Direct conversion of mouse fibroblasts into induced neural stem cells

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.

Generation of Integration-free Induced Neural Stem Cells from Mouse Fibroblasts

“…As in our previous studies (10,17), only 2-5% of iNSCs from the early passage was SSEA1-positive ( Fig. 2C), although they already showed the homogenous morphology (data not shown).…”

“…Starting on the next day, the cells were cultured in NSC medium, which was replaced every other day with fresh medium until initial clusters were observed. To generate retroviral vector-mediated r-iNSCs, the MEFs were transduced with retroviral particles and cultured as previously described (9,10). Briefly, 5 ϫ 10 4 fibroblasts were plated onto the gelatincoated 35-mm dish and incubated with ecotropic retroviruses for 48 h. After 48 h of incubation, the medium containing retroviral particles was replaced with NSC medium.…”

“…Briefly, 5 ϫ 10 4 fibroblasts were plated onto the gelatincoated 35-mm dish and incubated with ecotropic retroviruses for 48 h. After 48 h of incubation, the medium containing retroviral particles was replaced with NSC medium. To enrich the initial cluster of both e-iNSCs and r-iNSCs, non-reprogrammed fibroblasts or unwarranted cells were removed with a cell scraper as previously described (10). The initial iNSC clusters were observed around 4 weeks after initiation of reprogramming process.…”

“…Recently, Thier et al (12) have shown that the restricted expression of Oct4 by a tetracycline-dependent lentiviral vector, together with retrovirus-mediated overexpression of Sox2, Klf4, and c-Myc, could elicit the conversion of fibroblasts into iNSCs. Simultaneously, we have generated multipotent and self-renewing iNSCs from mouse fibroblasts by retrovirus-mediated overexpression of Brn4/Pou3f4, Sox2, Klf4, and c-Myc (BSKM) (9,10). All the iNSCs generated in these studies closely resemble their in vivo counterparts in terms of morphology, gene expression profile, epigenetic status, and self-renewing capacity.…”

“…We have previously shown that the retroviral infection of the defined set of transcription factors Brn4/ Pou3f4, Sox2, Klf4, and c-Myc (BSKM) could directly convert fibroblasts into iNSCs (9,10). Although the retrovirus-mediated iNSCs (r-iNSCs) exhibited molecular and cellular characteristics similar to those of control NSCs from brain tissue, the integration of exogenous factors in the iNSC genome might preclude the clinical translation of iNSCs.…”