"The liver is associated with many types of diseases which can only be treated by Orthotopic liver transplants. However, shortage of organ donors represents a major limitation for wide-application of this therapy. Additionally, expansion of fully-functional hepatocytes in vitro remains an important challenge for the drug discovery industry. Human Embryonic Stem cells (hESCs) offer a promising new solution to these problems. hESCs are able to grow indefinitely in vitro while maintaining their capacity to differentiate to all types of cells. Here we propose to develop standardized, animal-free culture conditions to promote the differentiation and expansion of fully-functional hepatocytes from hESCs using four complementary Workpackages (WPs): Development of novel tools: WP1 will develop 3-D growth matrices, lentivirus-based reporter systems, liver-specific endothelial cells and a miRNA platform to facilitate the sequential process of establishing standardized, animal-free conditions to produce ES-derived hepatocytes. Generation of Anterior Definitive Endoderm multipotent stem cells: WP2 will define culture conditions to drive differentiation of hESCs to ADE cells, the earliest progenitors of liver cells during mammalian development. Proof-of-principle of conditions will be validated using the various hESC lines available from partners. Differentiation and expansion of hepatic progenitors from multipotent ADE cells using defined conditions: WP3 will be devoted to the generation of hepatic progenitors from ADE cells. Function will be validated in vitro and in vivo using human foetal hepatic progenitors and HepaRG human hepatoma cells as positive controls. Generation of mature hepatocytes from ES-derived hepatic progenitors: WP4 will generate functional hepatocyte lines suitable for drug testing and future preclinical evaluation. Results of the LIV-ES project will provide new insights into stem cell biology and establish a rationale basis for cell-based therapies."