Feline Lindhout

2 34 RESULTS Characterization of developmental stages in human iPSC-derived neurons We first systematically assessed if the human iPSC-derived neurons proceed through the initial neurodevelopmental stages, which have previously been described in dissociated rat neurons (Dotti, Sullivan, and Banker 1988). The hiPSC-derived neuron cultures were obtained by neuronal induction of neuronal stem cells (NSCs) and maintained up to ~15 days (Axol Bioscience, Protocol Version 5.0, Human iPSC-derived Neural Stem Cells). hiPSC- derived neurons were transduced with FUGW-GFP lentivirus to visualize cell morphologies, and immunostained at different timepoints (day 1, 5 and 14) for proliferation marker Ki67 and NSC marker Nestin to identify NSCs (stage 1), neuron specific markers ß3-Tubulin and MAP2 to identify differentiated neurons (stage 2), and AIS markers AnkG and Trim46 to identify polarized neurons (stage 3) (Fig 1A,B). The presence of these markers indicated a clear developmental transition over time: stage 1 NSCs (~day 1) differentiated into stage 2 neurons with a characteristic neuronal morphology (~day 5), and subsequently developed axons containing AIS structures (~day 14) (Fig 1C-E). Accordingly, axon width showed a developmental decrease over time, whereas dendrite width remained relatively stable (Fig S1A,B). These observations indicate that hiPSC-derived neurons have a relatively prolonged development compared to dissociated rat neurons, consistent with the protracted development of the human brain (Dotti, Sullivan, and Banker 1988; Petanjek et al. 2011). This further supports the emerging evidence showing species-dependent differences in developmental timing of human and non-human neurons in vivo and in vitro (Shi, Kirwan, and Livesey 2012; Espuny-Camacho et al. 2013; Nicholas et al. 2013; Otani et al. 2016; Linaro et al. 2019). We further analyzed the structural AIS organization in axons of hiPSC- derived neurons by quantifying the average fluorescence intensity profiles of Trim46 and AnkG (Fig 1F,G). Consistent with previous reports in dissociated rat neurons, we found that Trim46 and AnkG localization largely overlapped, with the peak of AnkG intensity located ~6 µm more distally than the peak Trim46 intensity (Fig 1F,G) (van Beuningen et al. 2015). The AIS structure was also enriched for voltage-gated sodium channels (NaV), which strongly overlapped with AnkG structures (Fig S1C). In summary, these data demonstrate that human iPSC-derived neurons follow the characteristic sequence of developmental stages during neuronal polarization, which occurs at a relatively slower rate than non- human neurons. Action potential firing of polarized human iPSC-derived neurons To determine whether the polarized human iPSC-derived neurons are able to fire APs, we performed whole-cell patch clamp recordings of stage 3 neurons. We observed AP firing upon positive somatic current stimulation in nearly all cells (59/61). Neuronal excitability was quantified as the number of APs in response to increasing current stimuli (steps of 5 pA; 400 ms) in 54 neurons (Fig 1H,I). Of these, 22 neurons (41%) fired multiple times upon higher current stimulation, while 32 neurons (59%) fired only once independent of stimulus strength (Fig S1D). Neurons that fired one AP showed more immature intrinsic