Hepatic differentiation from embryonic stem cells in vitro

作     者：胡安斌 蔡继业 郑启昌 何晓青 潘运龙 李凌松 

作者机构：Department of General Surgery College Huaqiao Hospital Stem Cell Research Center 

出 版 物：《Chinese Medical Journal》 (中华医学杂志（英文版）)

年 卷 期：2003年第116卷第12期

页      面：1893-1897页

核心收录：

学科分类：1002[医学-临床医学] 100210[医学-外科学(含：普外、骨外、泌尿外、胸心外、神外、整形、烧伤、野战外)] 10[医学] 

基　　金：ThisworkwassupportedbytheNationalKeyResearchFoundationofChina ( 973 ) (No 2 0 0 1CB5 10 10 1)andtheNationalNaturalScienceFoundationofChina (No 3 0 2 3 0 3 5 0 No 60 2 780 14 ) 

主　　题：Animals Cell Differentiation Cells, Cultured Embryo Hepatocytes Liver Mice Mice, Inbred BALB C Research Support, Non-U.S. Gov't Stem Cells 

摘      要：OBJECTIVE: To investigate an method for hepatic differentiation from embryonic stem cells (ES cells) in vitro and the resulting differentiation ratio, in order to develop a procedure for producing a new type of hepatocyte for hepatocyte replacement therapy in the treatment of liver failure. METHODS: ES cells from Balb/C mice were cultured and maintained in an undifferentiated state in gelatin-coated dishes using Dulbecco s modified Eagle s medium (DMEM) containing 1000 U/ml leukemia inhibitory factor (LIF). Then, LIF was withdrawn from the DMEM to allow the ES cells to develop into embryonic bodies (EBs). EBs were plated onto tissue culture dishes, and growth factors such as acidicfibroblast growth factor (aFGF) and hepatocyte growth factor (HGF) were added to the medium to promote directional differentiation. The course of development and differentiation was observed dynamically using an inversion microscope. The expression of hepatic proteins, such as alpha-fetoprotein (AFP), albumin (ALB), cytokeratin 8 (CK8), cytokeratin 18 (CK18), in cytoplasm was analyzed by immunocytochemistry (ICC). The concentration of ALB in the medium was determined dynamically by radioimmunoassay (RIA). RESULTS: ES cells replicated as clones, without differentiating, in DMEM containing LIF. They developed into EBs in medium without LIF. Our ICC assay showed that differentiating cells did not express hepatic proteins, such as AFP, ALB, CK8, and CK18 until day 7, day 9, day 11, and day 11, respectively (up to 2 days later when growth factors are not present). The concentration of AFP in the medium was first detected on day 8, at a concentration of 3.4 ng/ml, and increased to 22.8 ng/ml by day 15. The concentration of ALB in the medium was 0.2 micro g/ml on day 11, and increased to 2.2 micro g/ml by day 15. ALB-positive cells under ICC manifest morphological structures were consistent with normal mouse hepatocytes. The differentiation ratio of hepatocytes in the ES cell differentiation syst