Combination of different factors directly induces human embryonic stem cells into hepatocyte-like cells

Abstract

Objective To establish an efficient culture system of inducing human embryonic stem cells (hESCs) into hepatocyte-like cells.Methods For hepatic differentiation,hESCs were cultured in basement membrane extract coated plates in culture system supplemented with 100 μg/L activin A for 3 days.The differentiated cells were then cultured in medium containing 20 μg/L bone morphogenetic protein-4 (BMP-4) and 10 μg/L basic fibroblast growth factor (bFGF) for 4 days,and were further differentiated in medium containing 10 μg/L hepatocyte growth factor (HGF) for 5 days.Finally,cells were matured in medium containing 10 μg/L oncostatin M (OSM) and 1 × 10-7 mol/L Dex for another 4 days.At the sixteenth day of cell differentiation,reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence examinations were carried out to detect the hepatic-specific genes and proteins expression of differentiated cells.Periodic acid schiff (PAS) and Indocyanine green (ICG) tests were performed to determine the hepatic-specific functions of differentiated cells.Results During the induction of hESCs,the induced cells showed a significant hepatocyte-like morphology.RT-PCR results showed positive gene expression of alpha fetal protein (AFP),cytokeratin 7 (CK7),cytokeratin 19 (CK19),hepatocyte nuclear factor 4α (HNF4α) and AAT.Immunofluorescence results showed hepatic-specific markers such as ALB,AFP,CK19 and CYP7A1 were also positively expressed.PAS reaction and cellular uptake of ICG indicated the differentiated hESC possessed the hepatic functions of glycogen synthesis and indocyanine grenn uptake.Conclusion The combination of different inducing factors such as activin A,BMP-4,bFGF,HGF,OSM and Dex could successfully induce hESCs to differentiate into functional hepatocyte-like cells. Key words: Embryonic stem cells; Hepatocyte-like cells ; Differentiation