

    HE Nianª²Hai, ZHAO Wenª²Li, WANG Yuª²Ming

    Institute of Infectious Diseases of Chinese PLA, Southwest Hospital, Third Military Medical University, Chongqing 400038, China

    ¡¾Abstract¡¿ AIM: To establish and optimize the induction and differentiation condition for human fetal marrow mesenchymal stem cell (MMSCs) differentiation into hepatocyteª²like cells, observe the differentiation procedure and identify the phenotypes of MMSCs. METHODS: MMSCs, isolated from fetal marrow, were induced in vitro as the following six groups: control group, induction groups 1-5. Morphological changes and cytobiological characteristics of the induced MMSCs were observed during induction. Expression of hepatocyte markers such as ALB and CK18 was measured with immunohistochemistry. RESULTS: Cells in control group kept the long spindle shape, with shape agent as 4.5¡À1.0 and small size, proliferated quickly, submitted typical fibroblast whirlpool or flamboyancy growth. Five to seven branches were observed at the late culture period, no cell rounded, and the rate of dikaryocyte was less than 0.1%. They gradually aged, the amount of granules increased, and there was no expression of ALB and CK18. Branches of cells in all induction groups decreased during culture, and 1-3 branches were common. Cells without matrix kept long spindle shape growth, and part of the induction cells with matrix changed from long to round in shape and shape agent reduced. AZA pretreatment inhibited the cell hyperplasia, and cell changed from long to short in shape. PDGFª²BB pretreatment had no inhibition to cell proliferation, and cells branched easily. Immunohistochemical staining in all the induction groups showed that ALB and CK18 expressed positively at day 21. In induction groups 2 to 5, the rate of cell rounding and dikaryocytes and ALB and CK18 positive cells were higher than those in control group and induction group 1 (P<0.05). CONCLUSION: Under noª²serumª²culture condition, high ratio hepatocyteª²like cells in induced MMSCs can be obtained by the condition of induction group 4: in which 10 g/L Matrigel is used as matrix, MMSCs are pretreated with 2.5 mmol/L AZA for 10-12 h, and HGF 10 ¦Ìg/L+FGF4 10 ¦Ìg/L, used as promoters, are added into HGM medium. ......