To generate neurons directly from fibroblasts, we optimized an Ascl1/Ngn2 (AN)-based and small molecule-enhanced iN protocol (Figure 2A) (Ladewig et al., 2012; Liu et al., 2013). Fibroblast identity was verified and the cells were lentivirally transduced to express rtTA and a 2A-peptide-linked transcript coding for Ngn2 and Ascl1, resulting in transgenic, but silent and expandable, AN fibroblasts (Figures 2B and S1). For iN conversion, cells were transferred to conversion media containing small molecular inhibitors for TGFβ/SMAD and GSK3β signaling as well as enhancers of intracellular cyclic AMP (cAMP) (Figure 2B). Under these conditions, fibroblasts underwent marked morphological changes, with typical neuronal morphologies appearing around week 2 of conversion and becoming more pronounced after 3 to 6 weeks. Neuronal cells stained positive for βIII-tubulin, phosphorylated human Tau (hTau), MAP2ab, and the mature neuronal marker NeuN, and most cells showed punctate staining for the glutamateric synaptic marker vGlut1 (Figure 2C). Non-neuronal cells showed typical fibroblast morphologies and Vimentin expression; cell division was not involved in the iN process (Figure S1). Following gentle relocation onto a layer of astrocytes during week 4 of
