We generated a stable iPSC line expressing NGN2 that can be induced by doxycycline (Dox) and drives the differentiation toward iNeurons (Zhang et al., 2013). We then performed scRNA-seq (10× Genomics) at multiple time points during directed differentiation (Figure 1A). After filtering the data of astrocytes, multiplets, and cells with insufficient unique molecular identifiers (UMIs), a total of 6,764 cells (day 0 [d0], 1,412 cells; d1, 2,688 cells; d5, 524 cells; d14, 1,515 cells; d28, 625 cells) were included in the analysis. We combined all time course data and reconstructed the differentiation path of NGN2-iNs (Figures 1B–1D). Surprisingly, we found at least four transcriptionally distinct cell populations at the d28 time point. One population is marked by DCN/COL5A1 and we interpret this cluster as an off-target mesenchymal population (Figure 1C). In addition, we observed three different neuronal clusters that express high levels of pan-neuronal genes (MAP2, NCAM1) yet are molecularly distinct (Figure 1C). Two clusters have high expression of PRPH, an intermediate neurofilament that is highly expressed in neurons of the peripheral nervous system and some central nervous system regions
