Fig. 4

Functional characterization and high-content analysis of miMNs. (A) Microscopic image of a patched miMN (Bar = 10 μm). (B) Typical rebound potentials after steps of hyperpolarization in miMNs. (C and D) miMNs displayed repetitive action potentials after depolarization commands. The action potentials were reversibly blocked by TTX (C) and TEA (D). Traces underneath each panel indicate current steps. (E) Interspike intervals (ISI) to depolarization current injections in miMNs are significantly delayed (n = 6, p = 0.001, t-test). The first ISI indicates the ISI between the first and second spike following depolarization commands, whereas the last ISI indicates the ISI between the last two spikes after depolarization commands being withdrawn. (F) A brightfield image shows miMNs attached on the electrode underneath the MEA plate (Bar = 100 μm). The right panels show spiking waveforms (top) recorded by one electrode and spontaneous spiking activity of miMNs (bottom) with +/− TTX treatment (0.5 μM). (G) Control miMNs from iPSC1 and iPSC3 iPSCs (referred to as control miMN 1 and 2, respectively) and ALS miMNs from iPSC2 and iPSC4 iPSCs (referred to as ALS miMN 1 and 2, respectively), were plated to the MEA plate at day 4 of differentiation. Their spontaneous spiking was recorded at indicated days in vitro. ALS miMNs showed more spiking activity than control miMNs (3 technical replicates for each miMN line, *: p < 0.05, linear regression with clustered data, ALS vs control miMNs). (H and I) miMNs from iPSC1 at day 4 of differentiation were cultured in the 1536-well plate for 2 days, and subjected to 24 h H₂O₂ treatment followed by Calcein AM staining, neurite tracing (H) and neurite length quantification (I). Neurite length was normalized to nuclei numbers (6 wells for each condition as technical replicates, *: p < 0.01, one-way ANOVA with Tukey’s HSD pos-hoc test, compared to the untreated control). Data represents Mean ± SEM.