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Production of hepatocyte-like cells from human pluripotent stem cells

Nature Protocols volume 8, pages 430–437 (2013)Cite this article

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Abstract

Large-scale production of hepatocytes from a variety of genetic backgrounds would be beneficial for drug screening and to provide a source of cells to be used as a substitute for liver transplantation. However, fully functional primary hepatocytes remain difficult to expand in vitro, and circumventing this problem by using an alternative source of cells is desirable. Here we describe a 25-d protocol to direct the differentiation of human pluripotent stem cells into a near-homogenous population of hepatocyte-like cells. As cells progress through this protocol, they express genes in a chronological manner similar to that described during in vivo hepatic development. The protocol relies on culture systems devoid of serum, feeders or complex extracellular matrices, which enable molecular analyses without interference from unknown factors. This approach works efficiently with human embryonic stem cells and human induced pluripotent stem cells and was recently used to model liver diseases in vitro.

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Figure 1: Protocol for differentiating hPSCs into hepatocytes.
Figure 2: Microphotographs showing endoderm differentiation of hPSCs.
Figure 3: Microphotographs showing hepatic specification.
Figure 4: Hepatocyte-like cells generated after 20 d of differentiation.

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References

  1. Fisher, R.A. & Strom, S.C. Human hepatocyte transplantation: worldwide results. Transplantation 82, 441–449 (2006).

    Article  Google Scholar 

  2. Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Rashid, S.T. & Vallier, L. Induced pluripotent stem cells—alchemist's tale or clinical reality? Expert Rev. Mol. Med. 12, 25 (2010).

    Article  Google Scholar 

  5. Rashid, S.T. et al. Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells. J. Clin. Invest. 120, 3127–3136 (2010).

    Article  CAS  Google Scholar 

  6. Yusa, K. et al. Targeted gene correction of alpha1-antitrypsin deficiency in induced pluripotent stem cells. Nature 478, 391–394 (2011).

    Article  CAS  Google Scholar 

  7. Mikkola, M. et al. Distinct differentiation characteristics of individual human embryonic stem cell lines. BMC Dev. Biol. 6, 40 (2006).

    Article  Google Scholar 

  8. Bader, D. et al. α-Fetoprotein in the early neonatal period—a large study and review of the literature. Clin. Chim. Acta. 349, 15–23 (2004).

    Article  CAS  Google Scholar 

  9. Stevens, J.C. et al. Developmental expression of the major human hepatic CYP3A enzymes. J. Pharmacol. Exp. Ther. 307, 573–582 (2003).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Brons, I.G. et al. Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191–195 (2007).

    Article  CAS  Google Scholar 

  12. Yang, Y. et al. The E47 transcription factor negatively regulates CD5 expression during thymocyte development. Proc. Natl. Acad. Sci. USA 101, 3898–3902 (2004).

    Article  CAS  Google Scholar 

  13. Vallier, L., Reynolds, D. & Pedersen, R.A. Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. Dev. Biol. 275, 403–421 (2004).

    Article  CAS  Google Scholar 

  14. Yamada, T. et al. In vitro differentiation of embryonic stem cells into hepatocyte-like cells identified by cellular uptake of indocyanine green. Stem Cells 20, 146–154 (2002).

    Article  Google Scholar 

  15. Teo, A.K. et al. Pluripotency factors regulate definitive endoderm specification through eomesodermin. Genes Dev. 25, 238–250 (2011).

    Article  CAS  Google Scholar 

  16. Rodriguez, R.T. et al. Manipulation of OCT4 levels in human embryonic stem cells results in induction of differential cell types. Exp. Biol. Med. (Maywood) 232, 1368–1380 (2007).

    Article  CAS  Google Scholar 

  17. Aguilar-Gallardo, C. et al. 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 Cell Dev. Biol. Anim. 46, 317–326 (2010).

    Article  Google Scholar 

  18. Vallier, L. et al. Signaling pathways controlling pluripotency and early cell fate decisions of human induced pluripotent stem cells. Stem Cells 27, 2655–2666 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was funded by a Medical Research Council (MRC) Senior Non-clinical Fellowship (T.T. and L.V.); the Cambridge Hospitals National Institute for Health Research Biomedical Research Center (L.V.); the EU grant LivES and the Evelyn Trust (N.R.F.H.); and the Children's Liver Disease Foundation (C.-P.S.).

Author information

Authors and Affiliations

  1. Department of Surgery, Wellcome Trust–Medical Research Council Stem Cell Institute, Anne McLaren Laboratory for Regenerative Medicine, University of Cambridge, Cambridge, UK

    Nicholas R F Hannan, Charis-Patricia Segeritz & Ludovic Vallier

  2. Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA

    Thomas Touboul

  3. Wellcome Trust Sanger Institute, Hinxton, UK

    Ludovic Vallier

Authors
  1. Nicholas R F Hannan
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  2. Charis-Patricia Segeritz
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  3. Thomas Touboul
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  4. Ludovic Vallier
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Contributions

N.R.F.H. and L.V. contributed to the protocol concept, design, interpretation, validation and optimization; C.-P.S. and T.T. contributed to protocol optimization and validation.

Corresponding author

Correspondence to Ludovic Vallier.

Supplementary information

Supplementary Table 1

Primer sequences for qPCR analyses of genes expressed during differentiation of artificial hepatocytes. (PDF 220 kb)

Supplementary Figure 1

Characterization of hESC-derived hepatocyte-like cells and comparison with primary hepatocytes. (A) Expression of hepatic markers during differentiation of hESCs. qPCR analyses showing the progressive decrease in AFP expression as cells start to mature and at the same time increase expression of the hepatic makers (Albumin and A1AT) during maturation. Primary Hepatocytes (Biopredicts) were used as positive control. Data are presented as the average of 3 biological replicates and error bars indicate standard deviation. (B) ELISA analyses showing increase in Albumin and A1AT secretion during maturation of hepatocytes like cells. Primary Hepatocytes (Biopredicts) were used as positive control. Data are presented as the average of 3 biological replicates and error bars indicate standard deviation. Medium only was used as negative control (C) hPSCs derived hepatocytes cells also display detoxifying activity associated with cytochrome P450 family members such as CyP3A4. This activity can be further induced by chemical such as dexamethasone. (D) Cells also display the ability to take up and metabolise indocyanine green as described by Yamada et el 200214. Scale bar: 200 μm (PDF 433 kb)

Supplementary Figure 2

Comparison of hPSC-derived hepatocytes to human primary fetal hepatocytes. Foetal hepatocytes differentiated from hPSC's share an expression profile very similar to in-vivo derived primary human foetal hepatocytes. AFP, ALB, APOF and CYP3A4 show very similar expression levels when compared between the two cell types, while A1AT does appear to be generally higher along with several other mature metabolic genes such as such as tyrosine amino-transferase (TAT), tryptophan 2,3-dioxygenase (TDO2) and transthyretin (TTR). (PDF 319 kb)

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Hannan, N., Segeritz, CP., Touboul, T. et al. Production of hepatocyte-like cells from human pluripotent stem cells. Nat Protoc 8, 430–437 (2013). https://doi.org/10.1038/nprot.2012.153

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