The expression levels of DE markers in the si-HHEX-transfected cells were upregulated in hepatoblast differentiation from DE cells.

<p>(<b>A</b>) hESCs (H9) were differentiated into DE cells according to the protocol described in the <i>Materials and Methods</i> section. The DE cells were transfected with 50 nM si-control or si-HHEX on day 4, and cultured in the medium containing 20 ng/ml BMP4 and 20 ng/ml FGF4 until day 9. On day 9, the gene expression levels of hepatoblast markers (<i>AFP</i>, <i>EpCAM</i>, <i>TTR</i>, <i>HNF4α</i>, and <i>PROX1</i>) in si-control- or si-HHEX-transfected cells were examined by real-time RT-PCR. The gene expression levels in the si-control-transfected cells were taken as 1.0. (<b>B</b>) On day 9, the percentage of AFP-positive cells was measured by using FACS analysis to examine the hepatoblast differentiation efficiency. (<b>C</b>) The gene expression levels of DE (<i>EOMES</i>, <i>FOXA2</i>, <i>GATA4</i>, <i>GATA6</i>, <i>GSC</i>, and <i>SOX17</i>), pancreatic (<i>PDX1</i>, <i>NKX2.2</i>, and <i>NKX6.1</i>), intestinal (<i>CDX2</i> and <i>KLF5</i>), and pluripotent markers (<i>NANOG</i> and <i>OCT3/4</i>) in the si-control- or si-HHEX-transfected cells were examined by real-time RT-PCR. The gene expression levels in the si-control-transfected cells were taken as 1.0. (<b>D</b>) On day 9, the percentage of cells positive for the DE markers (CXCR4 and EOMES) was examined by using FACS analysis. All data are represented as means ± SD (<i>n = </i>3). *<i>p</i><0.05, **<i>p</i><0.01.