In order to probe the gene expression network changes that may contribute to the functional differences we observed between D398 and N398 human neurons (Fig. 2), we conducted gene expression profiling by RNAseq, using replicate cultures from single donors from each variant group (Fig. 3). We included in our analysis RNAseq data from control DA cultures published by the Studer lab (mDA)35, iPSC cultures, and hESC-derived neural stem cells (NSC) at two stages of differentiation (producing glutamatergic neurons, day 0 [NSC0] and day 5 [NSC5])36. Clustering analysis shows that D398 and N398 cultures are more similar to DA neuron cultures than to cortical glutamatergic NSC (Fig. 3A). We find similar expression levels of several mRNAs known to be associated with DA neurons in midbrain-like DA, D398, and N398 cultures (Fig. 3B). CORIN, FOXA1, LMX1A, NR4A2, and PITX3 mRNAs are all prominent and similar to levels found in previously-published mDA cultures. EN1 levels are lower, but well above the limit of detection, and were also observed in cultures of excitatory neuron precursors (NSC0 and NSC5). Expression plots for several other groups
