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 unclear if this culture paradigm is indicative of CNS functionality. Modifications of the NGN2-iN protocol by adding developmental patterning factors to the culture medium can steer neuron differentiation to a desired path (Nehme et al., 2018). Our data support a continued effort into identifying combinatorial transcription factor overexpression systems (Ravasi et al., 2010) and medium conditions that can support precise neuron cell type engineering. Furthermore, comprehensive human nervous system reference cell atlases are required to understand the identity of cell states that emerge in in-vitro-engineered neuron systems. Single-cell genomics and comparisons with high-dimensional reference atlases should become a field gold standard to assess the heterogeneity and precision of in vitro engineered neurons.
