Article

Long-term expansion of alveolar stem cells derived from human iPS cells in organoids

  • Nature Methods volume 14, pages 10971106 (2017)
  • doi:10.1038/nmeth.4448
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Abstract

The stable expansion of tissue-specific stem cells in vitro has contributed to research on several organs. Alveolar epithelial type II (AT2) cells function as tissue stem cells in the lung, but robust models for studying human AT2 cells are lacking. Here we report a method for the efficient generation and long-term expansion of alveolar organoids (AOs) harboring SFTPC+ alveolar stem cells derived from human induced pluripotent stem cells (hiPSCs). hiPSC-derived SFTPC+ cells self-renewed, with transcriptomes and morphology consistent with those of AT2 cells, and were able to differentiate into alveolar epithelial type I (AT1)-like cells. Single-cell RNA-seq of SFTPC+ cells and their progenitors demonstrated that their differentiation process and cellular heterogeneity resembled those of developing AT2 cells in vivo. AOs were applicable to drug toxicology studies recapitulating AT2-cell-specific phenotypes. Our methods can help scientists overcome the limitations of current approaches to the modeling of human alveoli and should be useful for disease modeling and regenerative medicine.

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Change history

  • Corrected online 14 November 2017

    In the version of this article initially published, there were errors in the Online Methods section. Specifically, incorrect concentrations were given for 8-Br-cAMP, 3-isobutyl-1-methylxanthine, and KGF, as used for induction and passage of alveolar stem cells in fibroblast-dependent organoids. The errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank K. Osafune, K. Okita, K. Takahashi and S. Yamanaka (Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan) for providing iPSC lines. We thank F. Chen, H. Date and all the members of the Department of Thoracic Surgery, Kyoto University (Kyoto, Japan), for providing surgical specimens of human adult lung. We thank K. Okamoto-Furuta and H. Kohda (Center for Anatomical Studies, Kyoto University, Kyoto, Japan) for electron microscopy studies. We thank T. Horiuchi and K. Imamura (Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan) for scRNA-seq. We thank K. Ando and K. Seyama (Juntendo University Faculty of Medicine and Graduate School of Medicine, Tokyo, Japan) for technical guidance on the isolation of human adult alveolar epithelial cells. We thank N. Inagaki and D. Tanaka (Department of Diabetes, Endocrinology and Nutrition, Kyoto University, Kyoto, Japan) for kindly providing anti-human ABCA3 antibody, and M. Hagiwara, Y. Okuno and all the members of Medical Research Support Center, Kyoto University (Kyoto, Japan), for helpful discussion and supporting use of Gene Spring software and fluorescence studies. We also thank Y. Maeda and A. Inazumi for technical support. This work was supported by MEXT of Japan (Grant-in-Aid for Scientific Research (KAKENHI) numbers 15K21114 and 17H05084 to S.G., 15H04318 and 16H06279 to Y.S., and 22249031 and 15H02537 to M.M.), the Japan Agency for Medical Research and Development (grants KU-A032, 16bm0704008h0001 and 17bm0704008h0002 to S.G.), KANAE Foundation for the Promotion of Medical Science (to S.G.), Takeda Science Foundation (to S.G.), and in part by Daiichi Sankyo (research grant to T.H. and M.M.).

Author information

Affiliations

  1. Department of Respiratory Medicine, Kyoto University, Kyoto, Japan.

    • Yuki Yamamoto
    • , Shimpei Gotoh
    • , Yohei Korogi
    • , Satoshi Konishi
    • , Satoshi Ikeo
    • , Naoyuki Sone
    • , Tadao Nagasaki
    • , Hisako Matsumoto
    • , Shigeo Muro
    • , Isao Ito
    • , Toyohiro Hirai
    •  & Michiaki Mishima
  2. Department of Drug Discovery for Lung Diseases, Kyoto University, Kyoto, Japan.

    • Shimpei Gotoh
  3. Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan.

    • Masahide Seki
    •  & Yutaka Suzuki
  4. Division of Genome Biology, National Cancer Center Research Institute, Tokyo, Japan.

    • Takashi Kohno
  5. Division of Genome Biology, National Cancer Center Research Institute, Tokyo, Japan.

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Contributions

Y.Y. and S.G. conceived and designed the study. Y.Y., S.G., Y.K., M.S., S.K., S.I., N.S. and T.N. conducted the experiments. Y.Y., S.G., M.S., T.K. and Y.S. analyzed the data. Y.Y. and S.G. wrote the manuscript after fruitful discussion with and under the supervision of M.S., H.M., S.M., I.I., T.H., T.K., Y.S. and M.M.

Competing interests

Kyoto University has applied for a patent related to the method of alveolar cell differentiation from induced pluripotent stem cells discussed in this paper.

Corresponding author

Correspondence to Shimpei Gotoh.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–15 and Supplementary Tables 3, 5, 10 and 11.

  2. 2.

    Life Sciences Reporting Summary

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Protocol

    Methods of generating human pluripotent stem cell (PSC)-derived alveolar stem cells and their expansion

Excel files

  1. 1.

    Supplementary Table 1

    Genes that were significantly upregulated inhiPSC-derived SFTPC+ cells compared with expression in SFTPC– cells and CPMhi progenitor cells, as determined by microarray

  2. 2.

    Supplementary Table 2

    Genes associated with preconditioning of VAFE cells

  3. 3.

    Supplementary Table 4

    Genes that were significantly upregulated in adult AT2 cells compared with their expression in FD-SFTPC+ cells

  4. 4.

    Supplementary Table 6

    Genes with significantly different expression between AT1-marker-high and AT1-marker-low cells, as determined by single-cell RNA-seq

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    Supplementary Table 7

    Genes whose expression changed in GNE7915-treated or amiodarone-treated P3 FD-SFTPC+ cells

  6. 6.

    Supplementary Table 8

    GO terms associated with genes that responded to treatment with GNE7915 and/or amiodarone

  7. 7.

    Supplementary Table 9

    Performance of the methods in the present study compared with that in the previous study