(a) FSK but not any other tested small molecules synergizes with NGN2 to generate Tuj1+ cells from IMR-90 fetal fibroblasts. GFP indicates cells transduced with NGN2-expressing virus. FSK, forskolin; RA, retinoic acid; VPA, valproic acid; NaB, sodium n-butyric acid; TSA, trichostatin A; SB, SB431542; DM, dorsomorphin. (b) Quantification of Tuj1+ cells 5 days post chemical treatments (means ± s.e.m., n=40 randomly selected 20x fields from triplicate samples). (c) DM promotes conversion efficiency, neuronal survival and maturation. Cells were counted 10 days post treatment (means ± s.e.m., n=30 random 20x fields from triplicate samples). (d) Time-dependent maturation of converted neurons by NGN2/FSK/DM (means ± s.e.m., n=10 random 20x fields from triplicate samples). (e-f) Representative morphology of induced neurons at 7 or 30 dpi. (g-l) Converted cells express markers for mature neurons. (m-n) Induced neurons express glutamatergic (vGlut1) and presynaptic (synaptotagmin 1, SYT1) markers, which showed a punctate staining pattern. (o) GABA is rarely detected in converted neurons. Scales, 50 μm for (a, e-k, n-o), 20 μm for (l, m).