Skip to main content

Development of a Scalable Three-Dimensional Culture of Human Induced Pluripotent Stem Cells-Derived Liver Organoids

Part of the Methods in Molecular Biology book series (MIMB,volume 2455)

Abstract

Human induced pluripotent stem cells (hiPSCs) represent a powerful tool for the generation of specialized cells to be used in regenerative medicine as well as hepatocellular repopulation tool to treat liver metabolic diseases such as nonalcoholic steatohepatitis (NASH). Here we describe a strategy to obtain fully functional liver organoids from hiPSCs in a scalable manner. Our approach uses a two-step process, with a first step involving the scalable formation of homogeneous and uniform-sized human embryoid bodies (hEBs), followed by the application of a four-step liver differentiation protocol for the derivation of liver organoids that possess all the features of primary human hepatocytes. This chapter will also illustrate the characterization of the liver organoids by directed biomolecular techniques.

Key words

  • Differentiation
  • Human induced pluripotent stem cells (hiPSCs)
  • Liver differentiation
  • Liver organoids
  • Human embryoid bodies (hEBs)
  • Regenerative medicine

This is a preview of subscription content, access via your institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-0716-2128-8_12
  • Chapter length: 17 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   169.00
Price excludes VAT (USA)
  • ISBN: 978-1-0716-2128-8
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Hardcover Book
USD   219.99
Price excludes VAT (USA)
Fig. 1
Fig. 2
Fig. 3

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Snykers S, De Kock J, Tamara V et al (2011) Hepatic differentiation of mesenchymal stem cells: in vitro strategies. Methods Mol Biol 698:305–314

    CAS  CrossRef  Google Scholar 

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

    CAS  CrossRef  Google Scholar 

  3. Asgari S, Moslem M, Bagheri-Lankarani K et al (2013) Differentiation and transplantation of human induced pluripotent stem cell-derived hepatocyte-like cells. Stem Cell Rev Rep 9:493–504

    CAS  CrossRef  Google Scholar 

  4. Hay DC, Zhao D, Ross A et al (2007) Direct differentiation of human embryonic stem cells to hepatocyte-like cells exhibiting functional activities. Cloning Stem Cells 9:51–62

    CAS  CrossRef  Google Scholar 

  5. Soderdahl T, Kuppers-Munther B, Heins N et al (2007) Glutathione transferases in hepatocyte-like cells derived from human embryonic stem cells. Toxicol In Vitro 21:929–937

    CrossRef  Google Scholar 

  6. Schwartz RE, Linehan JL, Painschab MS et al (2005) Defined conditions for development of functional hepatic cells from human embryonic stem cells. Stem Cells Dev 14:643–655

    CAS  CrossRef  Google Scholar 

  7. Rambhatla L, Chiu CP, Kundu P et al (2003) Generation of hepatocyte-like cells from human embryonic stem cells. Cell Transplant 12:1–11

    CrossRef  Google Scholar 

  8. Cai J, Zhao Y, Liu Y et al (2007) Directed differentiation of human embryonic stem cells into functional hepatic cells. Hepatology 45:1229–1239

    CAS  CrossRef  Google Scholar 

  9. Subramanian K, Owens DJ, Raju R et al (2014) Spheroid culture for enhanced differentiation of human embryonic stem cells to hepatocyte-like cells. Stem Cells Dev 23:124–131

    CAS  CrossRef  Google Scholar 

  10. Kim WR, Lake JR, Smith JM et al (2018) OPTN/SRTR 2016 annual data report: liver. Am J Transplant 18(Suppl 1):172–253

    CrossRef  Google Scholar 

  11. Dutkowski P, Linecker M, DeOliveira ML et al (2015) Challenges to liver transplantation and strategies to improve outcomes. Gastroenterology 148:307–323

    CrossRef  Google Scholar 

  12. Hovaguimian F, Schlapfer M, Beck-Schimmer B (2014) Organ protection in allograft recipients: anesthetic strategies to reduce postoperative morbidity and mortality. Current Opin Organ Transplant 19:121–130

    CAS  CrossRef  Google Scholar 

  13. Ilmaz S, Ozgor D, Dirichan A et al (2014) Complications after transplantation of a hepatic fragment from a living donor: the results of surgical interventions in 600 donors. Klin Khir 3:16–18

    Google Scholar 

  14. Ohashi K (2008) Liver tissue engineering: the future of liver therapeutics. Hepatol Res 38(Suppl 1):S76–S87

    CAS  CrossRef  Google Scholar 

  15. Pettinato G, Vanden Berg-Foels WS et al (2014) ROCK inhibitor is not required for embryoid body formation from singularized human embryonic stem cells. PLoS One 9:e100742

    CrossRef  Google Scholar 

  16. Pettinato G, Wen X, Zhang N (2014) Formation of well-defined embryoid bodies from dissociated human induced pluripotent stem cells using microfabricated cell-repellent microwell arrays. Sci Rep 4:7402

    CAS  CrossRef  Google Scholar 

  17. Pettinato G, Wen X, Zhang N (2015) Engineering strategies for the formation of Embryoid bodies from human pluripotent stem cells. Stem Cells Dev 24:1595–1609

    CAS  CrossRef  Google Scholar 

  18. Pettinato G, Ramanathan R, Fisher RA et al (2016) Scalable differentiation of human iPSCs in a multicellular spheroid-based 3D culture into hepatocyte-like cells through direct Wnt/beta-catenin pathway inhibition. Sci Rep 6:32888

    CAS  CrossRef  Google Scholar 

  19. Collin de l’Hortet A, Takeishi K et al (2016) Liver-regenerative transplantation: regrow and reset. Am J Transplant 16:1688–1696

    CrossRef  Google Scholar 

  20. Ferrer JR, Chokechanachaisakul A, Wertheim JA (2015) New tools in experimental cellular therapy for the treatment of liver diseases. Curr Transplant Rep 2:202–210

    CrossRef  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Giuseppe Pettinato .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Verify currency and authenticity via CrossMark

Cite this protocol

Pettinato, G., Perelman, L.T., Fisher, R.A. (2022). Development of a Scalable Three-Dimensional Culture of Human Induced Pluripotent Stem Cells-Derived Liver Organoids. In: Sarkar, D. (eds) Non-Alcoholic Steatohepatitis. Methods in Molecular Biology, vol 2455. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2128-8_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2128-8_12

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2127-1

  • Online ISBN: 978-1-0716-2128-8

  • eBook Packages: Springer Protocols