Extended NGN2 Expression in iPSCs Dramatically Enhances Purity of Neuronal Cultures

iPSC-derived neuronal cultures can provide valuable insights into the pathogenesis of neurological disease. However, single-cell iPSC clones expressing NGN2 and mCherry exhibit spontaneous loss of mCherry fluorescence, raising questions about the homogeneity of neurons derived from what appear to be heterogeneous iPSCs. We find that mCherry silencing does not influence iNeurons with two lines of evidence. First, using single-cell proteomics, we found that spontaneous mCherry silencing does not drive heterogeneity in iPSCs. Second, bulk proteomics and immunofluorescence analysis indicated that iNeurons from iPSCs expressing or lacking mCherry both resemble cortical glutamatergic neurons. The primary confounding factor in iNeuron generation was that suboptimal neuronal conversion led to cell aggregates comprised of actively proliferating neuronal progenitor cells and astrocytes as the culture developed. Our results indicate that extended NGN2 dosage substantially improves neuron purity.