Additional file 1 of Electric field stimulation boosts neuronal differentiation of neural stem cells for spinal cord injury treatment via PI3K/Akt/GSK-3β/β-catenin activation
Creators: name: Liu, Qian
name: Telezhkin, Vsevolod
name: Jiang, Wenkai
name: Gu, Yu
name: Wang, Yan
name: Hong, Wei
name: Tian, Weiming
name: Yarova, Polina
name: Zhang, Gaofeng
name: Lee, Simon Ming-yuen
name: Zhang, Peng
name: Zhao, Min
name: Allen, Nicholas D.
name: Hirsch, Emilio
name: Penninger, Josef
name: Song, Bing
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: FOS: Biological sciences
subject: Pharmacology
subject: Developmental Biology

description: Additional file 1: Table S1. Proportions and percentages of Q33n1 hiPSC-derived NPCs which demonstrated each of the different types of induced action potentials for electric field (EF) stimulated and non-stimulated control (NoEF) at weeks 1-3. None = no significant voltage excursions from baseline; Attempting Single = voltage excursions which do not overshoot 0 mV; Single = one excursion only, which overshoots 0 mV; Attempting Train = several excursions, but only one which overshoots 0 mV; Train = several excursions, with more than one which overshoots 0 mV. Table S2. Analysis of passive and active parameters of induced action potentials of Q33n1 hiPSC-derived NPSs stimulated with an electric field (EF) and nonstimulated (NoEF) at weeks 1-3. * P &lt; 0.05, *** P &lt; 0.001. *-*** were considered as significantly different from correspondent values of NoEF Q33n1 hiPSC-derived groups of NPSs. Abbreviations: Membrane potential (Vm), input resistance (Rin), whole cell capacitance (Cp), I Namax and I Kmax are Na+ and K+ currents, respectively. n: cell number. Figure S1. Safety of EF stimulation on NSCs. A. The MTT assay demonstrated lower absorbance with EF stimulation on NSCs for 4 to 7 days. * P &lt; 0.05 was considered as significantly different between EF and NoEF groups. B. The morphology observation displayed healthy and active status of both NSCs with and without EF stimulation. But the smaller cell population and longer axon process were detected in the EF stimulated group. Scale bar: 20 µm. Figure S2. EF promoted neuronal differentiation of hiPSC-derived NPCs. A-B. The EF stimulation for 14 days in vitro induced synaptophysin and βIII-tubulin up-regulation in hiPSC-derived NPCs. Scale bar: 20 µm. C. Pie charts displaying percentage and proportion of Q33n1 hiPSC-derived neurons, EF stimulated (upper panel) and noEF (lower panel), cultured in vitro for 1-3 weeks exhibiting: no activity - Quiet (red), Attempting activity - (amber) or Spontaneous activity (green). D. Exemplar traces of membrane potential of Q33n1 hiPSC-derived neurons which demonstrates each different type of activity (Quiet, Attempting and Spontaneous). E. Mean ± S.E.M. values of membrane potential of EF stimulated (filled squares) and noEF (empty circles) Q33n1 hiPSC-dervied neurons at weeks 1-3 of culturing in vitro. The synaptophysin+ and βIII-tubulin+ positive cell count and percentage data were presented as mean ± SD. * P &lt; 0.05 and ** P &lt; 0.01 were considered as significantly different between EF and NoEF groups. Figure S3. Effect of EF treatment on voltage-gated Na+ current activation and inactivation characteristics during hiPSC differentiation (A). Exemplar family of whole cell currents (upper) during the activation/inactivation voltage protocol (lower). Inset (right) illustrate Na+ currents. Peak Na+ current activation and inactivation levels are shown by the red and blue arrows, respectively. (B-G). Mean activation and inactivation curves of whole-cell Na+ currents recorded in EF treated Q33n1-hiPSC derived neuronscultured in vitro at weeks 1 -3 (B, D, F) and noEF control (C, E, G). Activation curves are depicted by the filled squares and inactivation curves are shown by the empty circles. On each panel individual Vm values (filled upward triangles) and mean Vm values (red arrow on abscissa) are also shown. Voltages of half maximal action (Va50) and half maximal inactivation (Vi50) are also indicated in each panel, along with h factors, mean crossing points (downward arrows) and number of cells (n) recorded. Figure S4. The differentiating potential of PI3Kγ -/-and PI3KγKD/KD NSCs. The PI3Kγ -/-and PI3KγKD/KD NSCs were plated on slides for monolayer culture, differentiation induction and immunofluorescence. For the differentiation induction, 0.1% FBS was added to the culture medium for 7 days. A. MAP2 and GFAP positive cells in the PI3Kγ -/-and PI3KγKD/KD NSCs with 7-day differentiation induction. B. Cellcount ratio of MAP2+ cells from the induced PI3Kγ -/-and PI3KγKD/KD NSCs. C. Cellcount ratio of GFAP+ cells from the induced PI3Kγ -/-and PI3KγKD/KD NSCs. Scale bar: 20 µm. * P &lt; 0.05 was considered as significantly different between EF and NoEF groups.