Efficient Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Three-Dimensional Microscale Culture

  • Ran-Ran Zhang
  • Takanori TakebeEmail author
  • Leina Miyazaki
  • Maho Takayama
  • Hiroyuki Koike
  • Masaki Kimura
  • Masahiro Enomura
  • Yun-Wen Zheng
  • Keisuke Sekine
  • Hideki Taniguchi
Part of the Methods in Molecular Biology book series (MIMB, volume 1210)


Human induced pluripotent stem cells (iPSCs) represent a novel source of hepatocytes for drug development, disease modeling studies, and regenerative therapy for the treatment of liver diseases. A number of protocols for generating functional hepatocytes have been reported worldwide; however, reproducible and efficient differentiation remained challenging under conventional two-dimensional (2D) culture. In this study, we describe an efficient differentiation protocol for generating functional hepatocyte-like cells from human iPSC-derived homogenous hepatic endoderm cells combined with three-dimensional (3D) microscale culture system. First, hepatic endoderm cells (iPSC-HEs) were directly differentiated using two-step approaches, and then cultured in the 3D micropattern plate. Human iPSC-HEs quickly reaggregated and formed hundreds of round-shaped spheroids at day 4 of cell plating. The size distribution of iPSC-HEs derived spheroids was relatively uniform around 100–200 μm in diameter. After 14 days, iPSC-HEs efficiently differentiated into hepatocyte-like cells in terms of hepatic maker gene expression compared with conventional 2D approach. We conclude that our scalable and three-dimensional culture system would be one promising approach to generate a huge number of hepatocyte-like cells from human iPSCs aiming at future industrial and clinical applications.

Key words

Human induced pluripotent stem cells Hepatocyte-like cells Three-dimensional culture Spheroid 



We thank F. Kawamata, S Funayama, N. Hijikata, and N. Sasaki for kindly providing technical support; Y. Ueno and all of the members of our laboratory for help with several comments. This work was supported by the Grants-in-Aid of the Ministry of Education, Culture, Sports, Science, and Technology of Japan to T. Takebe (no. 24106510, 24689052), N. Koike (no. 22390260), and H. Taniguchi (no. 21249071, 25253079). This work was also supported by grants to H. Taniguchi from the Strategic Promotion of Innovative Research and Development (S-innovation, 62890004) of the Japan Science and Technology Agency (JST); by the Specified Research Grant of the Takeda Science Foundation and a grant from the Japan IDDM network to H. Taniguchi; and by a grant of the Yokohama Foundation for Advanced Medical Science to T. Takebe.


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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Ran-Ran Zhang
    • 1
  • Takanori Takebe
    • 1
    Email author
  • Leina Miyazaki
    • 1
  • Maho Takayama
    • 1
  • Hiroyuki Koike
    • 1
  • Masaki Kimura
    • 1
  • Masahiro Enomura
    • 1
  • Yun-Wen Zheng
    • 1
  • Keisuke Sekine
    • 1
  • Hideki Taniguchi
    • 1
  1. 1.Department of Regenerative MedicineYokohama City University Graduate School of MedicineYokohamaJapan

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