title: Selective enrichment of hepatocytes from mouse embryonic stem cells with a culture system containing cholestatic serum.
abstract: OBJECTIVE
  There is increasing evidence indicating that embryonic stem (ES) cells are capable of differentiating into hepatocyte-like cells in vitro. However, it is necessary to improve the differentiation efficiency so as to promote the clinical application. Here, we report an efficient culture system to support hepatocyte 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 storage 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 selective medium containing cholestatic serum, numerous epithelial cells resembling hepatocytes were observed. The RT-PCR analysis showed that undifferentiated ES cells did not express any hepatic-specific markers; however, in the presence of sodium butyrate and conditional selective medium containing cholestatic serum, hepatic differentiation markers were detected. Immunofluorescence staining showed that those ES-derived hepatocytes were alpha-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 enriched ES-derived hepatocyte-like cells by inducing lineage differentiation and enhancing lineage proliferation.
  
  
  CONCLUSIONS
  The conditional selective medium containing 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.
authors.affiliation: Department of Hepatobiliary Surgery, The Second Affiliated Hospital of Sun Yat-sen University, Guangzhou 510120, China. j-min@yeah.net
authors.forename: Jun
authors.initials: J
authors.lastname: Min
authors.forename: Chang-zhen
authors.initials: CZ
authors.lastname: Shang
authors.forename: Ya-jin
authors.initials: YJ
authors.lastname: Chen
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Lu
authors.initials: L
authors.lastname: Liu
authors.forename: Xiao-geng
authors.initials: XG
authors.lastname: Deng
authors.forename: Mei
authors.initials: M
authors.lastname: Yang
authors.forename: Dong-ping
authors.initials: DP
authors.lastname: Chen
authors.forename: Jun
authors.initials: J
authors.lastname: Cao
authors.forename: Er-wei
authors.initials: EW
authors.lastname: Song
authors.forename: Ji-sheng
authors.initials: JS
authors.lastname: Chen
mesh_terms: D000818:Animals; D001483:Base Sequence; D001769:Blood; D002779:Cholestasis / Q000473:pathology*; D017077:Culture Media, Conditioned; D017931:DNA Primers; D053595:Embryonic Stem Cells / Q000166:cytology*; D022781:Hepatocytes / Q000166:cytology*; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D017077:Culture Media, Conditioned; D017931:DNA Primers