Electrophysiological characterization of sourced human iPSC-derived motor neurons

Abstract

Induced pluripotent stem cell (iPSC)-derived motor neurons provide a powerful platform for studying motor neuron diseases. These cells enable human-specific modeling of disease mechanisms and high-throughput drug screening. While commercially available iPSC-derived motor neurons offer a convenient alternative to time-intensive differentiation protocols, their electrophysiological properties and maturation require comprehensive evaluation to validate their utility for research and therapeutic applications. In this study, we characterized the electrophysiological properties of commercially available iPSC-derived motor neurons. Immunofluorescence confirmed the expression of motor neuron-specific biomarkers, indicating successful differentiation and maturation. Electrophysiological recordings revealed stable passive membrane properties, maturation-dependent improvements in action potential kinetics, and progressive increases in repetitive firing. Voltage-clamp analyses confirmed the functional expression of key ion channels, including high- and low-voltage-activated calcium channels, TTX-sensitive and TTX-insensitive sodium channels, and voltage-gated potassium channels. While the neurons exhibited hallmark features of motor neuron physiology, high input resistance, depolarized resting membrane potentials, and limited firing capacity suggest incomplete electrical maturation. Altogether, these findings underscore the potential of commercially available iPSC-derived motor neurons as a practical resource for MND research, while highlighting the need for optimized protocols to support prolonged culture and full maturation.

Keywords: Motor neuron; action potential; calcium current; iPSC; potassium current; sodium current.

Figure 1.

Immunostaining of iPSC-derived motor neurons for neuronal markers. (a) Confocal images of cryopreserved motor neurons stained for motor neuron late-stage precursor markers, Tuj1 (green) and MNX1/HB9 (red) at DIV 2 (top panels), DIV 10 (middle panels), and DIV 14 (bottom panels). (b) Corresponding fluorescence intensity quantification. (c) The cells were also stained for mature motor neuron markers, CHAT (green) and pan-neuronal maker MAP2 (red) at DIV 5 (top panels), DIV 10 (middle panels), and DIV 14 (bottom panels). (d) Corresponding fluorescence intensity quantification. Scale applies to both panels. Statistical analysis was conducted using ANOVA followed by Tukey’s multiple comparison test.

Figure 2.

Passive membrane properties of iPSC-derived motor neurons. (a) Resting membrane potential values at two developmental stages: DIV 5–8 (black circles) and DIV 9–15 (blue circles). (b) Representative voltage traces in response to hyperpolarizing current injections and corresponding I/V relationship used to assess the input resistance. (c) Corresponding input resistance values of iPSC-derived motor neurons.

Figure 3.

Single action potential analysis of iPSC-derived motor neurons. (a) Representative voltage traces of single action potentials recorded at two developmental stages: DIV 5–8 (black circles) and DIV 9–15 (blue circles). Actions potentials were elicited by 5-ms depolarizing current pulses of 280 pA, applied from a holding current adjusted to maintain a resting membrane potential of approximately −70 mV. Corresponding (b) Threshold potential values, (c) Peak amplitude values, (d) Rise time values, (e) Spike time values, (f) Half-width values, and (g) Decay time values.

Figure 4.

Repetitive firing analysis of iPSC-derived motor neurons. A Representative voltage traces recorded from iPSC-derived motor neurons at development stage DIV 5–8 (left panels), elicited by 500-ms depolarizing current pulses of 20 pA. The holding current adjusted to maintain a resting membrane potential was approximately −70 mV. The traces demonstrate different firing patterns: 1 AP (purple), 2–3 APs (orange), and > 4 APs (brown). Corresponding phase plots are shown in the right panels. B same as (A), but for iPSC-derived motor neurons at development stage DIV 9–15. C corresponding percentage of cells exhibiting different firing patterns: no response (black), 1 AP (purple), 2–3 APs (orange), and > 4 APs (brown).

Figure 5.

Voltage-activated calcium current analysis of iPSC-derived motor neurons. A Representative high-voltage activated (HVA) calcium current traces recorded from iPSC-derived motor neurons at DIV 10. Currents were elicited by 150-ms depolarizing steps ranging from −70 mV to +60 mV, applied from a holding potential of −80 mV. B corresponding mean I/V relationship for HVA calcium currents. C Representative voltage-activated calcium current traces recorded in the presence of a cocktail of HVA channel blockers (2 μM ω-conotoxin GVIA, 600 nM ω-agatoxin IVA, 200 nM SNX-482, and 10 μM nifedipine) to isolate low-voltage-activated (LVA, T-type) calcium currents. Currents were recorded from a holding membrane potential of −100 mV. A representative LVA current trace recorded at −30 mV is shown in blue. D corresponding mean I/V relationship for LVA currents (blue). Grey dots correspond to the remaining HVA conductance that was not fully blocked by the cocktail of HVA channel inhibitors. E mean maximal macroscopic conductance values (G_max) for HVA (black circles) and LVA (blue circles) currents, obtained by fitting the I/V curves with a modified Boltzmann equation (eq. 1). F mean normalized voltage-dependence of activation for HVA and LVA currents. G mean half-activation potential values obtained from the activation curve fits using a modified Boltzmann equation (eq. 2). H Representative time course of HVA calcium current amplitude in response to sequential application of various HVA channel blockers. The inset shows representative HVA calcium current traces in response to the various blockers. I mean maximal inhibition of HVA calcium currents by each blocker.

Figure 6.

Voltage-activated sodium and potassium current analysis of iPSC-derived motor neurons. A Representative voltage-activated sodium current traces recorded from iPSC-derived motor neurons at DIV 10. Currents were elicited by 20-ms depolarizing steps ranging from −70 mV to +50 mV, applied from a holding potential of −100 mV. Traces are shown before (black) and after (blue) application of 1 mm TTX. B corresponding mean I/V relationships for sodium currents, with and without TTX. C corresponding mean maximal macroscopic conductance values (G_max) for Nav currents before (black circles) and after TTX application. D Representative voltage-activated potassium current traces recorded from iPSC-derived motor neurons at DIV 10. Currents were elicited by 400-ms depolarizing steps ranging from −40 mV to +80 mV, applied from a holding membrane potential of −60 mV. E corresponding mean I/V relationship for potassium currents.