There is increasing evidence to suggest that embryonic stem cells (ESCs) are capable of differentiating into hepatocytes in vitro. In this study, we used a combination of cytokines and sodium butyrate in a novel three-step procedure to efficiently direct the differentiation of mouse ESCs into hepatocytes. Mouse ESCs were first differentiated into definitive endoderm cells by 3 days of treatment with Activin A. The definitive endoderm cells were then differentiated into hepatocytes by the addition of acidic fibroblast growth factor (aFGF) and sodium butyrate to the culture medium for 5 days. After 10 days of further in vitro maturation, the morphological and phenotypic markers of hepatocytes were characterized using immunohistochemistry, immunoblotting, and reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, the cells were tested for functions associated with mature hepatocytes, including glycogen storage and indocyanine green uptake and release, and the ratio of hepatic differentiation was determined by counting the percentage of albumin-positive cells. In the presence of medium containing cytokines and sodium butyrate, numerous epithelial cells resembling hepatocytes were observed, and approximately 74% of the cells expressed the hepatic marker, albumin, after 18 days in culture. RT-PCR analysis and immunohistochemistry showed that these cells expressed adult liver cell markers, and had the abilities of glycogen storage and indocyanine green uptake and release. We have developed an efficient method for directing the differentiation of mouse ESCs into cells that exhibit the characteristics of mature hepatocytes. This technique will be useful for research into the molecular mechanisms underlying liver development, and could provide a source of hepatocytes for transplantation therapy and drug screening.

OBJECTIVES Histone deacetylase (HDAC) is an important therapeutic target in cancer. Two of the main anticancer mechanisms of HDAC inhibitors are induction of terminal differentiation and inhibition of cell proliferation. To investigate the role of HDAC in maintenance of self-renewal and cell proliferation, we treated mesenchymal stem cells (MSCs) that originated from adipose tissue or umbilical cord blood with valproic acid (VPA) and sodium butyrate (NaBu). MATERIALS AND METHODS Human MSCs were isolated from mammary fat tissue and cord blood. We performed MTT assay and flow cytometry-based cell cycle analysis to assess self-renewal of MSCs. In vitro differentiation assays into osteogenic, adipogenic, neurogenic and chondrogenic lineages were conducted to investigate MSC multipotency. Immunocytochemistry, Western blot and reverse transcription-polymerase chain reaction were used to interrogate molecular pathways. RESULTS VPA and NaBu flattened the morphology of MSCs and inhibited their growth. VPA and NaBu activated the transcription of p21(CIP1/WAF1) by increasing the acetylation of histone H3 and H4 and eventually blocked the cell cycle at G2/M phase. The expression level of p16(INK4A), a cdk inhibitor that is closely related to cellular senescence, was not changed by HDAC inhibitor treatment. We performed controlled differentiation into bone, fat, cartilage and nervous tissue to elucidate the role of HDAC in the pluripotency of MSC to differentiate into functional tissues. VPA and NaBu decreased the efficiency of adipogenic, chondrogenic, and neurogenic differentiation as visualized by specific staining and reverse transcription-polymerase chain reaction. In contrast, osteogenic differentiation was elevated by HDAC inhibitor treatment. CONCLUSION HDAC activity is essential for maintaining the self-renewal and pluripotency of MSCs.