Stem Cell Reviews and Reports

, Volume 12, Issue 1, pp 90–104 | Cite as

One Standardized Differentiation Procedure Robustly Generates Homogenous Hepatocyte Cultures Displaying Metabolic Diversity from a Large Panel of Human Pluripotent Stem Cells

  • Annika Asplund
  • Arvind Pradip
  • Mariska van Giezen
  • Anders Aspegren
  • Helena Choukair
  • Marie Rehnström
  • Susanna Jacobsson
  • Nidal Ghosheh
  • Dorra El Hajjam
  • Sandra Holmgren
  • Susanna Larsson
  • Jörg Benecke
  • Mariela Butron
  • Annelie Wigander
  • Karin Noaksson
  • Peter Sartipy
  • Petter Björquist
  • Josefina Edsbagge
  • Barbara Küppers-MuntherEmail author


Human hepatocytes display substantial functional inter-individual variation regarding drug metabolizing functions. In order to investigate if this diversity is mirrored in hepatocytes derived from different human pluripotent stem cell (hPSC) lines, we evaluated 25 hPSC lines originating from 24 different donors for hepatic differentiation and functionality. Homogenous hepatocyte cultures could be derived from all hPSC lines using one standardized differentiation procedure. To the best of our knowledge this is the first report of a standardized hepatic differentiation procedure that is generally applicable across a large panel of hPSC lines without any adaptations to individual lines. Importantly, with regard to functional aspects, such as Cytochrome P450 activities, we observed that hepatocytes derived from different hPSC lines displayed inter-individual variation characteristic for primary hepatocytes obtained from different donors, while these activities were highly reproducible between repeated experiments using the same line. Taken together, these data demonstrate the emerging possibility to compile panels of hPSC-derived hepatocytes of particular phenotypes/genotypes relevant for drug metabolism and toxicity studies. Moreover, these findings are of significance for applications within the regenerative medicine field, since our stringent differentiation procedure allows the derivation of homogenous hepatocyte cultures from multiple donors which is a prerequisite for the realization of future personalized stem cell based therapies.


Hepatocyte differentiation Human induced pluripotent stem cells Human embyronic stem cells Liver Toxicity Cellular therapy 



We thank other members of Takara Bio Europe AB and Skövde University who have provided assistance and technical help through-out this project.

Supported by the IMI-JU project MIP-DILI (grant 115336), the Seventh Framework Program project InnovaLiv (grant 278152), and the Knowledge Foundation (grants 2010/0069, 2012/0310, 2013/89).

Disclosure of Potential Conflicts of Interest

All authors are or have been employed by Takara Bio Europe AB (former Cellartis AB) when this study was performed.

Author Contributions

Annika Asplund: Collection, assembly, analysis, and interpretation of data, manuscript writing.

Anders Aspegren: Conception and design, data analysis and interpretation.

Arvind Pradip, Mariska van Giezen, Marie Rehnström, Susanna Jacobsson, Nidal Ghosheh, Dorra El Hajjam, Sandra Holmgren, Susanna Larsson, Jörg Benecke, Mariela Butron, Annelie Wigander: Collection, assembly and analysis of data.

Helena Choukair, Karin Noaksson: Analysis and interpretation of data.

Peter Sartipy, Josefina Edsbagge: Interpretation of data, manuscript writing.

Petter Björquist: Conception and design, manuscript writing.

Barbara Küppers-Munther: Conception and design, assembly, analysis, and interpretation of data, manuscript writing.

All: Final approval of manuscript

Supplementary material

12015_2015_9621_Fig7_ESM.gif (416 kb)
Suppl. Figure 1

Homogenous expression of the hepatic marker HNF4α in hepatocyte cultures derived from 16 hPSC lines. Immunocytochemical staining of hepatocyte cultures derived from 12 hiPSC and 4 hESC lines after 28 days of differentiation for the hepatic marker HNF4α. Nuclear counterstaining with DAPI. On average 93.8 % ± 0.7 (SEM) of DAPI-stained nuclei are immuno-positive for HNF4α. Percentages for the individual hPSC lines are given in the respective HNF4α-picture. The scale bars represent 100 μM. Abbreviations: DAPI = 4′,6-diamidino-2-phenylindol; HNF4α = hepatocyte nuclear factor 4α. (GIF 415 kb)

12015_2015_9621_MOESM1_ESM.tif (10.8 mb)
High Resolution Image (TIFF 11048 kb)
12015_2015_9621_Fig8_ESM.gif (107 kb)
Suppl. Figure 2

Expression of hepatic markers in hPSC-derived hepatocytes after 28 days of differentiation. Immunocytochemical stainings of hepatocyte cultures derived from the hiPSC lines ChiPSC4 and P11012, and the hESC lines SA121 and SA461 for the hepatic markers Cytokeratin 18 (CK18), α1-Antitrypsin (a1-AT), and Albumin (Alb). Scale bars represent 100 μM. Abbreviations: α1-AT = α1-Antitrypsin; Alb = Albumin; CK18 = Cytokeratin 18. (GIF 106 kb)

12015_2015_9621_MOESM2_ESM.tif (2.7 mb)
High Resolution Image (TIFF 2782 kb)
12015_2015_9621_Fig9_ESM.gif (98 kb)
Suppl. Figure 3

Inter-individual variation of mRNA expression of hepatic genes in freshly isolated primary human hepatocytes. QPCR analysis of mRNA expression of the hepatic markers α1-Antitrypsin and Albumin (A,B), the drug-metabolizing enzymes CYP1A1, 1A2, 2C9, 3A4, 3A5, and 3A7 (C-H), the phase II enzymes GSTA1-1 and UGT2B7 (I,J), and the transporters NTCP and OATP1B1 (K,L) in freshly isolated human primary hepatocytes from 7 different donors. Expression levels are normalized to CEBPα serving as a house-keeping gene and a calibrator mix (set as 1) and presented as relative quantification. Abbreviations: α1-AT = α1-Antitrypsin; CEBPα = CCAAT/enhancer binding protein α; CYP = Cytochrome P450 enzyme; GSTA1-1 = glutathione-S-transferase A1-1; hphep = human primary hepatocytes; NTCP = Sodium taurocholate co-transporting polypeptide; OATP1B1 = organic anion transporter family, member 1B1; UGT2B7 = UDP-glucuronosyltransferase 2B7. (GIF 97 kb)

12015_2015_9621_MOESM3_ESM.tif (1.7 mb)
High Resolution Image (TIFF 1722 kb)
12015_2015_9621_Fig10_ESM.gif (1.4 mb)
Suppl. Figure 4

Homogenous hepatocyte cultures repeatedly derived from 9 hPSC lines. A Representative phase contrast pictures of homogenous hepatocyte populations obtained in repeated experiments from the hiPSC lines ChiPSC4, ChiPSC17, ChiPSC22, ChiPSC18, P11012, P11032, and the hESC lines SA121, SA461, Val9 on between day 21 and 28 after start of differentiation. Per repeated differentiation experiment one picture is shown. The scale bar represents 100 μm. (GIF 1444 kb)

12015_2015_9621_MOESM4_ESM.tif (69.9 mb)
High Resolution Image (TIFF 71546 kb)


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Annika Asplund
    • 1
    • 2
  • Arvind Pradip
    • 1
    • 3
  • Mariska van Giezen
    • 1
  • Anders Aspegren
    • 1
  • Helena Choukair
    • 1
  • Marie Rehnström
    • 1
  • Susanna Jacobsson
    • 1
  • Nidal Ghosheh
    • 2
  • Dorra El Hajjam
    • 1
  • Sandra Holmgren
    • 1
    • 4
  • Susanna Larsson
    • 1
  • Jörg Benecke
    • 1
  • Mariela Butron
    • 1
  • Annelie Wigander
    • 1
  • Karin Noaksson
    • 1
  • Peter Sartipy
    • 2
    • 5
  • Petter Björquist
    • 1
    • 6
  • Josefina Edsbagge
    • 1
  • Barbara Küppers-Munther
    • 1
    • 2
    Email author
  1. 1.Takara Bio Europe AB (former Cellartis AB)GothenburgSweden
  2. 2.Systems Biology Research Center, School of BioscienceUniversity of SkövdeSkövdeSweden
  3. 3.Novo Nordisk A/SBagsværdDenmark
  4. 4.Institute of Biomedicine, Sahlgrenska AcademyUniversity of GothenburgGothenburgSweden
  5. 5.AstraZeneca R&D, GMD CVMD GMedMölndalSweden
  6. 6.NovaHep ABGothenburgSweden

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