Hepatic Maturation of hES Cells by Using a Murine Mesenchymal Cell Line Derived from Fetal Livers

Hepatocytes derived from human embryonic stem (hES) cells are a potential cell source for regenerative medicine. However, the successful differentiation of hES cells into mature hepatocytes has been difficult to achieve because the definitive mechanisms governing hepatocyte differentiation have not yet been well defined. The CD45⁻ CD49f  �  Thy1⁺ gp38⁺ mesenchymal cells that reside in murine fetal livers induce hepatic progenitor cells to differentiate into mature hepatocytes by direct cell–cell contact. A cell line named MLSgt20 was also successively established from these mesenchymal cells. The MLSgt20 cells possess the ability to promote the hepatic maturation of not only murine ES cells but also hES cell-derived endodermal cells. hES cells were treated with a two-step procedure for hepatic maturation; first, hES cells were differentiated into endodermal cells or hepatic progenitor cells, and second, hES cell-derived endodermal cells were matured into functional hepatocytes by co-culture with MLSgt20 cells, forming cell aggregates. The hES cell-derived hepatocyte-like cells possess hepatic functions. In this chapter, we describe a two-step protocol for the hepatic maturation of hES cells utilizing the MLSgt20 cells.