Aim:There is increasing evidence indicating that embryonic stem (ES) cells arecapable of differentiating into hepatocyte-like cells in vitro.However,it is neces-sary to improve the differentiation efficiency so as to promote the clinicalapplication.Here,we report an efficient culture system to support hepatocytedifferentiation from ES cells by utilizing cholestatic serum.Methods:One weekafter the induction of E14 mouse ES cells into hepatocytes with sodium butyrate,cholestatic serum was added into the culture system at various concentrationsand hepatocyte-like cells were induced to proliferate.The morphological andphenotypic markers of hepatocytes were characterized using light microscopy,immunocytochemistry,and RT-PCR,respectively.The function of glycogen stor-age of the differentiated cells was detected by Periodic acid-Schiff (PAS) reaction,and the ratio of hepatic differentiation was determined by counting the albuminand PAS-positive cells.Results:In the presence of conditional selective mediumcontaining cholestatic serum,numerous epithelial cells resembling hepatocyteswere observed.The RT-PCR analysis showed that undifferentiated ES cells didnot express any hepatic-specific markers;however,in the presence of sodiumbutyrate and conditional selective medium containing cholestatic serum,hepaticdifferentiation markers were detected.Immunofluorescence staining showed thatthose ES-derived hepatocytes were α-fetoprotein,albumin,and cytokeratin 18positive,with the ability of storing glycogen.Further determination of the hepaticdifferentiation ratio showed that the application of cholestatic serum efficientlyenriched ES-derived hepatocyte-like cells by inducing lineage differentiation andenhancing lineage proliferation.Conclusion:The conditional selective mediumcontaining cholestatic serum is optimal to selectively enrich hepatocyte-like cellsfrom mixed differentiated ES cells,which may provide a novel method to improvethe hepatic differentiation ratio of ES cells. Aim: There is increasing evidence indicating that embryonic stem (ES) cells are capable of differentiating into hepatocyte-like cells in vitro. However, it is neces-sary to improve the differentiation efficiency so as to promote the clinicalapplication. Here, we report an efficient culture system to support hepatocyted differentiation from ES cells by utilizing cholestatic serum. Methods: One week after the induction of E14 mouse ES cells into hepatocytes with sodium butyrate, cholestatic serum was added into the culture system at various concentrations and hepatocyte-like cells were induced to proliferate. The morphological and phenotypic markers of hepatocytes were characterized using light microscopy, immunocytochemistry, and RT-PCR, respectively. The function of glycogen stor-age of the differentiated cells was detected by Periodic acid-Schiff (PAS) reaction, and the ratio of hepatic differentiation was determined by counting the albumin and PAS-positive cells. Results: In the presence of conditional sel The RT-PCR analysis showed that undifferentiated ES cells didnot express any hepatic-specific markers; however, in the presence of sodiumbutyrate and conditional selective medium containing cholestatic serum, hepatic differentiation markers were detected. Immunofluorescence staining showed that those ES-derived hepatocytes were α-fetoprotein, albumin, and cytokeratin 18 positive, with the ability of storing glycogen. Further determination of the hepatic differentiation ratio showed that the application of cholestatic serum efficiently edited ES-derived hepatocyte-like cells by inducing lineage differentiation andenhancing lineage proliferation. Conlusion: The conditional selective medium con- chining cholestatic serum is optimal to selectively enrich hepatocyte-like cells from mixed differentiated ES cells, which may provide a novel method to improve the hepatic differentiation ratio of ES cells.