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The role of epigenetic modifiers in extended cultures of functional hepatocyte-like cells derived from human neonatal mesenchymal stem cells

Abstract

The development of predictive in vitro stem cell-derived hepatic models for toxicological drug screening is an increasingly important topic. Herein, umbilical cord tissue-derived mesenchymal stem cells (hnMSCs) underwent hepatic differentiation using an optimized three-step core protocol of 24 days that mimicked liver embryogenesis with further exposure to epigenetic markers, namely the histone deacetylase inhibitor trichostatin A (TSA), the cytidine analogue 5-azacytidine (5-AZA) and dimethyl sulfoxide (DMSO). FGF-2 and FGF-4 were also tested to improve endoderm commitment and foregut induction during Step 1 of the differentiation protocol, being HHEX expression increased with FGF-2 (4 ng/mL). DMSO (1%, v/v) when added at day 10 enhanced cell morphology, glycogen storage ability, enzymatic activity and induction capacity. Moreover, the stability of the hepatic phenotype under the optimized differentiation conditions was examined up to day 34. Our findings showed that hepatocyte-like cells (HLCs) acquired the ability to metabolize glucose, produce albumin and detoxify ammonia. Global transcriptional analysis of the HLCs showed a partial hepatic differentiation degree. Global analysis of gene expression in the different cells revealed shared expression of gene groups between HLCs and human primary hepatocytes (hpHeps) that were not observed between HepG2 and hpHeps. In addition, bioinformatics analysis of gene expression data placed HLCs between the HepG2 cell line and hpHeps and distant from hnMSCs. The enhanced hepatic differentiation observed was supported by the presence of the hepatic drug transporters OATP-C and MRP-2 and gene expression of the hepatic markers CK18, TAT, AFP, ALB, HNF4A and CEBPA; and by their ability to display stable UGT-, EROD-, ECOD-, CYP1A1-, CYP2C9- and CYP3A4-dependent activities at levels either comparable with or even higher than those observed in primary hepatocytes and HepG2 cells. Overall, an improvement of the hepatocyte-like phenotype was achieved for an extended culture time suggesting a role of the epigenetic modifiers in hepatic differentiation and maturation and presenting hnMSC-HLCs as an advantageous alternative for drug discovery and in vitro toxicology testing.

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Abbreviations

Ac-LDL:

Acetylated low-density lipoprotein

AFP:

α-Fetoprotein

ALB:

Albumin

5-AZA:

5-Azacytidine

CK-18:

Cytokeratin 18

CK-19:

Cytokeratin 19

CYP:

Cytochrome P-450

DMSO:

Dimethyl sulfoxide

c/EBPα:

CCAAT/enhancer binding protein

ECOD:

7-Ethoxycoumarin-O-deethylase

EROD:

7-Ethoxyresorufin-O-deethylase

FGF-2:

Fibroblast growth factor-2

FGF-4:

Fibroblast growth factor-4

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

HHEX:

Hematopoietically expressed homeobox protein

HLC:

Hepatocyte-like cell

HNF-4α:

Hepatocyte nuclear factor-4α

hnMSC:

Human neonatal mesenchymal stromal cell

hpHep:

Human primary hepatocyte

hrHep:

Human rat hepatocyte

IPA:

Ingenuity pathway analysis

3-MC:

3-Methylcholantherene

MRP-2:

Multidrug resistance protein-2

4-MU:

4-Methylumbelliferone

OATP-C:

Organic anion-transporting polypeptide C

TAT:

Tyrosine aminotransferase

PCA:

Principal component analysis

TSA:

Trichostatin A

UCX® :

hnMSCs isolated from the umbilical cord matrix

UGT:

Uridine 5′-diphosphate glucuronosyltransferase

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Acknowledgements

The authors thank Alexandra Medeiros for technical support on immunochemistry assays. This work was supported by Fundação para a Ciência e a Tecnologia (FCT, Portugal) through research grants (PTDC/SAU-TOX/110457/2009, PEst-OE/SAU/UI4013/2011, UID/DTP/04138/2013, SFRH/BD/87508/2012 to M.C., PD/BD/114280/2016 to S.P.C. and SFRH/BPD/96719/2013 and IF/00846/2015 to J.P.M.) and by Human Frontier Science Program (Young Investigator grant to J.L.R). We also acknowledge the support of the COST action BM1305 (A FACTT: Action to focus and accelerate cell-based tolerance-inducing therapies). The work herein presented was performed at iMed.ULisboa and Karolinska Institutet.

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Correspondence to J. P. Miranda.

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Conflict of interest

H.C. and P.C. are shareholders of ECBio; J.M.S. is employee of ECBio. The other authors declare that they have no conflict of interests.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This study was approved by the Ethics Committee of the Hospital Dr. José de Almeida (Cascais, Portugal), in scope of a research protocol between ECBio (Research & Development in Biotechnology, S.A.) and HPP Saúde (Parcerias Cascais, S.A.). Umbilical cord donations, with written informed consents, as well as umbilical cord procurement, were made according to Directive 2004/23/EC of the European Parliament and of the Council of 31 March 2004 on setting standards of quality and safety for the donation, procurements, testing, processing, preservation, storage and distribution of human tissues and cells. All applicable international, national and/or institutional guidelines for the care and use of animals were followed.

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Cipriano, M., Correia, J.C., Camões, S.P. et al. The role of epigenetic modifiers in extended cultures of functional hepatocyte-like cells derived from human neonatal mesenchymal stem cells. Arch Toxicol 91, 2469–2489 (2017). https://doi.org/10.1007/s00204-016-1901-x

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  • DOI: https://doi.org/10.1007/s00204-016-1901-x

Keywords

  • hnMSC
  • Hepatocyte-like cells
  • CYP metabolism
  • Drug transporters
  • Epigenetic modifiers