Supplementary MaterialsLegends. research have been deposited to GEO with the accession code: “type”:”entrez-geo”,”attrs”:”text”:”GSE112381″,”term_id”:”112381″GSE112381 and the BioGPS platform (http://biogps.org/dataset/BDS_00016/). Abstract The transcriptional programs that establish neuronal identity evolved to produce the rich diversity of neuronal cell types that arise sequentially during development. Remarkably, transient expression of certain transcription factors can also endow non-neural cells with neuronal properties. The relationship between reprogramming factors and the transcriptional networks that produce neuronal identity and diversity remains largely unknown. Here, from a screen Itga2b of 598 pairs of transcription factors, we identify 76 pairs of transcription factors that induce mouse fibroblasts to differentiate into cells with neuronal features. By comparing the transcriptomes of these induced Umbelliferone neuronal cells (iN cells) with those of endogenous neurons, we define a core cell-autonomous neuronal signature. The iN cells also exhibit diversity; each transcription Umbelliferone factor pair produces iN cells with unique transcriptional patterns that can predict their pharmacological responses. By linking distinct transcription factor input codes to defined transcriptional outputs, this study delineates cell-autonomous features of neuronal identity and diversity and expands the reprogramming toolbox to facilitate engineering of induced neurons with desired patterns of gene expression and related functional properties. Reporting summary. Further information on experimental design is available in the Nature Research Reporting Summary linked to this paper. Neurons comprise a conspicuously diverse but clearly recognizable cell type. All neurons share defining features such as electrical excitability and synaptic connectivity. However, in even the simplest organisms, neurons also exhibit extensive diversity that affords each species its unique sensory modalities, behaviours and cognitive capabilities. The extent to which this diversity reflects the action of intrinsic cellular programs or depends on environmental and developmental cues is a central question in neuroscience. Despite the elaborate sequential mechanisms that specify cell identity during development, recent studies have shown that transient overexpression of transcription factors can stably reprogram cells from one lineage to another without cell division, including the direct conversion of fibroblasts into iN cells using three transcription factors1C3. This discovery has enabled engineering of iN cells that resemble various endogenous subtypes, typically by adding transcription factors to the orginal neuron-inducing factors3C10. The majority of these protocols included achaete-scute homolog 1 (ASCL1, encoded by the gene), suggesting that this may be an essential factor11. However, we showed that replacing ASCL1 with neurogenin 1 (encoded by = 3 wells, 2 104 fibroblasts per well). c, MEFs were transfected with vectors encoding to generate iN cells. Immunofluorescence showing co-labelling of TUJ1+ (red) candidate iN cells with tauCeGFP (green), MAP2 (green) and synapsin (green) with nuclei in blue (DAPI) from = 5, 5 and 3 independent experiments, left to right, respectively. Scale bars, 100 m. d, Percentage of TUJ1+ cells that co-express tauCeGFP (= 574), MAP2 (= 574) or synapsin (= 293) for iN cells induced by (N3.P1, = 5, 5 and 3 independent experiments, respectively), (N3.O4, = 4, 4 and Umbelliferone 3 independent experiments, respectively), (A2.B3c, = 3, 3 Umbelliferone and 3 independent experiments, respectively), (ND2.B3c, = 4, 4 and 3 independent experiments, respectively) and (Atoh1.B3c, = 3, 3 and 3 independent experiments, respectively). is also known as under whole-cell patch-clamp conditions at maximum current injection (top) and current steps until the first induction of action potentials (middle), with current traces (bottom). c, iN cells generated with five transcription factor pairs exhibit current-induced action potentials in the majority of cells: (N3. P1, 15 of 15 cells), (N3.O4; 10 of 10 cells), (A2.B3c; 15 of 16 cells), (ND2.B3c; 10 of 10 cells) and (Atoh1.B3c; 8 of 9 cells). AP, action potential. d, Current trace showing EPSCs from an iN cell generated with (N3.O4, Umbelliferone top) and (ND2.B3c, bottom). f, Quantification of voltage sag (Vsag) behaviour for candidate iN cells that exhibited current-induced action potentials: N3.P1 (= 15 cells), N3.O4 (= 10), A2.B3c (= 15), ND2.B3c (= 10) and Atoh1.B3c (= 8). Voltage sag is plotted as the slope of the voltage sag versus current. Coloured points correspond to the plotted cells. Data are mean s.d., *= 0.0207, one-way ANOVA, Tukeys multiple comparison test. Both MEFs and human embryonic fibroblast-like cells (HEFs) derived from iPSCs can be reprogrammed with pairs of mouse transcription factors12C14. Here we show.