5E). Although BMP signaling did not induce Hex, a functional Hex gene is required for establishment of the liver fate, because BMP-4 was unable to induce Alb expression in Hex−/− endoderm (Fig. 5F). In contrast, BMP-4 did induce Tcf1 expression in the absence of Hex, although the levels were not as high as those observed in the wild-type population (Fig. 5G). Findings from these analyses suggest that Hex and BMP-4 can regulate Tcf1 expression independently, but optimal levels of expression require both pathways. We also evaluate if Hex can induce

BMP-4 mRNA levels. However, Hex did not affect the gene expression levels of BMP-4 (data not shown). To determine if BMP-4 and Hex have an impact on the hepatoblast stage of development, we analyzed the different populations for expression of Dlk1. Tanimizu et al.26 have shown that Dlk1 is expressed GSK2118436 cost on progenitors with hepatoblast potential as fetal liver cells sorted for this marker displayed both hepatocyte and biliary epithelial potential. Dlk1 message was detected Birinapant in day 10 EBs cultured in the absence of BMP-4 and Dox induction. Addition of BMP-4, but not the induction of Hex, increased the levels of Dlk1 expression. The relatively high levels of Dlk1 observed in the absence of BMP-4 appear to be a result of activin signaling, because substantially lower levels were detected in cells differentiated in

the absence of activin. Induction with BMP-4 doubled the Grape seed extract expression levels of Dlk1 in either the absence of presence of activin. Finally, neither factor induced significant levels of Dlk1 in the absence of a functional Hex gene. The directed differentiation of ESCs in culture is emerging as a powerful model system for studying mammalian development in vitro as well as a renewable source of functional cell types for transplantation for future cell-based therapy and for drug discovery and toxicology testing.27 Of the different cell populations that can be generated

from these pluripotent stem cells, hepatocytes are of particular interest because hepatocyte-based therapy has been considered as a new generation and effective treatment mode for liver diseases28 and the liver is a primary target organ of drug toxicity.29 A number of reports have documented the efficient generation of immature hepatocytes from both mouse and human ESCs,16–18 demonstrating that specification of this lineage can be studied in this model system. The most successful approaches to date have translated developmental biology to the culture dish and recreated the key aspects of the normal hepatic developmental program in the differentiation cultures. Although these studies collectively show that it is possible to generate populations with hepatic characteristics, the cells that do develop in the cultures remain immature.