Ion Channels:

Application Note

NaV, KV, GABA A
Cells:
iCell ® Neurons

Stem cell derived neurons recorded on
Nanion’s Patchliner

Tools:Patchliner

Nanion Technologies GmbH, Munich

Summary
Stem cell–derived neurons provide a novel and uniqueinvestigation into voltage- and ligand-gated ion channels

model for studying human drug targets in their physiologically

expressed in these cells was undertaken using the Patchliner.

relevant environment of terminally differentiated, postmitotic

The presence of NaV, KV and GABAA receptor mediatedproteins modulate the physiology and pharmacology ofneuronal proteins1,2. Therefore, assays that investigate neuronaltoxicity, drug effects, or basic cellular functions of neurons
can mostly benefit from the development of human neurons
derived from induced pluripotent stem cells (hiPSC-neurons). In
2011, Fujifilm Cellular Dynamics International (CDI) announced
the commercial launch of human iCell® Neurons for use inneuroscience drug discovery, neurotoxicity screens, and other
health research. This was the first commercially available
iPSC-derived neuronal type available3. It was a mixture of
GABAergic and glutamatergic post-mitotic neurons which
rapidly regenerate post-thaw4. Over the years, Fujifilm CellularDynamics International has developed a range of hiPSCneurons with either healthy or diseased models. These canbe used to study a number of different neurodegenerative
diseases including Alzheimer’s Disease, one of the leading
causes on death in the United States5.
iCell® Neurons were the first commercially available hiPSCneuronal cell type which could be provided in the quantity,

currents were detected in these cells and the results agreed
well with manual patch clamp data using the same cells6.

Results
Voltage-gated Na+ (NaV) currents of an individual cell are

shown in Figure 1A. Not every cell showed expression of a NaV

current and in this example the current is quite large. Typically,
when observed, the maximum peak amplitude was between-100 and -500 pA. The current voltage plot of 54 cells is shownin Figure 1B. A full concentration response curve to TTX wasperformed on 5 cells, revealing an IC50 for TTX of 12 ± 6 nM(Figure 1C,D). The NaV current is TTX sensitive although theexact Na+ channel subtype present was not yet investigatedfurther.
An outward voltage-gated K+ (KV) current was also seen in mostcells. There appeared to be two populations of KV currents, aA

B

800

quality, and purity required for life science research. These

600

neurons were used on the Patchliner, a fully automated patch

400

clamp device for recording from up to 8 cells simultaneously.

1 nA

After plating, cells developed neuronal outgrowth within

200
5 ms

24 hours and could be kept in culture for 2 weeks. An-80

iCell Neuron. B IV plot from an average of®

54 cells. C: NaV current blocked by increasing
concentrations of TTX. D Concentration response
curve for TTX inhibition for an average of 5 cells.

-40
-200

D 1.0 TTX
Control
1 nM TTX
10 nM TTX
100 nM TTX
1µM TTX
0.5 nA
5 ms

40Voltage (mV)IC = 12 ± 6 nM (n = 5)50
0.8 Max= 0.9 ± 0.1

1-(I/Imax)

CFigure 1: A NaV current recorded from an example

Imax (pA)cells. It has been increasingly recognized that associated

0.6Hill = 0.9 ± 0.3

0.4
0.20.01 pM 0.1 nM10 nM 1 µM
[TTX]

A

Figure 2: A Inactivating KV current recorded fromB

an iCell® neuron. B Corresponding IV plot from an

300

500 pA
100

50 ms

CGABA

Dcurrent activated by 500 ms application of 30 μM
GABA. D Action potential elicited from an iCell®

Imax (pA)200

average of 121 cells. C GABAA receptor mediatedneuron following a 2 ms depolarizing current
pulse.

-60 -40 -20
20 40
Voltage (mV)

0 mV

200 pA

20 mV

500 ms-85 mV

10 ms

non-inactivating (not shown) and an inactivating type (FigureMethods2A), although in most cases it was not possible to clearlyCells

distinguish the 2 populations6. A current-voltage plot for an

Human iPS cell-derived neurons (iCell® Neurons) from Cellular

average of 121 cells is shown in Figure 2B. The current could beDynamics International were used.

blocked by increasing concentrations of TEA (data not shown)6
but was not fully blocked even with the highest concentration

Electrophysiology measurementsof TEA (10 mM)6. GABAA receptors were activated in iCell®

Cells were received as frozen aliquots and were plated and

Neurons using a single concentration of GABA (30 µM) applied

cultured according to the manufacturer’s instructions. Cells

to the cells for ~500 ms (Figure 2C). The current could be blocked

were harvested according to Nanion’s standard protocols.

by increasing concentrations of bicuculline with an IC50 = 632 ±Cells were resuspended in external recording solution

149 nM (n = 5)6. In current clamp mode, action potentials wereand stored in the CellHotel of the Patchliner before being

elicited using a 2 ms depolarizing current pulse (Figure 2D).

dispensed into each well of the NPC-16 chip. Internal andexternal solution compositions are available upon request.

In summary, iCell® Neurons

from Fujifilm Cellular Dynamics

Whole cell patch clamp recordings were conductedInternational can be successfully used on the Patchliner toaccording to Nanion’s standard procedure for the Patchliner.

combine a cellular neuronal model with higher throughput

For NaV currents, cells were stepped from a holding potential

automated electrophysiology (see Ref. 6). Such a cell modelof -100 mV to -80 mV for 20 ms and then increasing in 10 mV

provides an alternative to primary neuronal cell cultures forincrements with each sweep up to 60 mV. For TTX application,

studying neuronal toxicity, disease research and drug discovery.

cells were stepped from -100 mV to -20 mV for 10 ms repeated

References

every 5 s. For KV currents, cell were stepped from a holding

potential of -80 mV to -60 mV for 200ms and then increasing
in 20 mV increments with each sweep up to 60 mV. For GABA

1. Zolles, G., et al., 2009. Neuron. 62(6): 8143–825.
2. Bett, G.C., Rasmusson, R.L. 2008. J. Physiol. 586(4): 929-950.

experiments, cells were held at a constant holding potentialof -70 mV and GABA was applied for ~500 ms using a stacked
solutions protocol. Bicuculline was pre-incubated for at least 30

3. https://www.technologynetworks.com/cell-science/news/

s before co-application with 30 µM GABA. For current clamp

cellular-dynamics-announces-commercial-launch-of-icell-recordings, cells were switched from voltage clamp to current

neurons-for-neuroscience-drug-discovery-190143 (accessedclamp mode using the ‘gentle switch’ method of the HEKA15.10.2023)

EPC10 amplifier. Action potentials were elicited using a 2 ms

4. Berry, B.J., 2015. Biotechnol. Prog. 31(6): 1613–1622.

depolarizing current pulse.

5. Heron, M. 2017. National Vital Statistics Reports. 68(6):https://www.cdc.gov/nchs/data/nvsr/nvsr68/nvsr68_06-508.
pdf (accessed 15.10.2023).
6. Haythornthwaite A, et al., 2011. J Biomol Screen. 17:
1264–1272.

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