Cell fate engineering of neural subtypes from human iPSCs using defined TFs provides extraordinary new inroads into disease modeling and therapy screening using human cells. Methods to rapidly generate mature human neurons are exciting and transformative for these endeavors. It has been established that the NGN2-iN protocol is able to reprogram stem cells to general neural fates, with less heterogeneity and higher consistency across multiple stem cell lines compared with traditional reprogramming strategies mediated by small-molecule inhibitors/activators. However, our analysis suggests that the emergent neuron population is heterogeneous, with the heterogeneity being consistent across different cell lines. We are unable to assign the neuron populations to a particular identity with high confidence. We note that this may be due to the fact that current single-cell and spatial transcriptome reference atlases are incomplete. However, without a specific matrix and guiding molecules it may be expected that neurons are not able to establish the molecular profile observed in vivo with high precision. Our data show that multiple NGN2-iN subpopulations are more similar to neurons of the PNS than CNS, and it is
