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

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Stem Cells and Tissue Repair

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1210))

Abstract

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.

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Acknowledgements

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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Authors and Affiliations

  1. Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, 3-9, Fukuura, Kanazawa-ku, Yokohama, Kanagawa, 236-0004, Japan

    Ran-Ran Zhang, Takanori Takebe, Leina Miyazaki, Maho Takayama, Hiroyuki Koike, Masaki Kimura, Masahiro Enomura, Yun-Wen Zheng, Keisuke Sekine & Hideki Taniguchi

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  1. Ran-Ran Zhang
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  2. Takanori Takebe
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  3. Leina Miyazaki
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  4. Maho Takayama
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  5. Hiroyuki Koike
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  6. Masaki Kimura
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  7. Masahiro Enomura
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  8. Yun-Wen Zheng
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  9. Keisuke Sekine
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  10. Hideki Taniguchi
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Correspondence to Takanori Takebe .

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Editors and Affiliations

  1. Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon, USA

    Chrissa Kioussi

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Zhang, RR. et al. (2014). Efficient Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Three-Dimensional Microscale Culture. In: Kioussi, C. (eds) Stem Cells and Tissue Repair. Methods in Molecular Biology, vol 1210. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1435-7_10

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  • DOI: https://doi.org/10.1007/978-1-4939-1435-7_10

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1434-0

  • Online ISBN: 978-1-4939-1435-7

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