Enrichment of Hepatocytes Differentiated from Mouse Embryonic Stem Cells as a Transplantable Source

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Abstract

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.

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