We next set out to narrow down the number of transcription factors required for generation of iN cells. Given their important roles in neuronal cell fate determination23–26 we first tested the bHLH transcription factors Ascl1, (also known as Mash1) and Neurod1 individually. Surprisingly, we observed occasional Tuj1-, TauEGFP-positive cells exhibiting a simple mono- or bipolar morphology after infection with only Ascl1 (Supplementary Fig. 2b). However, 19F-iN cells exhibited more complex morphologies, which indicated that the activity of Ascl1 alone was not sufficient to recapitulate the full activity of the 19F pool (compare to Fig. 1d,e). We therefore tested the neuron-inducing activity of Ascl1 in combination with each of the remaining eighteen candidate genes (Supplementary Fig. 2a). Five genes (Brn2, Brn4, Myt1l, Zic1, and Olig2) substantially potentiated the neuron-inducing activity of Ascl1 (Supplementary Fig. 2a–b). Importantly, none of these five genes generated iN cells when tested individually (data not shown). Next, we tested whether combinatorial expression of these factors with Ascl1 could further increase the induction of neuron-like cells by infecting TauEGFP MEFs with a pool of Brn2, Myt1l, Zic1, Olig2,
