Human embryonic stem cells (hESCs) are a valuable source of pluripotential primary cells. To date, however, their homogeneous cellular differentiation to specific cell types in vitro has proven difficult. Wnt signaling has been shown to play important roles in coordinating development, and we demonstrate that Wnt3a is differentially expressed at critical stages of human liver development in vivo. The essential role of Wnt3a in hepatocyte differentiation from hESCs is paralleled by our in vitro model, demonstrating the importance of a physiologic approach to cellular differentiation. Our studies provide compelling evidence that Wnt3a signaling is important for coordinated hepatocellular function in vitro and in vivo. In addition, we demonstrate that Wnt3a facilitates clonal plating of hESCs exhibiting functional hepatic differentiation. These studies represent an important step toward the use of hESC-derived hepatocytes in high-throughput metabolic analysis of human liver function.definitive endoderm ͉ function ͉ hepatocyte ͉ drug metabolism ͉ high throughput H uman embryonic stem cells (hESCs) are derived from the inner cell mass of preimplantation embryos and demonstrate pluripotency in vitro and in vivo (1, 2). Such attributes allow hESCs to be differentiated down all germ lineages in large numbers and offer significant advantages over their adult stem cell counterparts, which are generally limited in their capacity to differentiate and proliferate (3). Although these hESCs provide a valuable source of adult differentiated cells, homogeneous cellular differentiation to specific germ layers has proven difficult to achieve. One potential explanation for this failure is that cells do not receive sequential developmental cues that they do in vivo.Wnt signaling has been shown to play an important role in hESC self-renewal and differentiation and stimulates numerous intracellular signal transduction cascades, including the canonical pathway regulating gene expression in the nucleus and what seems to be a network of noncanonical pathways regulating many other aspects of cell biology [reviewed by Cadigan and Liu (4)]. In the absence of Wnt signaling, ␤-catenin is targeted for degradation; however, active Wnt signaling inhibits ␤-catenin destruction, resulting in its nuclear translocation (5, 6). After nuclear localization, ␤-catenin dimerizes with the nuclear proteins from the T cell factor/lymphoid enhancer factor (TCF/ LEF) family and transactivates gene expression. TCF/LEFs are not only present in transcriptional activator complexes; they also play a role in corepressor complex assembly (6).The important role played by Wnt signaling during gastrulation in vivo is evidenced by gene knockouts or dominant negatives (7, 8). Wnt3-mediated Brachyury expression is also important for migration of precursor cells through the anterior region of the primitive streak (PS). The subsequent specification of the anterior region of the PS to mesoderm or endoderm is likely to depend on the duration and magnitude of Nodal signaling (...

With the advent of induced pluripotent stem cell (iPSC) technology, it is now feasible to generate iPSCs with a defined genotype or disease state. When coupled with direct differentiation to a defined lineage, such as hepatic endoderm (HE), iPSCs would revolutionize the way we study human liver biology and generate efficient "off the shelf" models of human liver disease. Here, we show the "proof of concept" that iPSC lines representing both male and female sexes and two ethnic origins can be differentiated to HE at efficiencies of between 70%-90%, using a method mimicking physiological relevant condition. The iPSC-derived HE exhibited hepatic morphology and expressed the hepatic markers albumin and E-cadherin, as assessed by immunohistochemistry. They also expressed alpha-fetoprotein, hepatocyte nuclear factor-4a, and a metabolic marker, cytochrome P450 7A1 (Cyp7A1), demonstrating a definitive endodermal lineage differentiation. Furthermore, iPSC-derived hepatocytes produced and secreted the plasma proteins, fibrinogen, fibronectin, transthyretin, and alpha-fetoprotein, an essential feature for functional HE. Additionally iPSC-derived HE supported both CYP1A2 and CYP3A4 metabolism, which is essential for drug and toxicology testing. Conclusion: This work is first to demonstrate the efficient generation of hepatic endodermal lineage from human iPSCs that exhibits key attributes of hepatocytes, and the potential application of iPSC-derived HE in studying human liver biology. In particular, iPSCs from individuals representing highly polymorphic variants in metabolic genes and different ethnic groups will provide pharmaceutical development and toxicology studies a unique opportunity to revolutionize predictive drug toxicology assays and allow the creation of in vitro hepatic disease models. (HEPATOLOGY 2010;51:329-335.) H uman induced pluripotent stem cells (iPSCs) are reprogrammed mature somatic fibroblasts which represent a pluripotent cell population able to generate all primary cell types in vitro. [1][2][3] The ability to derive iPSCs from an indefinite range of genotypes makes them an attractive resource on which to model liver function reflecting the complexity of polygenic influences on metabolism in vitro. Another facet of iPSC technology is the ability to study the impact of gene polymorphisms in a native chromatin setting and model gene interactions with precision. Therefore iPSC-derived models hold great potential to develop a detailed understanding of human liver disease and metabolism including drug toxicity (for a review, see Dalgetty et al. 4