Feline Lindhout

2 32 ABSTRACT The differentiation of neuronal stem cells into polarized neurons is a well-coordinated process which has mostly been studied in classical non-human model systems, but to what extent these findings are recapitulated in human neurons remains unclear. To study neuronal polarization in human neurons, we cultured hiPSC-derived neurons, characterized early developmental stages, measured electrophysiological responses, and systematically profiled transcriptomicandproteomicdynamicsduring thesesteps. Theneuron transcriptome and proteome shows extensive remodeling, with differential expression profiles of ~1,100 transcripts and ~2,200 proteins during neuronal differentiation and polarization. We also identified a distinct axon developmental stage marked by the relocation of axon initial segment proteins and increased microtubule remodeling from the distal (stage 3a) to the proximal (stage 3b) axon. This developmental transition coincides with action potential maturation. Our comprehensive characterization and quantitative map of transcriptome and proteome dynamics provides a solid framework for studying polarization in human neurons. INTRODUCTION Neuronal development is a complex multistep process in which neurons undergo dramatic morphological changes, including migration, axon outgrowth, dendritogenesis, and synapse formation. Much of the fundamental knowledge about neuronal development is based on experimental studies in non-human model systems, such as Drosophila , C. Elegans , mice and rats (Zhao and Bhattacharyya 2018). However, to what extent the knowledge obtained in animal models can be extrapolated to human neuronal development remains largely unclear. Moreover, analysis of human-specific characteristics is hindered by the difficulty in obtaining human brain tissue. The generation of human induced pluripotent stem cells (iPSCs) has provided a critical step forward for studying the development and function of human neuronal cells. In recent years, many labs have used human iPSC-derived neuronal cultures to study fundamental neurobiological questions. This has contributed to our understanding of processes such as neuronal polarity, spine development and synaptic plasticity in human cells. For example, human iPSC-derived model systems have been used to study dynamic changes in gene expression during early neurogenesis, and to study polarization of neuronal progenitors (Compagnucci et al. 2015; Grassi et al. 2020). In addition, human synaptic transmission and plasticity have been studied at single cell level in hiPSC-derived neurons, and human-specific protein functions have been shown to regulate excitatory synaptic transmission specifically in human neurons (Meijer et al. 2019; Marro et al. 2019). These examples illustrate how the use of human iPSC-derived neurons as a model system can lead to novel findings for human neurodevelopment. One of the classic model systems to study neuronal development are dissociated rat hippocampal neurons, developed by Banker and collaborators (Dotti, Sullivan, and Banker