Efficient Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Three-Dimensional Microscale Culture
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 wordsHuman 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.
- 9.Acikgöz A, Giri S, Cho M-G, Bader A (2013) Morphological and functional analysis of hepatocyte spheroids generated on poly-HEMA-treated surfaces under the influence of fetal calf serum and nonparenchymal cells. Biomolecules 3(1):242–269. doi: 10.3390/biom3010242 PubMedCentralPubMedCrossRefGoogle Scholar
- 16.Takayama K, Kawabata K, Nagamoto Y, Kishimoto K, Tashiro K, Sakurai F, Tachibana M, Kanda K, Hayakawa T, Furue MK, Mizuguchi H (2013) 3D spheroid culture of hESC/hiPSC-derived hepatocyte-like cells for drug toxicity testing. Biomaterials 34(7):1781–1789. doi: 10.1016/j.biomaterials.2012.11.029 PubMedCrossRefGoogle Scholar
- 17.Xia L, Sakban RB, Qu Y, Hong X, Zhang W, Nugraha B, Tong WH, Ananthanarayanan A, Zheng B, Chau IY-Y, Jia R, McMillian M, Silva J, Dallas S, Yu H (2012) Tethered spheroids as an in vitro hepatocyte model for drug safety screening. Biomaterials 33(7):2165–2176. doi: 10.1016/j.biomaterials.2011.12.006 PubMedCrossRefGoogle Scholar
- 21.Ogawa S, Surapisitchat J, Virtanen C, Ogawa M, Niapour M, Sugamori KS, Wang S, Tamblyn L, Guillemette C, Hoffmann E (2013) Three-dimensional culture and cAMP signaling promote the maturation of human pluripotent stem cell-derived hepatocytes. Development 140(15):3285–3296PubMedCentralPubMedCrossRefGoogle Scholar
- 22.Kawasaki H, Suemori H, Mizuseki K, Watanabe K, Urano F, Ichinose H, Haruta M, Takahashi M, Yoshikawa K, Nishikawa S-I (2002) Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity. Proc Natl Acad Sci 99(3):1580–1585PubMedCentralPubMedCrossRefGoogle Scholar
- 25.Takebe T, Zhang R-R, Koike H, Kimura M, Yoshizawa E, Enomura M, Koike N, Sekine K, Taniguchi H (2014) Generation of a vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature Protocols 9(2):396–409Google Scholar