Reasoning that multiple transcription factors would likely be required to reprogram fibroblasts to a neuronal fate, we cloned a total of nineteen genes that are specifically expressed in neural tissues, play important roles in neural development, or have been implicated in epigenetic reprogramming (Supplementary Table 1). A pool of lentiviruses containing all nineteen genes (19F pool) was prepared to infect mouse embryonic fibroblasts (MEFs) from TauEGFP knock-in mice, which express EGFP specifically in neurons 21,22 (see Fig. 1a for experimental outline). Great care was taken to exclude neural tissue for the MEF isolation, and we were unable to detect evidence for the presence of neurons or neural progenitor cells in these cultures using immunofluorescence, fluorescence activated cell sorting (FACS), and RT-PCR analyses (Supplementary Fig. 1). However, uninfected MEFs did contain rare Tuj1-positive, TauEGFP-negative cells with fibroblast-like morphology, indicative of weak Tuj1 (i.e. β-III-tubulin) expression in non-neuronal cells (Fig. 1b,c; Supplementary Figure 1a). In contrast, 32 days after infection with the 19F pool, we detected Tuj1-positive cells with typical neuronal morphologies and bright TauEGFP fluorescence (Fig. 1d,e). Thus, some combination(s) of the genes in the 19F pool was capable of converting MEFs into induced neuronal (iN) cells.
