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

Abstract

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.

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References

  1. 1.

    Kola, I., & Landis, J. (2004). Can the pharmaceutical industry reduce attrition rates? Nature Reviews Drug Discovery, 3, 711–715.

    Article  CAS  PubMed  Google Scholar 

  2. 2.

    Lee, W. M. (2003). Acute liver failure in the United States. Seminars in Liver Disease, 23, 217–226.

    Article  CAS  PubMed  Google Scholar 

  3. 3.

    Hewitt, N. J., Gomez-Lechon, M. J., Houston, J. B., et al. (2007). Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies. Drug Metabolism Reviews, 39, 159–234.

    Article  CAS  PubMed  Google Scholar 

  4. 4.

    Gomez-Lechon, M. J., Donato, M. T., Castell, J. V., et al. (2004). Human hepatocytes in primary culture, the choice to investigate drug metabolism in man. Current Drug Metabolism, 5, 443–462.

    Article  CAS  PubMed  Google Scholar 

  5. 5.

    Li, A. P., Lu, C., Brent, J. A., et al. (1999). Cryopreserved human hepatocytes: characterization of drug-metabolizing enzyme activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability, and drug-drug interaction potential. Chemico-Biological Interactions, 121, 17–35.

    Article  CAS  PubMed  Google Scholar 

  6. 6.

    Richert, L., Liguori, M. J., Abadie, C., et al. (2006). Gene expression in human hepatocytes in suspension after isolation is similar to the liver of origin, is not affected by hepatocyte cold storage and cryopreservation, but is strongly changed after hepatocyte plating. Drug Metabolism and Disposition, 34, 870–879.

    Article  CAS  PubMed  Google Scholar 

  7. 7.

    Rodriguez-Antona, C., Donato, M. T., Boobis, A., et al. (2002). Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells. Xenobiotica, 32, 505–520.

    Article  CAS  PubMed  Google Scholar 

  8. 8.

    Gerets, H. H. J., Tilmant, K., Gerin, B., et al. (2012). Characterization of primary human hepatocytes, HepG2 cells and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins. Cell Biology and Toxicology, 28, 69–87.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  9. 9.

    Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., et al. (1998). Embryonic stem cell lines derived from human blastocysts. Science, 282, 1145–1147.

    Article  CAS  PubMed  Google Scholar 

  10. 10.

    Takahashi, K., Tanabe, K., Ohnuki, M., et al. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131, 861–872.

    Article  CAS  PubMed  Google Scholar 

  11. 11.

    Baxter, M. A., Rowe, C., Alder, J., et al. (2010). Generating hepatic cell lineages from pluripotent stem cells for drug toxicity screening. Stem Cell Research, 5, 4–22.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  12. 12.

    Sartipy, P., & Björquist, P. (2011). Concise review: human pluripotent stem cell-based models for cardiac and hepatic toxicity assessment. Stem Cells, 29, 744–748.

    Article  CAS  PubMed  Google Scholar 

  13. 13.

    Li, A. P. (2008). Human hepatocytes as an effective alternative experimental system for the evaluation of human drug properties: general concepts and assay procedures. ALTEX, 25, 33–42.

    PubMed  Google Scholar 

  14. 14.

    Ek, M., Söderdahl, T., Küppers-Munther, B., et al. (2007). Expression of drug metabolizing enzymes in hepatocyte-like cells derived from human embryonic stem cells. Biochemical Pharmacology, 74, 496–503.

    Article  CAS  PubMed  Google Scholar 

  15. 15.

    Söderdahl, T., Küppers-Munther, B., Heins, N., et al. (2007). Glutathione transferases in hepatocyte-like cells derived from human embryonic stem cells. Toxicology In Vitro, 21, 929–937.

    Article  PubMed  Google Scholar 

  16. 16.

    Hay, D. C., Fletcher, J., Payne, C., et al. (2008). Highly efficient differentiation of human embryonic stem cells to functional hepatic endoderm requires Activin A and Wnt3a signaling. Proceedings of the National Academy of Sciences of the United States of America, 105, 2301–2306.

    Article  Google Scholar 

  17. 17.

    Hay, D. C., Zhao, D., Fletcher, J., et al. (2008). Efficient differentiation of hepatocytes from human embryonic stem cells exhibiting markers recapitulating liver development in vivo. Stem Cells, 26, 894–902.

    Article  CAS  PubMed  Google Scholar 

  18. 18.

    Song, Z., Cai, J., Liu, Y., et al. (2009). Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells. Cell Research, 19, 1233–1242.

    Article  PubMed  Google Scholar 

  19. 19.

    Basma, H., Soto-Gutiérrez, A., Yannam, G. R., et al. (2009). Differentiation and transplantation of human embryonic stem cell-derived hepatocytes. Gastroenterology, 136, 990–999.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  20. 20.

    Brolen, G., Sivertsson, L., Björquist, P., et al. (2010). Hepatocyte-like cells derived from human embryonic stem cells specifically via definitive endoderm and a progenitor stage. Journal of Biotechnology, 145, 84–94.

    Article  Google Scholar 

  21. 21.

    Duan, Y., Ma, X., Zou, W., et al. (2010). Differentiation and characterization of metabolically functioning hepatocytes from human embryonic stem cells. Stem Cells, 28, 674–686.

    Article  CAS  PubMed  Google Scholar 

  22. 22.

    Si-Tayeb, K., Noto, F. K., Nagaoka, M., et al. (2010). Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology, 51, 297–305.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  23. 23.

    Sullivan, G. J., Hay, D. C., Park, I. H., et al. (2010). Generation of functional human hepatic endoderm from human induced pluripotent stem cells. Hepatology, 51, 329–335.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  24. 24.

    Touboul, T., Hannan, N. R., Corbineau, S., et al. (2010). Generation of functional hepatocytes from human embryonic stem cells under chemically defined conditions that recapitulate liver development. Hepatology, 51, 1754–1765.

    Article  CAS  PubMed  Google Scholar 

  25. 25.

    Rashid, S. T., Corbineau, S., Hannan, N., et al. (2010). Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells. Journal of Clinical Investigation, 120, 3127–3136.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  26. 26.

    Ghodsizadeh, A., Taei, A., Totonchi, M., et al. (2010). Generation of liver disease-specific induced pluripotent stem cells along with efficient differentiation to functional hepatocyte-like cells. Stem Cell Reviews and Reports, 6, 622–632.

    Article  PubMed  Google Scholar 

  27. 27.

    Yildirimman, R., Brolén, G., Vilardell, M., et al. (2011). Human embryonic stem cell derived hepatocyte-like cells as a tool for in vitro hazard assessment of chemical carcinogenicity. Toxicological Sciences, 124, 278–290.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  28. 28.

    Funakoshi, N., Duret, C., Pascussi, J. M., et al. (2011). Comparison of hepatic-like cell production from human embryonic stem cells and adult liver progenitor cells: CAR transduction activates a battery of detoxification genes. Stem Cell Reviews, 7, 518–531.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  29. 29.

    Ulvestad, M., Nordell, P., Asplund, A., et al. (2013). Drug metabolizing enzyme and transporter protein profiles of hepatocytes derived from human embryonic and induced pluripotent stem cells. Biochemical Pharmacology, 86, 691–702.

    Article  CAS  PubMed  Google Scholar 

  30. 30.

    Kajiwara, M., Aoi, T., Okita, K., et al. (2012). Donor-dependent variations in hepatic differentiation from human induced pluripotent stem cells. Proceedings of the National Academy of Sciences of the United States of America, 109, 12538–12543.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  31. 31.

    Hannan, N. R., Segeritz, C. P., Touboul, T., et al. (2013). Production of hepatocyte-like cells from human pluripotent stem cells. Nature Protocols, 8, 430–437.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  32. 32.

    Hannan, N. R., Fordham, R. P., Syed, Y. A., et al. (2013). Generation of multipotent foregut stem cells from human pluripotent stem cells. Stem Cell Reports, 1, 293–306.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  33. 33.

    Mikkola, M., Olsson, C., Palgi, J., et al. (2006). Distinct differentiation characteristics of individual human embryonic stem cell lines. BMC Developmental Biology, 6, 40.

    PubMed Central  Article  PubMed  Google Scholar 

  34. 34.

    Osafune, K., Caron, L., Borowiak, M., et al. (2008). Marked differences in differentiation propensity among human embryonic stem cell lines. Nature Biotechnology, 3, 313–315.

    Article  Google Scholar 

  35. 35.

    Itaba, N., Wairagu, P. M., Aramaki, N., et al. (2014). Nuclear receptor gene alteration in human induced pluripotent stem cells with hepatic differentiation propensity. Hepatology Research, 44(14), 408–439.

    Article  Google Scholar 

  36. 36.

    Bock, C., Kiskinis, E., Verstappen, G., et al. (2011). Reference maps of human embryonic and induced pluripotent stem cell variation enable high-throughput characterization of pluripotent cell lines. Cell, 144, 439–452.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  37. 37.

    Heins, N., Englund, M. C., Sjöblom, C., et al. (2004). Derivation, characterization, and differentiation of human embryonic stem cells. Stem Cells, 22, 367–370.

    Article  PubMed  Google Scholar 

  38. 38.

    Sjögren-Jansson, E., Zetterström, M., Moya, K., et al. (2005). Large-scale propagation of four undifferentiated human embryonic stem cell lines in a feeder-free culture system. Developmental Dynamics, 233, 1304–1314.

    Article  PubMed  Google Scholar 

  39. 39.

    Caisander, G., Park, H., Frej, K., et al. (2006). Chromosomal integrity maintained in five human embryonic stem cell lines after prolonged in vitro culture. Chromosome Research, 14, 131–137.

    Article  CAS  PubMed  Google Scholar 

  40. 40.

    Aguilar-Gallardo, C., Poo, M., Gomez, E., et al. (2010). Derivation, characterization, differentiation, and registration of seven human embryonic stem cell lines (VAL-3, -4, -5, -6M, -7, -8, and -9) on human feeder. In Vitro Cellular and Developmental Biology - Animal, 46, 317–326.

    Article  PubMed  Google Scholar 

  41. 41.

    Funa, N. S., Schachter, K. A., Lerdrup, M., et al. (2015). Beta-Catenin regulates primitive streak induction through collaborative interactions with SMAD2/SMAD3 and OCT4. Cell Stem Cell, 16(6), 639–652.

    Article  CAS  PubMed  Google Scholar 

  42. 42.

    Holmgren, G., Sjögren, A. K., Barragan, I., et al. (2014). Long-term chronic toxicity testing using human induced pluripotent stem cell-derived hepatocytes. Drug Metabolism and Disposition, 42, 1401–1406.

    Article  PubMed  Google Scholar 

  43. 43.

    Sartipy, P. (2013). Advancing pluripotent stem cell culture: it is a matter of setting the standard. Stem Cells and Development, 22, 1159–1161.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  44. 44.

    Kim, K., Doi, A., Wen, B., et al. (2010). Epigenetic memory in induced pluripotent stem cells. Nature, 467, 285–290.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  45. 45.

    Ohi, Y., Qin, H., Hong, C., et al. (2011). Incomplete DNA methylation underlies a transcriptional memory of somatic cells in human induced pluripotent stem cells. Nature Cell Biology, 13, 541–549.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  46. 46.

    Bar-Nur, O., Russ, H. A., Efrat, S., et al. (2011). Epigenetic memory and preferential lineage-specific differentiation in induced pluripotent stem cell-derived from human beta-cells. Cell Stem Cell, 9, 17–23.

    Article  CAS  PubMed  Google Scholar 

  47. 47.

    Lee, S. B., Seo, D., Choi, D., et al. (2012). Contribution of hepatic lineage stage-specific donor memory to the differential potential of induced mouse pluripotent stem cells. Stem Cells, 30, 997–1007.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  48. 48.

    Ponsoda, X., Pareja, E., Gomez-Lechon, M. J., et al. (2001). Drug biotransformation by human hepatocytes. In vitro/in vivo metabolism by cells from the same donor. Journal of Hepatology, 34, 19–25.

    Article  CAS  PubMed  Google Scholar 

  49. 49.

    Kuehl, P., Zhang, J., Lin, Y., et al. (2001). Sequence diversity in CYP3A promoters and characterization of the genetic basis of polymorphic CYP3A5 expression. Nature Genetics, 27, 383–391.

    Article  CAS  PubMed  Google Scholar 

  50. 50.

    Takayama, K., Morisaki, Y., Kuno, S., et al. (2014). Prediction of interindividual differences in hepatic functions and drug sensitivity by using human induced pluripotent stem cell-derived hepatocytes. Proceedings of the National Academy of Sciences of the United States of America, 111, 16772–16777.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

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Acknowledgments

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

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Correspondence to Barbara Küppers-Munther.

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Suppl. Figure 1
figure7

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)

Suppl. Figure 2
figure8

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)

Suppl. Figure 3
figure9

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)

Suppl. Figure 4
figure10

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)

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Asplund, A., Pradip, A., van Giezen, M. et al. One Standardized Differentiation Procedure Robustly Generates Homogenous Hepatocyte Cultures Displaying Metabolic Diversity from a Large Panel of Human Pluripotent Stem Cells. Stem Cell Rev and Rep 12, 90–104 (2016). https://doi.org/10.1007/s12015-015-9621-9

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Keywords

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