Enrichment of hepatocytes differentiated from mouse embryonic stem cells as a transplantable source.
BACKGROUND
We previously reported that hepatocytes can be differentiated from embryonic stem (ES) cells by way of embryoid body (EB) formation and are transplantable into the mouse liver. However, the transplantation of EB-derived cells frequently resulted in teratoma formation in the recipient liver. In the present study, we eliminated the tumorigenic cells from EB outgrowths and examined the effects of enriched ES-cell-derived hepatocyte transplantation into an injured liver.


METHODS
On day 15 in culture, the EBs were partially disaggregated and subcultured. Hepatocytes in the subcultured cells were examined by the expression of hepatocyte markers. Undifferentiated cells contaminating in the EB-derived cells were eliminated by Percoll discontinuous gradient centrifugation. Furthermore, undifferentiated cells, endothelial cells, and macrophages were eliminated by magnetic cell sorting using platelet/endothelial cell adhesion molecule (PECAM)-1 and Mac-1 antibodies. These enriched ES-cell-derived hepatocytes were then transplanted into the injured mouse liver.


RESULTS
Percoll centrifugation and PECAM-1 antibodies eliminated the undifferentiated cells expressing Oct-3/4 from the EB-derived cells. ES-cell-derived hepatocytes showed expression of liver-related genes, synthesis of urea and glycogen, and structural characteristics during subculture. A transplantation study showed that the enriched ES-cell-derived hepatocytes integrated into the injured mouse liver and produced no teratomas. When the ES-cell-derived hepatocytes were transplanted into a CCl4-injured liver, the liver function was subsequently improved.


CONCLUSIONS
Functional hepatocytes can be differentiated from mouse ES cells by way of EB formation. The elimination of undifferentiated cells from the EBs provides transplantable cells for liver failure without tumorigenicity.
Kumashiro
Yuji
Y
Department of Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, Tokyo, Japan.
Asahina
Kinji
K
Ozeki
Rie
R
Shimizu-Saito
Keiko
K
Tanaka
Yujiro
Y
Kida
Yujiro
Y
Inoue
Kouji
K
Kaneko
Michinari
M
Sato
Tetsuji
T
Teramoto
Kenichi
K
Arii
Shigeki
S
Teraoka
Hirobumi
H
D000818:Animals; D002454:Cell Differentiation; D004622:Embryo, Mammalian / Q000166:cytology*; D005260:Female; D022781:Hepatocytes / Q000166:cytology / Q000637:transplantation*; D051379:Mice; D008810:Mice, Inbred C57BL; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000502:physiology; D013234:Stem Cells / Q000166:cytology*
D019408:Platelet Endothelial Cell Adhesion Molecule-1