Direct Reprogramming of Fibroblasts into Neural Stem Cells by Defined Factors

However, direct reprogramming of differentiated cells into somatic stem cell types has not been described yet4. Here we show that a combination of neural-specific transcription factors (Sox2, Klf4, c-Myc, Brn4/Pou3f4 or Sox2, Klf4, c-Myc, Brn4, E47/Tcf3) can induce a neural stem cell (NSC) fate on the fibroblasts. Induced neural stem cells (iNSCs) showed morphology, gene expression, epigenetic features, differentiation potential, and functionality similar to wild-type NSCs. Therefore, our data suggest that cell type-specific defined factors can induce specific stem cell identities on somatic cells. Fibroblasts (5 x 104 cells) were infected with retroviruses for two days, and cells were maintained in NSC media: DMEM/F-12 supplemented with N2 or B27 supplements (Gibco-BRL), 10 ng/ml EGF, 10 ng/ml bFGF (both from Invitrogen), 50 ug/ml BSA (Fraction V Gibco-BRL), and 1x penicillin/streptomycin/glutamine (Gibco-BRL). In order to establish stable iNSC lines, were either manually picked mature iNSC clumps or passaged and seeded the whole dishes of cells onto either gelatin- or laminin-coated dishes. RNA samples to be analysed on microarrays were prepared using QIAGEN RNeasy columns with on-column DNA digestion. 500 ng of total RNA per sample was used as input RNA into a linear amplification protocol (Ambion) involving synthesis of T7-linked double-stranded cDNA and 12 h of in-vitro transcription incorporating biotin-labelled nucleotides. Purified and labelled cRNA was hybridised onto MouseRef-8 v2 expression BeadChips (Illumina) for 18 h according to the manufacturer's instructions. After washing, as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using iScan reader (Illumina) and accompanying software. Samples were hybridised as biological replicates.