Supplemental Material, Supplemental_manuscript_20171121 - Human iPS Cell–based Liver-like Tissue Engineering at Extrahepatic Sites in Mice as a New Cell Therapy for Hemophilia B

<p>Supplemental Material, Supplemental_manuscript_20171121 for Human iPS Cell–based Liver-like Tissue Engineering at Extrahepatic Sites in Mice as a New Cell Therapy for Hemophilia B by Ryota Okamoto, Kazuo Takayama, Naoki Akita, Yasuhito Nagamoto, Daiki Hosokawa, Shunsuke Iizuka, Fuminori Sakurai, Hiroshi Suemizu, Kazuo Ohashi, and Hiroyuki Mizuguchi in Cell Transplantation</p

HHEX Promotes Hepatic-Lineage Specification through the Negative Regulation of Eomesodermin

<div><p>Human embryonic stem cells (hESCs) could provide a major window into human developmental biology, because the differentiation methods from hESCs mimic human embryogenesis. We previously reported that the overexpression of hematopoietically expressed homeobox (HHEX) in the hESC-derived definitive endoderm (DE) cells markedly promotes hepatic specification. However, it remains unclear how HHEX functions in this process. To reveal the molecular mechanisms of hepatic specification by HHEX, we tried to identify the genes directly targeted by HHEX. We found that HHEX knockdown considerably enhanced the expression level of eomesodermin (EOMES). In addition, HHEX bound to the HHEX response element located in the first intron of EOMES. Loss-of-function assays of EOMES showed that the gene expression levels of hepatoblast markers were significantly upregulated, suggesting that EOMES has a negative role in hepatic specification from the DE cells. Furthermore, EOMES exerts its effects downstream of HHEX in hepatic specification from the DE cells. In conclusion, the present results suggest that HHEX promotes hepatic specification by repressing EOMES expression.</p></div

Hepatoblast differentiation was promoted by knockdown of EOMES in the presence of BMP4.

<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 or si-EOMES on day 4, and then cultured with the medium containing BMP4 or FGF4. The percentage of AFP-positive cells was examined by FACS analysis on day 9. (<b>B</b>) The gene expression levels of hepatoblast markers (<i>AFP</i>, <i>EpCAM</i>, <i>TTR</i>, <i>HNF4α</i>, and <i>PROX1</i>) were measured by real-time RT-PCR on day 9. The gene expression levels in si-control-transfected cells were taken as 1.0. (<b>C</b>) The si-control- or si-EOMES-transfected cells were subjected to immunostaining with anti-AFP (green) antibodies. Nuclei were counterstained with DAPI (blue). The bar represents 50 μm. All data are represented as means ± SD (<i>n = </i>3). *<i>p</i><0.05, **<i>p</i><0.01.</p

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.</p

Temporal analysis of endogenous gene expression levels of EOMES and HHEX in hepatoblast differentiation from hESCs.

<p>(<b>A</b>) The schematic protocol for hepatoblast differentiation from hESCs (H9) is shown. (<b>B</b>) The temporal gene expression levels of <i>HHEX</i>, <i>AFP</i>, <i>SOX17</i> and <i>EOMES</i> were examined by real-time RT-PCR in hepatoblast differentiation. The gene expression levels in undifferentiated hESCs were taken as 1.0. (<b>C</b>) To examine the hepatoblast differentiation efficiency, the percentage of AFP-positive cells was measured by FACS analysis. (<b>D</b>) The HHEX protein-binding frequencies of the regions around the HRE of the EOMES gene and a negative control gene (β-ACTIN) were measured by ChIP-qPCR analysis. The results are presented as the percent input of anti-HHEX samples compared with those of anti-IgG samples. All data are represented as means ± SD (<i>n = </i>3).</p

HHEX suppresses EOMES expression by binding to the HRE located in the first intron of EOMES.

<p>(<b>A</b>) An overview of the EOMES mRNA precursor and the location of the putative HRE are presented. The HRE is located in the first intron of EOMES. (<b>B</b>) Luciferase reporter assays were performed to examine the regulation of EOMES expression by HHEX. HeLa cells were cotransfected with both firefly luciferase reporter plasmids (pControl-Luc, p5’ EOM-Luc, or p5’ EOM-mut-Luc) and effecter plasmids (control plasmids (pHMEF5) or HHEX expression plasmids (pHMEF5-HHEX)). The details of the luciferase reporter assays are described in the <i>Materials and Methods</i> section. The luciferase activities in the pControl-Luc- and pHMEF5-cotransfected cells were taken as 1.0. All data are represented as means ± SD (<i>n = </i>3). <i>*</i>, <i>p</i><0.05.</p

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.</p