We next determined if the NGN2-iN heterogeneity results from heterogeneous iPSC populations used to induce iNs, or if the heterogeneity was specific to the particular iPSC line. We established a single iPSC clone (409B2 monoclonal) from the parent 409B2 line and additionally generated a polyclonal NGN2-inducible line from another individual (Sc102a1). We induced these lines and analyzed the resulting transcriptomes at d35 of differentiation (Figure 2A). We found that significant heterogeneity is still observed in an integrated analysis of the scRNA-seq data, with six molecularly distinct clusters containing cells from each of the starting iPSC lines. We note that the three main neural subtypes and off-target cells are all observed for each line (Figures 2B–2E). We searched for the top DE genes in the six clusters, and observed distinct gene expression patterns for each cluster (Figure 2F; Table S2). These data suggest that NGN2-based neural reprogramming is intrinsically heterogeneous, independent of the purity of the starting cell population, and that the heterogeneity is detected in neurons from multiple iPSC lines.
