Generation of Brain Microvascular Endothelial-Like Cells from Human Induced Pluripotent Stem Cells by Co-Culture with C6 Glioma Cells

<div><p>The blood brain barrier (BBB) is formed by brain microvascular endothelial cells (BMECs) and tightly regulates the transport of molecules from blood to neural tissues. <i>In vitro</i> BBB models from human pluripotent stem cell (PSCs)-derived BMECs would be useful not only for the research on the BBB development and function but also for drug-screening for neurological diseases. However, little is known about the differentiation of human PSCs to BMECs. In the present study, human induced PSCs (iPSCs) were differentiated into endothelial cells (ECs), and further maturated to BMECs. Interestingly, C6 rat glioma cell-conditioned medium (C6CM), in addition to C6 co-culture, induced the differentiation of human iPSC-derived ECs (iPS-ECs) to BMEC-like cells, increase in the trans-endothelial electrical resistance, decreased in the dextran transport and up-regulation of gene expression of tight junction molecules in human iPS-ECs. Moreover, Wnt inhibitors attenuated the effects of C6CM. In summary, we have established a simple protocol of the generation of BMEC-like cells from human iPSCs, and have demonstrated that differentiation of iPS-ECs to BMEC-like cells is induced by C6CM-derived signals, including canonical Wnt signals.</p></div

Effects of inhibitors of the canonical Wnt pathway on the differentiation of hiPS-ECs into BMEC-like cells by treatment of C6CM.

<p>(A) Expression of <i>Axin-2</i> mRNA was examined in iPSECs, iPSEC-mono and iPSEC-C6CM by qRT-PCR analysis. (B, C) TEER values and permeability coefficients of iPSEC-mono, iPSEC-C6CM or Dkk1- or XAV939-treated iPSEC-C6CM were measured. All results shown are the mean of three independent experiments with the indicated standard deviations (S.D.).</p

Differentiation of human iPSCs into ECs.

<p>(A) EC differentiation of human iPSCs. The detailed differentiation procedure is described in the Materials and Methods section. (B) Human iPSC-derived EBs (day 6, day 9 and day 12) were stained with anti-CD34 and anti-CD144 antibodies, and were then subjected to flow cytometric analysis. Representative results from one of the three independent experiments are shown. (C) Total RNA was extracted from undifferentiated human iPSCs (day 0) and human iPSC-derived EBs (day 6 and day 9). Then, qRT-PCR analysis was performed. Results shown are the mean of three independent experiments with the indicated standard deviations (S.D.). * p < 0.05, ** p < 0.01. (D-G) Sorted CD34⁺ cells were cultured with FGF2, ECGS, and heparin on fibronectin-coated plates. They showed an endothelial-like morphology under these culture conditions (D). These cells were stained positive for CD31 and vWF (E). They were also capable of forming tube-like structures on Matrigel (F) and demonstrated acetylated-LDL uptake (G). The scale bar indicates 300 μm (D) or 100 μm (E, F, G).</p

Induction of BMEC-like properties of hiPS-ECs by treatment with C6CM.

<p>(A) hiPS-EC monolayers were transferred onto non-cell culture (iPSEC-mono), C6-cell culture (iPSEC-C6) or C6CM (iPSEC-C6CM) in 24-well plates. Then, the TEER value of each hiPS-EC monolayer was measured at indicated days. (B) The permeability coefficient for FD was measured in hiPS-ECs before (iPSECs) and after 5-day mono-culture (iPSEC-mono), 5-day co-culture with C6 cells (iPSEC-C6) or 5-day culture in C6CM (iPSEC-C6CM). (C) Expressions of tight junction-related genes (<i>Claudin-5</i>, <i>Occuludin</i> and <i>ZO-1</i>) were examined in iPSECs, iPSEC-mono, iPSEC-C6 and iPSEC-C6CM. (D) Expressions of transporter genes (<i>P-gp</i>, <i>Bcrp</i>, <i>Mrp-4</i> and <i>Glut1</i>) were examined in iPSECs, iPSEC-mono, iPSEC-C6 and iPSEC-C6CM by RT-PCR analysis. (E) The iPSEC-C6 was treated with CSA or MK571, and then the permeability coefficient for Rhodamin 123 was investigated in them. All results shown are the mean of three independent experiments with the indicated standard deviations (S.D.). * p < 0.05.</p

Induction of BMEC-like properties in hiPS-ECs by co-culture with C6 cells.

<p>(A) hiPS-ECs and HUVECs were cultured on fibronectin-coated inserts. When confluent, these inserts were transferred onto non-cultured 24-well plates (iPSEC-mono or HUVEC-mono) or C6 cell-cultured 24-well plates (iPSEC-C6 or HUVEC-C6). Then, TEER values for hiPS-ECs and HUVECs were measured at indicated days. (B, C) Expressions of tight junction-related genes (<i>Claudin-5</i>, <i>Occludin</i> and <i>ZO-1</i>) were examined by qRT-PCR analysis before (iPSECs, HUVECs) and after (iPSEC-mono, iPSEC-C6, HUVEC-mono, HUVEC-C6) 5 days of culture. (D) The permeability coefficient for FD was investigated in 5-day co-cultured hiPS-ECs and HUVECs. (E) Expressions of transporter genes (<i>P-gp</i>, <i>Bcrp</i>, <i>Mrp-4</i> and <i>Glut1</i>) were examined in 5-day mono- or co-cultured hiPS-ECs by RT-PCR analysis. (F) The 5-day co-cultured hiPS-ECs were treated with CSA (an inhibitor of P-gp) or MK571 (an inhibitor of MRP-4), and then the permeability coefficient for Rhodamin 123 (a specific substance of P-gp) was measured. All results shown are the mean of three independent experiments with the indicated standard deviations (S.D.). * p < 0.05.</p

Primer list used in Semi-quantitative PCR.

<p>Primer list used in Semi-quantitative PCR.</p

Primer list used in quantitative real-time PCR.

<p>Primer list used in quantitative real-time PCR.</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

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

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