Hepatoblast differentiation is inhibited by EOMES, which functions downstream of HHEX.

<p>(<b>A</b>) The hESCs (H9) were differentiated into the DE cells according to the protocol described in the <i>Materials and Methods</i> section. The hESC-derived DE cells were transfected with 50 nM si-control, si-EOMES, or si-HHEX on day 4, and then cultured with the medium containing BMP4 and FGF4. The gene expression levels of hepatoblast markers (<i>AFP</i>, <i>EpCAM</i>, <i>TTR</i>, and <i>HNF4α</i>) were measured by real-time RT-PCR on day 9. The gene expression levels in si-control- and si-HHEX-transfected cells were taken as 1.0. (<b>B</b>) The percentage of AFP-positive cells was examined by FACS analysis on day 9. All data are represented as means ± SD (<i>n = </i>3). *<i>p</i><0.05, **<i>p</i><0.01. (<b>C</b>) HHEX promotes the hepatic specification from the hESC-derived DE cells by negatively regulating EOMES expression. A model of the hepatic specification from the hESC-derived DE cells by HHEX is presented. In the hESC-derived DE cells, HHEX represses EOMES expression. In this way, HHEX promotes the hepatic specification from the hESC-derived DE cells.