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Direct conversion of mouse fibroblasts into induced neural stem cells


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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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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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.

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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).

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