We further analyzed NGN2-iN developmental trajectories after additional integration and clustering of all time course data (Figures S3A and S3B). NGN2 induction resulted in major gene expression changes early on in programming (d0, d1, and d5), likely driven by the immediate downstream targets of NGN2. Based on the observation of rapid transition from iPSCs to cells committed to a neuronal fate (Figures 1B and S3B), we hypothesized that directed differentiation bypasses early transitional states that are usually observed in vivo to reach neuronal states. To test the hypothesis, we ordered NGN2-iNs in pseudotime based on transcriptome similarities and compared the resulting trajectories with development of neurons in brain organoids (Figures S3C and S3D) (Kanton et al., 2019). We observed an escape from the early developmental stages from pluripotency directly into neural precursor stages, skipping multiple intermediate stages, including neuroectoderm and neuroepithelium induction, supporting a more direct differentiation model (Figure S3D).
