Ngn2 induces diverse neuronal lineages from human pluripotency
Human neurons engineered from induced pluripotent stem cells (iPSCs) through Neurogenin 2 (Ngn2) overexpression are widely used to study the mechanisms controlling neuronal differentiation and to model human neurodevelopmental and neurological diseases. However, the differentiation paths and heterogeneity of neurons that are produced through this cell fate programming method have not been fully explored, thus limiting the ability to interpret the results gained from averaging across an ensemble of cells. Here we used single-cell transcriptomics to dissect the cell states that emerge during Ngn2 overexpression across a time course from pluripotency to neuron functional maturation. We find that there is substantial molecular heterogeneity in the neuron types that are generated, with at least two populations that express genes associated with neurons of the peripheral nervous system. Neuron heterogeneity is observed across multiple iPSC clones and lines from different individuals. We find that neuron fate acquisition is sensitive to Ngn2 expression level and the duration of Ngn2 forced expression. Our data reveals that Ngn2 dosage can regulate neuron fate acquisition, and that Ngn2-iN heterogeneity can confound results that are sensitive to neuron type.