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Direct conversion of mouse fibroblasts into induced neural stem cells

Nature Protocols volume 9, pages 871–881 (2014)Cite this article

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Abstract

Terminally differentiated cells can be directly converted into different types of somatic cells by using defined factors, thus circumventing the pluripotent state. However, low reprogramming efficiency, along with the absence of proliferation of some somatic cell types, makes it difficult to generate large numbers of cells with this method. Here we describe a protocol to directly convert mouse fibroblasts into self-renewing induced neural stem cells (iNSCs) that can be expanded in vitro, thereby overcoming the limitations associated with low reprogramming efficiency. The four transcription factors required for direct conversion into iNSCs (Sox2, Klf4, Myc (also known as c-Myc) and Pou3f4 (also known as Brn4)) do not generate a pluripotent cell state, and thus the risk for tumor formation after transplantation is reduced. By following the current protocol, iNSCs are observed 4–5 weeks after transduction. Two additional months are required to establish clonal iNSC cell lines that exhibit retroviral transgene silencing and that differentiate into neurons, astrocytes and oligodendrocytes.

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Figure 1: Direct conversion of MEFs into iNSCs.
Figure 2: Establishment of clonal iNSC lines.
Figure 3: Nonclonal iNSC cultures are heterogeneous.

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Acknowledgements

We thank C. Ortmeier and M. Sinn for technical assistance, and M. Stehling for FACS sorting. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0013885) and also by a grant from the Next-Generation BioGreen 21 program (PJ009038), Rural Development Administration, Republic of Korea.

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Author notes

  1. Sung Min Kim and Hannah Flaßkamp: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul, Republic of Korea

    Sung Min Kim, Seung Chan Lee & Dong Wook Han

  2. Department of Animal Biotechnology, Konkuk University, Seoul, Republic of Korea

    Sung Min Kim, Sung Ho Lee & Hoon Taek Lee

  3. Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Münster, Germany

    Hannah Flaßkamp, Marcos Jesús Araúzo-Bravo, Hans R Schöler & Natalia Tapia

  4. Division for Neurodegenerative Diseases, Department of Neurology, Dresden University of Technology, Dresden, Germany

    Andreas Hermann & Alexander Storch

  5. Center for Regenerative Therapies Dresden,

    Andreas Hermann & Alexander Storch

  6. Dresden University of Technology,, Dresden, Germany

    Andreas Hermann & Alexander Storch

  7. Group of Computational Biology and Bioinformatics, Biodonostia Health Research Institute, San Sebastián, Spain

    Marcos Jesús Araúzo-Bravo

  8. IKERBASQUE, Basque Foundation for Science, Bilbao, Spain

    Marcos Jesús Araúzo-Bravo

  9. Department of Internal Medicine, School of Medicine, Konkuk University, Seoul, Republic of Korea

    Eun Hye Seo

  10. Department of Microbiology, School of Medicine, Konkuk University, Seoul, Republic of Korea

    Seung Hyun Lee

  11. Center for Regenerative Therapies Dresden, Dresden University of Technology, Dresden, Germany

    Alexander Storch

  12. Medical Faculty, University of Münster, Münster, Germany

    Hans R Schöler

Authors
  1. Sung Min Kim
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  10. Hoon Taek Lee
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  12. Natalia Tapia
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  13. Dong Wook Han
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Contributions

S.M.K. and H.F. were involved in protocol design as well as in the generation and characterization of iNSCs; A.H., S.C.L., Sung Ho Lee, E.H.S., Seung Hyun Lee, A.S. and H.T.L. carried out iNSC characterization; M.J.A.-B. analyzed the microarray analysis; H.R.S. edited the manuscript; and N.T. and D.W.H. were involved in protocol design, generation and characterization of iNSCs, and manuscript preparation.

Corresponding authors

Correspondence to Natalia Tapia or Dong Wook Han.

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The authors declare no competing financial interests.

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Kim, S., Flaßkamp, H., Hermann, A. et al. Direct conversion of mouse fibroblasts into induced neural stem cells. Nat Protoc 9, 871–881 (2014). https://doi.org/10.1038/nprot.2014.056

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