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Phthalazinone Pyrazole Enhances the Hepatic Functions of Human Embryonic Stem Cell-Derived Hepatocyte-Like Cells via Suppression of the Epithelial-Mesenchymal Transition

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Abstract

During liver development, nonpolarized hepatic progenitor cells differentiate into mature hepatocytes with distinct polarity. This polarity is essential for maintaining the intrinsic properties of hepatocytes. The balance between the epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) plays a decisive role in differentiation of polarized hepatocytes. In this study, we found that phthalazinone pyrazole (PP), a selective inhibitor of Aurora-A kinase (Aurora-A), suppressed the EMT during the differentiation of hepatocyte-like cells (HLCs) from human embryonic stem cells. The differentiated HLCs treated with PP at the hepatoblast stage showed enhanced hepatic morphology and functions, particularly with regard to the expression of drug metabolizing enzymes. Moreover, we found that these effects were mediated though suppression of the AKT pathway, which is involved in induction of the EMT, and upregulation of hepatocyte nuclear factor 4α expression rather than Aurora-A inhibition. In conclusion, these findings provided insights into the regulatory role of the EMT on in vitro hepatic maturation, suggesting that inhibition of the EMT may drive transformation of hepatoblast cells into mature and polarized HLCs.

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Abbreviations

AAT:

Alpha-1 antitrypsin gene

Ac-LDL:

Acetylated-low-density lipoprotein

ALB:

Albumin gene

Aurora-A:

Aurora-A kinase

bFGF:

Basic fibroblast growth factor

BrdU:

Bromodeoxyuridine

BSA:

Bovine serum albumin

CDF:

Carboxy-dichlorofluorescein

CDFDA:

5 (and 6)-carboxy-2,7-dichlorofluorescein diacetate

CK:

Cytokeratin

CYP:

Cytochrome P450

DMEM:

Dulbecco’s modified Eagle’s medium

DMSO:

Dimethylsulfoxide

DPBS:

Dulbecco’s phosphate-buffered saline

ELISA:

Enzyme-linked immunosorbent assay

EMT:

Epithelial-mesenchymal transition

FGF4:

Fibroblast growth factor 4

GSK-3β:

Glycogen synthase kinase-3β

HCM:

Hepatocyte culture medium

HGF:

Hepatocyte growth factor

HLC:

Hepatocyte-like cell

MET:

Mesenchymal-epithelial transition

MRP2:

Multidrug-resistance protein 2

OATP:

Organic anion-transporting polypeptide

OSM:

Oncostatin M

PAS:

Periodic acid-Schiff

PP:

Phthalazinone pyrazole

TDO2:

Tryptophan 2,3-dioxygenase gene

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Acknowledgements

The authors are grateful to CHA Stem Cell Institute (CHA University, Korea) for providing the CHA-hES15 cell line. This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning (MSIP), Republic of Korea (No. NRF-2012M3A9C7050138). This research was also supported by the Basic Science Research Program through the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning (MSIP), Republic of Korea (No. NRF-2015R1C1A2A01051471).

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Author notes

  1. Seokjoo Yoon and Han-Jin Park contributed equally to this work.

Authors and Affiliations

  1. Department of Predictive Toxicology, Korea Institute of Toxicology, Daejeon, 34114, Republic of Korea

    Young-Jun Choi, Hyemin Kim, Ji-Woo Kim, Chang-Woo Song, Seokjoo Yoon & Han-Jin Park

  2. Human and Environmental Toxicology, School of Engineering, University of Science and Technology, Daejeon, 34113, Republic of Korea

    Young-Jun Choi, Chang-Woo Song & Seokjoo Yoon

  3. Department of Biotechnology, Brain Korea 21, Korea University, Seoul, 02841, Republic of Korea

    Dae-Sung Kim

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  1. Young-Jun Choi
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Correspondence to Han-Jin Park.

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Electronic Supplementary Material

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12015_2017_9795_MOESM1_ESM.pptx

Supplementary Figure 1. Phase contrast images of HLCs after 5 days of treatment with DMSO or PP (0.1, 1, and 10 μM). Scale bars: 100 μm. (PPTX 1279 KB)

12015_2017_9795_MOESM2_ESM.pptx

Supplementary Figure 2. Relative gene expression levels of hepatic markers were analyzed on day 17 in DMSO control cells and Aurora A inhibitor-treated cells. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the DMSO control (analysis of variance followed by Bonferroni’s multiple comparison test). (PPTX 91 KB)

12015_2017_9795_MOESM3_ESM.pptx

Supplementary Figure 3. (a) qPCR analysis of Aurora-A after transfection with Aurora-A siRNAs on day 17. *** p < 0.001 compared with the DMSO control (analysis of variance followed by Bonferroni’s multiple comparison test). (b) Levels of total AKT and phospho-AKT1 were determined by western blotting after transfection with Aurora-A siRNAs on day 17 of differentiation. (PPTX 37 KB)

Supplementary material 4 (DOCX 14 KB)

Supplementary material 5 (DOCX 18 KB)

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Choi, YJ., Kim, H., Kim, JW. et al. Phthalazinone Pyrazole Enhances the Hepatic Functions of Human Embryonic Stem Cell-Derived Hepatocyte-Like Cells via Suppression of the Epithelial-Mesenchymal Transition. Stem Cell Rev and Rep 14, 438–450 (2018). https://doi.org/10.1007/s12015-017-9795-4

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