Quantitative mapping of transcriptome dynamics during polarization of human iPSC-derived neurons

Early neuronal development is a well-coordinated process in which neuronal stem cells differentiate into polarized neurons. This process has been well studied in classical non-human model systems, but to what extent this is recapitulated in human neurons remains unclear. To study neuronal polarization in human neurons, we cultured human iPSC-derived neurons, characterized early developmental stages, measured electrophysiological responses, and systematically profiled transcriptomic and proteomic dynamics during these steps. We found extensive remodeling of the neuron transcriptome and proteome, with altered mRNA expression of ~1,100 genes and different expression profiles of ~1,500 proteins during neuronal differentiation and polarization. We also identified a distinct stage in axon development marked by an increase in microtubule remodeling and apparent relocation of the axon initial segment from the distal to proximal axon. Our comprehensive characterization and quantitative map of transcriptome and proteome dynamics provides a solid framework for studying polarization in human neurons. ~100,000 hiPSC-derived NPCs were plated per well for bulk RNA sequencing samples. Prior to sample preparation, all equipment and surfaces were cleaned with RNaseZap (Sigma-Aldrich). Replicates of hiPSC-derived neurons were harvested at three different timepoints of differentiation (days 1, 3, and 7) with 200 µl Trizol (Invitrogen) per sample and stored at -80 °C until sequencing. RNA extraction, cDNA library preparation (CEL-Seq2 protocol), quality control for aRNA and cDNA, and sequencing on a NextSeq500 High output 1x75 bp paired end run with 2% sequencing depth were performed by Single Cell Discoveries (Utrecht, The Netherlands).