Article

Isolation and 3D expansion of multipotent Sox9+ mouse lung progenitors

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Abstract

Multiple adult tissues are maintained by stem cells of restricted developmental potential which can only form a subset of lineages within the tissue. For instance, the two adult lung epithelial compartments (airways and alveoli) are separately maintained by distinct lineage-restricted stem cells. A challenge has been to obtain multipotent stem cells and/or progenitors that can generate all epithelial cell types of a given tissue. Here we show that mouse Sox9+ multipotent embryonic lung progenitors can be isolated and expanded long term in 3D culture. Cultured Sox9+ progenitors transcriptionally resemble their in vivo counterparts and generate both airway and alveolar cell types in vitro. Sox9+ progenitors that were transplanted into injured adult mouse lungs differentiated into all major airway and alveolar lineages in vivo in a region-appropriate fashion. We propose that a single expandable embryonic lung progenitor population with broader developmental competence may eventually be used as an alternative for region-restricted adult tissue stem cells in regenerative medicine.

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Acknowledgements

We thank K. Chee, D.A. Silva and N. Talib (SIgN FACS Core); J. Cheng and H.Y. Ng (IMCB Histology Core); S. Lin and G. Wright (IMB Microscopy Unit); K.S. Keyau (Singapore General Hospital); and other A*STAR core facilities for infrastructure support. We also thank A. Bhinge, S.-C. Ng and Z. Wang for discussion; S. Namboori, E. Lim, S.H. Choo, S.P. Guan and J. Lee for technical assistance; and R. Ettikan for logistical support. This work was supported by the Singapore National Medical Research Council (NMRC/TCR/007-NCC/2013), the Singapore Agency for Science, Technology & Research (A*STAR) (1331AEG071; 1334I00052; 1334I00053; SPF 2012/001; H16/99/b0/003) (to B.L.) and the Stanford-UC Berkeley Siebel Stem Cell Institute (to K.M.L.).

Author information

Author notes

    • Bing Lim

    Present address: Translational Medicine Research Centre, Merck Sharp and Dohme, Singapore.

    • Kyle M Loh
    •  & Bing Lim

    These authors jointly directed this work.

Affiliations

  1. Stem Cell & Regenerative Biology Group, Genome Institute of Singapore, A*STAR, Singapore.

    • Massimo Nichane
    • , Monisha Ganesan
    • , Lay Teng Ang
    •  & Bing Lim
  2. Cancer Therapeutics & Stratified Oncology Group, Genome Institute of Singapore, A*STAR, Singapore.

    • Asif Javed
  3. The Jackson Laboratory for Genomic Medicine, Farmington, Connecticut, USA.

    • V Sivakamasundari
  4. Department of Biology, Clarkson University, Potsdam, New York, USA.

    • Petra Kraus
    •  & Thomas Lufkin
  5. Stanford Institute for Stem Cell Biology and Regenerative Medicine and the Stanford-UC Berkeley Siebel Stem Cell Institute, Stanford University School of Medicine, Stanford, California, USA.

    • Kyle M Loh

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Contributions

M.N. conceptualized this work; M.N. developed methodology; M.N. and M.G. carried out investigation; A.J. conducted formal analysis; A.J., V.S. and M.N. curated data; V.S., A.J. and M.N. carried out visualization; M.N. and K.M.L. wrote the original draft; M.N., A.J., V.S., K.M.L, L.T.A. and B.L. reviewed and edited the manuscript; B.L. and K.M.L. acquired funds; V.S., L.T.A., K.M.L., T.L. and P.K. provided resources; M.N. and B.L. supervised this work.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Massimo Nichane or Kyle M Loh or Bing Lim.

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Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–14, Supplementary Note 1 and Supplementary Tables 1–5

  2. 2.

    Life Sciences Reporting Summary

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Protocol

    3D culture of multipotent Sox9+ mouse embryonic lung progenitors: Isolation, Expansion and Cryopreservation