Feline Lindhout

Quantitative mapping of transcriptome and proteome dynamics during polarization of human iPSC-derived neurons 2 47 DISCUSSION To better understand neuronal differentiation and polarization in human cells, we performed an in-depth characterization of human iPSC-derived neurons during these developmental processes. We systematically assessed the early stages of human neurodevelopment in culture, including axon specification (transition stage 2-3), and performed transcriptomic and proteomic profiling during these steps. We describe previously undescribed intermediate stages of axonal outgrowth (stage 2a, 2b, 3a and 3b), which is characterized by a distal to proximal reorganization of the axonal microtubule network and relocation of AIS proteins. Development of polarized and functional human iPSC-derived neurons In this study we showed that NSCs consistently gave rise to polarized and functional human neurons, which was demonstrated by the loss of cell proliferation and NSC markers, and the appearance of neuron-specific and AIS markers. These neurons form a single axon with a functional AIS, and exhibit AP firing. As expected, AP properties and cell intrinsic variables appeared immature compared to other studies performed at later developmental stages (Bardy et al. 2016; Gunhanlar et al. 2018). Passive physiological properties were comparable to neurons recorded from ex vivo fetal cortical brain tissue (Moore et al. 2009). We consistently observed neurons that fired a single AP and neurons that fired multiple APs at different developmental stages. The reason for this difference remains unknown, but may indicate variation in the maturation, cell morphology or other factors that could contribute to cellular heterogeneity of the culture. Non-human neurons develop at faster rates: for example, rat dissociated neurons reach stage 3 after approximately 1.5 days in culture, and cortical development in maturation in mammals ranging from mouse to primate is both faster and less complex than in humans (Dotti, Sullivan, and Banker 1988; Clowry, Molnar, and Rakic 2010; Molnar and Clowry 2012; Silbereis et al. 2016; Marchetto et al. 2019). We found that human iPSC-derived neurons transition to stage 3 in approximately 7 days, which is consistent with the described prolonged development of human neurons in vivo and in vitro (Grabrucker et al. 2009). The slow rate of development is also reflected in the lack of spontaneous AP firing or incoming synaptic responses as mature synapses have likely not formed yet. Co-culturing with astrocytes could benefit synaptogenesis, as this has been shown to promote synaptic connectivity (Tang et al. 2013). The prolonged development of human iPSC-derived neurons allows studying neurobiological processes, such as AP maturation and AIS assembly, with higher temporal resolution. Quantitative profiles of transcriptomic and proteomic of early human neurodevelopment Quantitative transcriptomeanalysis identified549upregulatedgenes and614downregulated genes during early stages of neurodevelopment. As expected, and in agreement with changes observed in other differentiating cell types, GO term enrichment analysis showed strong downregulation of genes related to cell proliferation during terminal differentiation (An et al. 2014; Gao, Yourick, and Sprando 2014; Tripathi et al. 2014). Simultaneously, genes related to neurodevelopmental processes such as neurite formation and axonogenesis are