GeneSpring 6.2 (Silicon Genetics,

Inc., Redwood City, see more CA) was then used to evaluate the data obtained using CodeLink Expression Scanning Software. Total RNA was isolated from cell samples using the RNeasy mini kit (Qiagen). One microgram of purified, DNase-treated total RNA was used to synthesize biotinylated cRNA target preparation using the CodeLink™ Expression Assay Reagent Kit (Amersham) according to manufacturer’s instructions. Ten micrograms of fragmented cRNA was applied to the Uniset Mouse I Expression Bioarray, hybridized for 18 hours, and the arrays processed according to manufacturer’s instructions using Streptavidin-Alexa647 detection reagents. Slides were scanned using a GenePix 4000B scanner (Axon) and the images were analyzed with CodeLink™ Expression Analysis Software. To investigate the role played by Hex in hepatocyte lineage commitment during EB differentiation, we induced endoderm formation from wild-type (Hex+/+), heterozygous (Hex+/−), or Hex-deficient (Hex−/−) ESCs15 using the serum induction protocol that we have described.22 Consistent with our earlier findings, the hepatocyte genes Alb and Afp were both expressed in EBs generated from the Hex+/+ and Hex+/− ESCs. The levels of expression of both genes

were markedly reduced in the PLX-4720 solubility dmso Hex−/− EBs (Fig. 1A). These findings are in line with those from studies on the early embryo, demonstrating that Hex is required for development of the liver in vivo.13, 15 To further evaluate the role of Hex in hepatic specification in the ESC/EB model, we used an ESC line (AINV18)

that enables the regulated expression of a given gene under the control of a tet-inducible promoter. Using this system, we generated ESCs in which Hex expression was induced by the addition of the tetracycline analogue Dox (tet-Hex ESCs). Hex expression was induced in the cells by the addition of Dox (1 μg/mL) to the EB cultures either from days 6–10 or from days 6–22 of differentiation. Quantitative PCR analyses revealed that induction of Hex between days 6 and 10 of culture resulted in a significant up-regulation of Afp and Alb expression compared with the uninduced cultures (Fig. 1B,C). These levels of expression at day 14 represent 2.6% and MCE 2.5% of the expression found in the fetal liver, respectively. By contrast, when Hex was continuously expressed from day 6 to day 22, levels of Alb and Afp mRNA were diminished on day 22 (Fig. 1B,C), suggesting that prolonged Hex expression may disrupt hepatic differentiation or shift the tissue into another fate. Immunostaining showed the presence of clusters of Alb+ cells within the Hex-induced day 14 EBs (Fig. 1D). Hex induction also resulted in enhanced secretion of both Alb and transferrin by the EB-derived cells at day 14 of culture (Fig. 2A,B). These levels of secretion were 7.6% and 5.0% of that of day 14 fetal liver cells, respectively.