Journal
NATURE PROTOCOLS
Volume 9, Issue 4, Pages 871-881Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2014.056
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Funding
- National Research Foundation of Korea (NRF)
- Ministry of Education, Science and Technology [2011-0013885]
- Next-Generation BioGreen 21 program, Rural Development Administration, Republic of Korea [PJ009038]
- National Research Foundation of Korea [2011-0013885] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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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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