Selective enrichment of hepatocytes from mouse embryonic stem cells with a culture system containing cholestatic serum.
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.
Min
Jun
J
Department of Hepatobiliary Surgery, The Second Affiliated Hospital of Sun Yat-sen University, Guangzhou 510120, China. j-min@yeah.net
Shang
Chang-zhen
CZ
Chen
Ya-jin
YJ
Zhang
Lei
L
Liu
Lu
L
Deng
Xiao-geng
XG
Yang
Mei
M
Chen
Dong-ping
DP
Cao
Jun
J
Song
Er-wei
EW
Chen
Ji-sheng
JS
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
D017077:Culture Media, Conditioned; D017931:DNA Primers