Cells:

Application Note

EBiSC hiPSCcardiomyocytes
Tools:

Monitoring the effect of Triiodothyronine(T3) and Dexmethasone (Dex) on thecalcium handling property of hiPSCcardiomyocytes

CardioExcyte 96

Nanion Technologies GmbH, Munich.
Bayer AG. Hamamatsu Photonics.

Summaryby T3 and Dex treatment. Apart from changed expression ofHuman induced pluripotent stem cell derived

genes encoding relevant cardiac ion channels, shortening

cardiomyocytes (hiPSC-CMs) provide new avenues forof action potential duration (APD) in those cells has beendisease modelling, drug discovery and cell therapy.

observed2. Here, we have used electrical field potential

However, structural and functional immaturity of these(EFP) recordings, to investigate the effects of T3 and Dex oncells, poses a great challenge1. Therefore, there is a highly

population of hiPSC-CMs forming a functional monolayer.

unmet need for hiPSC-CMs with a mature phenotype.

By using transparent plates, we were able to corelate those

Recently, it has been reported that triiodothyronine (T3)signals with calcium transients.

and dexamethasone (Dex) play significant roles in hiPSC-Figure 1 shows EFP recordings performed on NSP-96

CMs maturation, by promoting the structural T-tubuletransparent CardioExcyte 96 plates. EFP recordings are widely

development2, enhancing the electrophysiologicalused as surrogates of action potential recordings, where thematuration3 and improving calcium handling ability.

field potential (FPD) duration – time from the sodium peakto the T-wave – corresponds to the APD in action potential

Testing for maturity of hiPSC-CMs, demands a holistic

recordings. We could observe that T3 and Dex induced shorter

approach and use of various techniques to monitor

FPDs in EBiSC hiPSC-CMs, which is consistent with the publishedfunctional readout of those cells. Exceptional benefit isresults that T3 and Dex could shorten the APD in hiPSC-CMs2.

seen in multiplexing various assays, and obtain valuable

Immediately after the electrical activity of the hiPSC-CMs wasinformation from exactly the same cells. Here, we haverecorded, in the NSP-96 transparent plates, cells were loaded

tested pro-maturation effects of T3 and Dex on EBiSC hiPSC-with calcium indicator dye (Calcium 6) and calcium transients

CMs, and monitored electrical activity, contractility andhave been recorded using FDSS/μCELL.

calcium transients. By using NSP-96 transparent plate, wewere able to conduct these measurements on the same
cell population, by multiplexing calcium imaging recordings
using FDSS/μCELL, after CardioExcyte 96 recordings haveAC-T3/Dex

Results

300

Electrophysiological properties hiPSC-CMs seem to be altered

200100B+T3/Dex
Figure 1. Effect of T3 and Dex on the EFP signalsof EBiSC hiPSC-CMs. Figure shows a typical EFP
signal recorded from EBiSC hiPSC-CMs on NSP-96
transparent plates, (A) without and with (B) T3and Dex treatment. C. Bar graph summarizes theshortening effect of T3 and Dex on FPD values.
Values represent mean ± SEM.

400

FP Duration (ms)been performed.

0-T3/Dex+T3/DexNSP 96-welltransparent plateFigure 2: Comparison of calcium transients,
recorded from standard imaging and NSP 96-well

B+ T3/Dex

2500

AMP

1500
1000100 RFU20 ms

PW D302000

500800300600

200C10020000+ T3/Dex- T3/Dex300

800+ T3/Dex

1002000PW D70

PW D30AMP100 RFU

100 RFU

20 msdurations as recorded on standard imaging 96represent mean ± SEM.

800
20020 msof T3 and Dex on amplitude, PW 30 and PW 70

1000

600

400

these plates, with (right) and without (left) T3 andwell plates (B) and NSP 96-well plates (D). Values

D- T3/Dexplates. A and C depict raw traced recorded fromDex treatment. Bar graphs summarize the effects

400

20 ms0NSP 96-well trasparent plate

400

PW D70- T3/Dex100 RFU

Standard 96-well plate

A600
40020000

As control for calcium imaging recordings, standard 96-well

References

imaging plates were loaded with dye as well, and recordedin parallel. Figure 2 shows the effect of T3 and Dex on thecalcium signals of hiPSC-CMs recorded from standard 96well imaging plate and NSP-96 transparent plate. Recordings
performed on standard normal 96-well imaging plate clearly

1. van den Berg C.W. et al. 2015. Development 142: 3231–3238.
2. Parikh S.S. et al. 2017. Circ. Res.; 121: 1323-1330
3. Wang L. et al. 2021. J Mol Cell Cardiol; 161:130-138

shows that T3 and Dex application increased the amplitude
of calcium signals, and also accelerated calcium transient
kinetics of treated hiPSC-CMs. Similar effects of T3 and Dexon the calcium handling properties of hiPSC-CMs have been
observed using NSP-96 transparent plate, as well. Calcium
imaging using NSP-96 transparent plates, confirmed that T3and Dex increased calcium amplitude and induced a faster
calcium decay in hiPSC-CMs.
In conclusion, by using NSP-96 transparent CardioExcyte96
plate, we were able to perform multiplex recordings ofcalcium transients and electrical activity from exactly thesame hiPSC-CMs. Our results confirmed previous findings,
that T3 and Dex influenced electrical activity of hiPSC-CMs.
Moreover, we could also show that T3 and Dex improved thecalcium handling ability of the same hiPSC-CMs. Increased
amplitude and a faster decay of calcium signals have been
observed in cells treated with T3 and Dex in both NSP-96
transparent plate and control standard 96-well imaging
plate, indicating that NSP-96 transparent plates can beused to successfully deliver information of multiple readouts,
crucial for monitoring maturation of hiPSC-CMs.

MethodsCells
We thank Bayer AG for providing iPSC-derived EBiSC
cardiomyocytes, and Hamamtsu for providing analysis software
for this collaborative work. We thank Dr. Wenying Xian for
performing experiments, analyzing data and her assistance withwriting the document. This work has received support from theEU / EFPIA / Innovative Medicines Initiative 2 Joint Undertaking(EBiSC2 grant n°821362).
Impedance and contractility measurements
EBiSC hiPSC-CMs have been seeded on the NSP-96
transparent plates with density of 5k cells/well , and formed
a synchronously beating monolayer 5 to 7 days afterplating. 100nM T3 and 1µM Dex have been applied to thecardiomyocytes to induce their maturation. Impedanceand EFP measurements were conducted according toNanion’s standard procedures for the CardioExcyte 96.
Calcium imaging measurements were conducted according
Hamamtsu´s standard procedures for the FDSS/μCELL, using
Calcium 6 as an indicator for 20 minutes at the incubatorand then started the calcium imaging with high speed
settings. Data was analyzed with DataControl 96 software andFDSS/μCELL software.

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