Ion Channels:

Application Note

NaV, K V, GABA A, glycineCells:
hiPSC-derived neurons(EBiSC-NEUR1)

The impact of astrocytes on thematuration of hiPSC-derived neuronsTools:
SyncroPatch 384

Nanion Technologies GmbH, Munich
Fraunhofer-Institute for Biomedical Engineering (IBMT), Würzburg

Summaryneurons (EBiSC-NEUR1) 6 and astrocytes (ScienCell, #1800) by

Human induced pluripotent stem cells (hiPSCs) areusing the SyncroPatch 384.

becoming increasingly used in biomedical research fordisease modelling and drug discovery. The use of stemcells for research reduces the need for animals, as wellas having the added advantages of being human inorigin and available in relatively high abundance. Oneof the challenges in the use of hiPSCs is their proposed
immaturity compared with native cells, potentially limiting
their usefulness as a model system. In order to address thislimitation, culture conditions including media supplements,

comparing the electrophysiological properties of the neurons

Results
hiPSC-derived neurons were either cultured alone (N) or cocultured with astrocytes (N+A) and the success rate for cellcapture and presence of NaV and KV currents was compared(Figure 1A). In addition, cell size, current amplitude of KV andNaV currents, and Vhalf of activation and inactivation of NaV

currents were measured and compared (Figure 1; Table 1).

or co-culture conditions are being investigated. Indeed,
in a recent study, the co-culture of hiPSC-derived atrial

Cm (pF)

NaV peak(nA)

Na Vhalf(act; mV)

Na Vhalf(inact;mV)KV peak(nA)cardiomyocytes with fibroblasts induced functional maturityof atrial (but not ventricular) cardiomyocytes 1.

N

6.2 ± 0.5(37)-1.0 ± 0.2(37)-17 (12)-56.5 ±
1.5 (14)0.9 ± 0.1(56)Stem cell-derived neurons have become important

N+A

18.4 ± 2.9(91)***-3.3 ± 2.9(91)***-22 (48)-58.9 ±
0.9 (65)

1.5 ± 0.1(81)***

tools in neuroscience research and a number of hiPSCderived neuronal types are currently available on themarket. Culture conditions are critical for maturation ofhiPSC-derived neurons and it was discovered early onthat commonly used culture media, e.g. DMEM-basal,

Table 1: Cell capacitance and current amplitudes of NaV and KV weresignificantly larger in neurons co-cultured with astrocytes. ***P<0.001.

Neurobasal or serum, while promoting neuronal survival,
could actually impair neurophysiological functions 2. In order
Amount of Wells (%)

In collaboration with the Fraunhofer Institute for Biomedical
Engineering (IBMT) we explored co-culture of hiPSC-derived

6040Neurons +Astrocytes

200.5 nAQC NaV KVParameter-80-60-40-200-200culture conditions. B Activation IV curves for hiPSC-1.5

neurons in different culture conditions, with example-2.0-0.5-1.0-2.0

20-0.5-1.0

20-1.55 ms

D

Voltage (mV)

N+A

different culture conditions.

-40

N+AN

culture conditions. D Example traces of KV currents in-60

Neurons-120 -100

traces at -10 mV. C Inactivation IV curve in different-80

NC(QC), and presence of NaV and KV current in different-120 -100

80

0Figure 1: A Bar graph of success rates for cell sealing

Voltage (mV)

B

100

IPeak (nA)

Awith astrocytes 3,4,5.

IPeak (nA)to promote maturation of neurons, they can be co-cultured

NN+A
0.2 nA

100 msAFigure 2: A GABA responses to a single application

Bof 100 μM GABA were recorded. B Glycine
responses to 500 μM glycine were also recorded
in some neurons. C GABA responses wereControl

Controlconcentration dependent and desensitization

100 µM GABA

500 µM Glycine

was observed at the highest concentration. The

50 pA

50 pA
1s

Normalized activationC

EC50 was 20.1 μM (n = 17 wells).

1s

GABA

1.0 N + A

EC50 = 20.1 µM (n = 17 wells)

0.84 µM GABA
11 µM GABA
33 µM GABA
100 µM GABA
300 µM GABA

0.60.40.20.2 nA1s

0.0
10-8

10-7

10-6

10-510-4[GABA (M)]

10-3

10-2

Although success rates for cell capture were similar for the 2culture conditions (60 - 70%), NaV currents were detected in 100%of N+A, compared with 60% of N, with a similar result for KVcurrents. In addition, NaV current amplitudes were significantly
larger in N+A vs N (P<0.001, unpaired Student’s t test), presumably
due to an observed increase in cell capacitance (Table 1). The
Vhalf of activation and inactivation of NaV currents, however,were similar between the two conditions, indicating that the NaV
subtypeisunchanged.

KV-mediatedcurrents

werealso

significantly larger in N+A vs N.

References
1. Brown, G.E., et al., 2024. Sci. Adv. 10: eadg1222.
2. Bardy C., et al., 2015. PNAS. 112 (20) E2725-E2734.
3. Hedegaard, A., et al., 2020. Stem Cell Reports. 15: 38–51.
4. Shih, P-Y., et al., 2021. Stem Cell Research. 54: 102386.
5. Supakul, S., et al., 2024. Inflammation and Degradation. 44: 8.
6. Wihan, J., et al. 2023. J Vis Exp. 2023 Mar 17:(193).

In preliminary experiments looking at different ligand-gated ionchannels, we could identify a GABA-mediated response andglycine-mediated responses in both N+A and N culture conditions(Figure 2). There appeared to be little difference in the number ofcells, nor the current amplitudes of GABA and glycine responses
recorded in the two conditions on the small number of cellstested so far. A GABA concentration response curve revealed anEC50 for GABA of 20.1 µM (n = 17 wells). Small responses toacetylcholine, Bz-ATP and glutamate were detected in a
proportion of N and N+A neurons (data not shown).
In summary, hiPSC-neurons cultured under different conditions,either with or without co-culture with astrocytes, could becaptured with excellent success rates (70%) and differences
could be observed in cell parameters and ion channel current
amplitudes. Further tests are required to determine effects onneuron excitability, by recording resting membrane potential
and action potentials in current clamp mode.

Methods
Cells

Cultured hiPSC-derived neurons (EBiSC, EBiSC-NEUR1) cultured
alone and co-cultured with astrocytes (ScienCell, #1800) werekindly provided by Fraunhofer IBMT.
Electrophysiology measurements
Cells were harvested according to Nanion’s cell harvesting
protocol for neurons. Cells were resuspended in external
recording solution and stored in the cooled CellHotelof the SyncroPatch 384, shaking at 15°C before use.
Internal and external solution compositions are available
upon request. Whole cell patch clamp recordings wereconducted according to Nanion’s standard procedure for
the SyncroPatch 384. Activation and inactivation IVs wererecorded using a triple step protocol in PatchControl 384
and analysed using the IV analysis tool of DataControl 384. KV
currents were recorded using a single step protocol. Ligands
GABA, glycine, acetylcholine, Bz-ATP and glutamate wereapplied using the ‘LigandPuff’ protocol, cells here held at-70 mV and briefly exposed to ligands (1 s). N and N+A run inparallel on the NPC-384 chips in each experiment.

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