Hepatoblast-like cells enriched from mouse embryonic stem cells in medium without glucose, pyruvate, arginine, and tyrosine.
In order to enrich hepatocytes differentiated from embryonic stem cells, we developed a novel medium. Since only hepatocytes have the activity of ornithine transcarbamylase, phenylalanine hydroxylase, galactokinase, and glycerol kinase, we expected that hepatocytes would be enriched in a medium without arginine, tyrosine, glucose, and pyruvate, but supplemented with ornithine, phenylanaline, galactose, and glycerol (hepatocyte-selection medium, HSM). Embryoid bodies were transferred onto dishes coated with gelatin in HSM after 4 days of culture. At 18 days after embryoid body formation, a single type of polygonal cell survived with an enlarged intercellular space and micorvilli. These cells were positive for indocyanine green uptake and for mRNAs of albumin, transthyretin, and alpha-feto protein, but negative for mRNAs of tyrosine aminotransferase, alpha1-antitrypsin, glucose-6-phosphatase, and phosphoenol pyruvate carboxykinase. Since cells in HSM were positive for cytokeratin (CK)8 and CK18 (hepatocyte markers) and for CK19 (a marker of bile duct epithelial cells), we concluded that they were hepatoblasts. They showed weaker expression of CCAAT/enhancer-binding protein (C/EBP)alpha than fetal liver (18.5 days of gestation) and expression of C/EBPbeta at a similar level to that of fetal liver. These data support our conclusion that HSM allows the selection of hepatoblast-like cells.
Tomizawa
Minoru
M
Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba City, Chiba, 260-8670, Japan. nihminor-cib@umin.ac.jp
Toyama
Yoshiro
Y
Ito
Chizuru
C
Toshimori
Kiyotaka
K
Iwase
Katsuro
K
Takiguchi
Masaki
M
Saisho
Hiromitsu
H
Yokosuka
Osamu
O
D000818:Animals; D001120:Arginine / Q000378:metabolism*; D018929:Cell Culture Techniques; D002460:Cell Line; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D051379:Mice; D008810:Mice, Inbred C57BL; D019289:Pyruvic Acid / Q000378:metabolism*; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism*
D003470:Culture Media; D014443:Tyrosine; D019289:Pyruvic Acid; D001120:Arginine; D005947:Glucose