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Self-assembled 3D spheroids and hollow-fibre bioreactors improve MSC-derived hepatocyte-like cell maturation in vitro

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Abstract

3D cultures of human stem cell-derived hepatocyte-like cells (HLCs) have emerged as promising models for short- and long-term maintenance of hepatocyte phenotype in vitro cultures by better resembling the in vivo environment of the liver and consequently increase the translational value of the resulting data. In this study, the first stage of hepatic differentiation of human neonatal mesenchymal stem cells (hnMSCs) was performed in 2D monolayer cultures for 17 days. The second stage was performed by either maintaining cells in 2D cultures for an extra 10 days, as control, or alternatively cultured in 3D as self-assembled spheroids or in multicompartment membrane bioreactor system. All systems enabled hnMSC differentiation into HLCs as shown by positive immune staining of hepatic markers CK-18, HNF-4α, albumin, the hepatic transporters OATP-C and MRP-2 as well as drug-metabolizing enzymes like CYP1A2 and CYP3A4. Similarly, all models also displayed relevant glucose, phase I and phase II metabolism, the ability to produce albumin and to convert ammonia into urea. However, EROD activity and urea production were increased in both 3D systems. Moreover, the spheroids revealed higher bupropion conversion, whereas bioreactor showed increased albumin production and capacity to biotransform diclofenac. Additionally, diclofenac resulted in an IC50 value of 1.51 ± 0.05 and 0.98 ± 0.03 in 2D and spheroid cultures, respectively. These data suggest that the 3D models tested improved HLC maturation showing a relevant biotransformation capacity and thus provide more appropriate reliable models for mechanistic studies and more predictive systems for in vitro toxicology applications.

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Abbreviations

2D:

Two dimensional

3D:

Three dimensional

3-MC:

Methylcholanthrene

5-AZA:

5-Azacytidine

AFP:

Alpha-fetoprotein

ALB:

Albumin

c/EBPα:

CCAAT/enhancer-binding protein

CK:

Cytokeratin

CYP:

Cytochrome P-450

DAPI:

4′,6-Diamidino-2-phenylindole

DMSO:

Dimethyl sulphoxide

ECM:

Extracellular matrix

EROD:

7-Ethoxyresorufin-O-deethylase assay

FGF:

Fibroblast growth factor

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

HbLCs:

Hepatoblast-like cells

hESCs:

Human embryonic stem cells

HGF:

Hepatocyte growth factor

HLCs:

Hepatocyte-like cells

HNF-4α:

Hepatocyte nuclear factor-4α

hnMSCs:

Human neonatal mesenchymal stem cells

hpHep:

Human primary hepatocytes

iPSCs:

Induced pluripotent stem cells

LDH:

Lactate dehydrogenase

MRP-2:

Multidrug resistance protein 2

MSCs:

Mesenchymal stem cells

MTS:

3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt

OATP-C:

Organic anion-transporting polypeptide C

OSM:

Oncostatin M

SC:

Stem cell

PAS:

Periodic acid Schiff’s staining

TAT:

Tyrosine aminotransferase

UCX® :

ECBio’s proprietary Umbilical Cord eXpanded hnMSCs isolated from the Wharton’s Jelly

UGT:

Uridine 5′-diphosphate glucuronosyltransferase

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Acknowledgments

We acknowledge Pharmacelsus GmbH (Dr. Ursula Muller-Vieira) for the MS quantifications and analyses. We also acknowledge the support of the COST action BM1305 (A FACTT: Action to Focus and Accelerate Cell-based Tolerance-inducing Therapies). This work was supported by FCT (Fundação para a Ciência e a Tecnologia) [EXPL/DTP-FTO/0308/2013, PTDC/SAU-TOX/110457/2009, UID/DTP/04138/2013, SFRH/BPD/96719/2013 and Ciência2008 to JPM, SFRH/BD/87508/2012 to M. Cipriano]. The work herein presented was performed at iMed.ULisboa, ECBio S.A. and BCRT.

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Authors and Affiliations

  1. Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal

    Madalena Cipriano, Nuno G. Oliveira, Matilde Castro & Joana P. Miranda

  2. Bioreactor Group, Berlin Brandenburg Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, 13353, Berlin, Germany

    Nora Freyer, Fanny Knöspel & Katrin Zeilinger

  3. ECBio S.A., Rua Henrique Paiva Couceiro, Nº 27, 2700-451, Amadora, Portugal

    Rita Barcia, Pedro E. Cruz, Helder Cruz & Jorge M. Santos

Authors
  1. Madalena Cipriano
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  2. Nora Freyer
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  3. Fanny Knöspel
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  4. Nuno G. Oliveira
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  5. Rita Barcia
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  6. Pedro E. Cruz
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  7. Helder Cruz
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  8. Matilde Castro
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  9. Jorge M. Santos
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  10. Katrin Zeilinger
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  11. Joana P. Miranda
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Corresponding author

Correspondence to Joana P. Miranda.

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Cipriano, M., Freyer, N., Knöspel, F. et al. Self-assembled 3D spheroids and hollow-fibre bioreactors improve MSC-derived hepatocyte-like cell maturation in vitro. Arch Toxicol 91, 1815–1832 (2017). https://doi.org/10.1007/s00204-016-1838-0

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