Liver Regeneration Using Cultured Liver Bud

  • Keisuke Sekine
  • Takanori Takebe
  • Hideki TaniguchiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1597)


Here, we describe a protocol to develop a three-dimensional (3D) liver bud-like tissue from human iPSCs in vitro. This method mainly consists of two parts: (1) hepatic endoderm (HE) differentiation from human iPSCs in 2D culture and (2) co-culturing iPSC-HE with endothelial and mesenchymal cells. First, iPSCs were differentiated into definitive endoderm (DE) cells, and the DE cells were differentiated into HE cells, which were then co-cultured with endothelial cells and mesenchymal cells on Matrigel-coated plastic plates or micropattern plates. The cells rapidly condensed to generate 3D tissue masses. We named these iPSC liver buds (iPSC-LBs) because they resemble the developing liver bud from the perspective of gene expression, cell proliferation, and cell proportion. This liver bud culture system provides a novel approach for future clinical applications, for drug development, and as a tool for studying human development.

Key words

Human-induced pluripotent stem cells Liver bud Three-dimensional culture Self-organization Multicellular interaction Regenerative medicine Drug development 


  1. 1.
    Mclaren A (2001) Ethical and social considerations of stem cell research. Nature 414:129–131. doi: 10.1038/35102194 CrossRefPubMedGoogle Scholar
  2. 2.
    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. doi: 10.1016/j.cell.2007.11.019 CrossRefPubMedGoogle Scholar
  3. 3.
    Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676. doi: 10.1016/j.cell.2006.07.024 CrossRefPubMedGoogle Scholar
  4. 4.
    Miura K, Okada Y, Aoi T et al (2009) Variation in the safety of induced pluripotent stem cell lines. Nat Biotechnol 27:743–745. doi: 10.1038/nbt.1554. pii: nbt.1554 [pii]\rCrossRefPubMedGoogle Scholar
  5. 5.
    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. doi: 10.1002/hep.23354 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Si-Tayeb K, Lemaigre FP, Duncan SA (2010) Organogenesis and development of the liver. Dev Cell 18:175–189. doi: 10.1016/j.devcel.2010.01.011 CrossRefPubMedGoogle Scholar
  7. 7.
    Zaret KS, Grompe M (2008) Generation and regeneration of cells of the liver and pancreas. Science 322:1490–1494. doi: 10.1126/science.1161431 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Franklin V, Khoo PL, Bildsoe H et al (2008) Regionalisation of the endoderm progenitors and morphogenesis of the gut portals of the mouse embryo. Mech Dev 125:587–600. doi: 10.1016/j.mod.2008.04.001 CrossRefPubMedGoogle Scholar
  9. 9.
    Arterbery AS, Bogue CW (2014) Endodermal and mesenchymal cross talk: a crossroad for the maturation of foregut organs. Pediatr Res 75:120–126. doi: 10.1038/pr.2013.201 CrossRefPubMedGoogle Scholar
  10. 10.
    Lokmane L, Haumaitre C, Garcia-Villalba P et al (2008) Crucial role of vHNF1 in vertebrate hepatic specification. Development 135:2777–2786. doi: 10.1242/dev.023010 CrossRefPubMedGoogle Scholar
  11. 11.
    Sekine K, Chen Y-R, Kojima N et al (2007) Foxo1 links insulin signaling to C/EBPalpha and regulates gluconeogenesis during liver development. EMBO J 26:3607–3615CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Kajiwara M, Aoi T, Okita K et al (2012) Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells. Proc Natl Acad Sci USA 109:12538–12543. doi: 10.1073/pnas.1209979109 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Grapin-Botton A (2008) Endoderm specification. Stem Book 3–4. doi:  10.3824/stembook.1.30.1
  14. 14.
    Matsuno K, Mae SI, Okada C, et al (2016) Redefining definitive endoderm subtypes by robust induction of human induced pluripotent stem cells. Differentiation 1–10. doi:  10.1016/j.diff.2016.04.002
  15. 15.
    Sekine K, Takebe T, Suzuki Y et al (2012) Highly efficient generation of definitive endoderm lineage from human induced pluripotent stem cells. Transplant Proc 44:1127–1129. doi: 10.1016/j.transproceed.2012.03.001 CrossRefPubMedGoogle Scholar
  16. 16.
    Collin de l’Hortet A, Takeishi K, Guzman-Lepe J et al (2016) Liver-regenerative transplantation: regrow and reset. Am J Transplant 16:1688–1696. doi: 10.1111/ajt.13678 CrossRefPubMedGoogle Scholar
  17. 17.
    Takebe T, Sekine K, Enomura M et al (2013) Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 499:481–484. doi: 10.1038/nature12271 CrossRefPubMedGoogle Scholar
  18. 18.
    Takebe T, Zhang R-R, Koike H et al (2014) Generation of a vascularized and functional human liver from an iPSC-derived organ bud transplant. Nat Protoc 9:396–409. doi: 10.1038/nprot.2014.020 CrossRefPubMedGoogle Scholar
  19. 19.
    Takebe T, Sekine K, Suzuki Y et al (2012) Self-organization of human hepatic organoid by recapitulating organogenesis in vitro. Transplant Proc 44:1018–1020. doi: 10.1016/j.transproceed.2012.02.007 CrossRefPubMedGoogle Scholar
  20. 20.
    Takebe T, Koike N, Sekine K et al (2012) Generation of functional human vascular network. Transplant Proc 44:1130–1133. doi: 10.1016/j.transproceed.2012.03.039 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Keisuke Sekine
    • 1
  • Takanori Takebe
    • 1
    • 2
    • 3
    • 4
  • Hideki Taniguchi
    • 1
    • 2
    Email author
  1. 1.Department of Regenerative MedicineYokohama City University Graduate School of MedicineYokohamaJapan
  2. 2.Advanced medical research centerYokohama City UniversityYokohamaJapan
  3. 3.PRESTO, Japan Science and Technology AgencyKawaguchiJapan
  4. 4.Department of Pediatrics, Cincinnati Children’s Hospital Medical CenterUniversity of CincinnatiCincinnatiUSA

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