Validation of hiPSC-Derived Atrial Cardiomyocytes

The potential for disease models of atrial fibrillation to be established from patients is offered with the development of atrial cardiomyocytes from iPSCs. Information may be provided on therapeutics that modify the phenotypic markers of cardiovascular disease and atrial fibrillation.

As one of the most common arrhythmias affecting the heart, drugs need to be developed that can target atrial arrhythmia. Fundamental differences in the electrophysiology of cardiac action potentials mean that current mouse models fail to translate in vitro.

Data on the molecular and electrophysiological characterization of Axol’s Human iPSC-derived Atrial Cardiomyocytes are presented here. The protein and gene expression, beat rate and action potential parameters are determined, and the functionality of the core cardiac and atrial-specific ion channels identified.

The expression of cardiac and atrial-specific markers troponin T, atrial myosin light chain 2 (MLC2a) and atrial natriuretic peptide (ANP) and key ion channels, Kv1.5 and Kir3.1/3.4 are revealed through molecular characterization of Axol’s Human iPSC-derived Atrial Cardiomyocytes.

Spontaneous action potentials are provoked, functional core cardiac ion channels, INa, ICa,L and IKr are expressed and a steady beat rate is exhibited with Axol’s Human iPSC-derived Atrial Cardiomyocytes.

The opportunity for the study of atrial-specific disorders (for example, atrial fibrillation) and the development of cell-based assays for identifying disease-modifying treatments are offered with the highly validated, physiologically relevant model, Axol’s Human iPSC-derived Atrial Cardiomyocytes.

Materials and Methods

Culture of hiPSC-derived atrial cardiomyocytes: Human iPSC-derived atrial cardiomyocytes (hiPSC-ACMs) (Axol Bioscience ltd., UK) were cultured at 8.0x105 cells/cm2 on a 384-channel 24-well multi-electrode array (MEA) chip (Alpha Med Scientific Inc., Japan) and a 24-well plate both coated with Axol SureBond Coating Solution (Axol Bioscience ltd., UK) at 37 °C in a 5% CO2/95% air atmosphere.

Immunofluorescent imaging: Immunostains (Troponin T, ANP, MLC2a and MLC2v along with DAPI counterstains) were applied to mature hiPSC-ACMs on 8 DIV. Images of the cardiomyocytes to characterize their morphology and receptor expression were obtained with immunofluorescent imaging using an EVOS Fl Auto (Life Technologies Corporation, UK).

Multi-electrode Arrays (MEA): The high-throughput MEA system was used to acquire spontaneous extracellular field potentials 37°C and extracellular potentials for 16 channels per well across 24-well plates (MED64 Presto, Alpha Med Scientific Inc., Japan) were simultaneously collected at a sampling rate of 20 kHz/channel before being stored on a personal computer. On most electrodes and across all wells, synchronized, spontaneous beats were first observed and recorded after four DIV. The experiment was terminated after 32 DIV when the hiPSC-ACMs became unviable.

Manual Patch Clamp (MPC): 7-10 days after cell seeding action potentials (AP) were recorded from hiPSC-ACMs. Perforated patch (100 μg/ml gramicidin) was used to take records at room temperature in current-clamp mode.

Data was acquired with EPC10 amplifiers and PatchMaster software (HEKA Elektronik, Germany). Digitisation at 20 kHz came after analog signals were low-pass filtered at 10 kHz. CAPA software (SSCE UG, Germany) was used to analyze spontaneous AP. AP parameters analyzed: maximum diastolic potential (MDP), upstroke velocity (dV/dtmax), AP amplitude (APA), AP duration at 20, 50 and 90% repolarization (APD20, APD50, APD90), and frequency (Freq). Data is presented as mean ± SEM. Paired student’s t-test of control values compared to the effect of compound application was used to determine significance. * P<0.05, ** P<0.01, *** P<0.001.

Electrophysiological analysis: MEA Symphony (Alpha Med Scientific Inc.) was used to acquire raw data. Clampfit (Molecular Devices, LLC, US) was used to produce trace plotting, beat extraction, beat count, waveform creation, interspike intervals (ISIs) and raster plots. GraphPad Prism (GraphPad Software Inc., US) was used to create graphs. Clampfit was used to automatically extract beats with a positive detection threshold of at least 300 μV compared to an average baseline noise of ±6 μV.

Clampfit was used to create waveforms from 25 ms pre-trigger to 400 ms post-trigger. These were averaged across at least a minute’s section of recording. Clampfit was used to determine FPAs automatically, beat counts, beat timings, ISIs and their standard deviation (SD).

Arrhythmia monitoring and identification: Identification of any arrhythmias within the cells was possible through daily monitoring of the beat rate under a microscope (Nikon Diaphot, Nikon, Japan), the appearance of after- depolarisations on the whole trace plot and within the waveform, unstable raster plots and large standard deviations for the ISIs.

1.Electrophysiological Characterization of hiPSC-Atrial Cardiomyocytes

Electrophysiology of Axol’s hiPSC-ACMs Axol’s hiPSC-ACMs produce typical atrial-like field action potential (fAPs) waveforms with no evidence of arrhythmias. A. Typical minute’s trace of fAPs recorded from hiPSC-ACMs from a single well (16 electrodes). Note stable, synchronous beating and no evidence of arrhythmia or after-depolarisations. B. Averaged waveform from 10-minute recording of fAPs recorded from hiPSC-ACMs by a single electrode showing typical atrial-like fAPs at full-scale (top) and zoomed-in to show the T-wave. No evidence of after-depolarisations within waveform. C. Raster plot of recorded beats from a single electrode from DIV4 to DIV29, showing rhythmic and stable beat rates. D. Interspike Intervals (±SD) for a single electrode from DIV4 to DIV29 showing stable ISIs with small SD. Image Credit: Axol Bioscience

Figure 1: Electrophysiology of Axol’s hiPSC-ACMs
Axol’s hiPSC-ACMs produce typical atrial-like field action potential (fAPs) waveforms with no evidence of arrhythmias. A. Typical minute’s trace of fAPs recorded from hiPSC-ACMs from a single well (16 electrodes). Note stable, synchronous beating and no evidence of arrhythmia or after-depolarisations. B. Averaged waveform from 10-minute recording of fAPs recorded from hiPSC-ACMs by a single electrode showing typical atrial-like fAPs at full-scale (top) and zoomed-in to show the T-wave. No evidence of after-depolarisations within waveform. C. Raster plot of recorded beats from a single electrode from DIV4 to DIV29, showing rhythmic and stable beat rates. D. Interspike Intervals (±SD) for a single electrode from DIV4 to DIV29 showing stable ISIs with small SD. Image Credit: Axol Bioscience

2. Expression of Cardiac Markers in hiPSC-ACM

Morphology and Immunocytochemistry of Axol’s hiPSC-ACMs Atrial cardiomyocytes express the cardiac and atrial-specific markers troponin T, atrial myosin light chain 2 (MLC2a) and atrial natriuretic peptide (ANP). The presence of these proteins was confirmed in hiPSC-ACMs.A. Immunocytochemistry data showed the expression of the cardiac- and atrial-specific proteins. Troponin T staining (red) confirmed the presence of cardiac myocytes which are responsible for sarcomere contraction. ANP is specifically secreted by atrial myocytes upon atrial stretching and MLC2a facilitates cardiac contractility. DAPI counterstain. B. Phase contrast images of hiPSC-ACMs at DIV 1, 8 and 14. Image Credit: Axol Bioscience

Figure 2: Morphology and Immunocytochemistry of Axol’s hiPSC-ACMs
Atrial cardiomyocytes express the cardiac and atrial-specific markers troponin T, atrial myosin light chain 2 (MLC2a) and atrial natriuretic peptide (ANP). The presence of these proteins was confirmed in hiPSC-ACMs.A. Immunocytochemistry data showed the expression of the cardiac- and atrial-specific proteins. Troponin T staining (red) confirmed the presence of cardiac myocytes which are responsible for sarcomere contraction. ANP is specifically secreted by atrial myocytes upon atrial stretching and MLC2a facilitates cardiac contractility. DAPI counterstain. B. Phase contrast images of hiPSC-ACMs at DIV 1, 8 and 14. Image Credit: Axol Bioscience

3. hiPSC-Atrial Cardiomyocyte Ion  Channel Pharmacology

Functional confirmation of atrial phenotype of Axol’s hiPSC-ACMs Manual patch clamp and pharmacological modulation was used to confirm the cardiac and atrial phenotype of Axol’s hiPSCs-ACMs. Key compounds were used to identify the three major cardiac currents: INa (B. Lidocaine, green), Ica,L (C. Nifedipine, blue) and IKr (D. E4031, red) and the atrial specific currents: IKur (E. 4-AP, blue) and IKACh (F. Carbachol, orange). IKur and IKACh are also targets for atrial fibrillation. Representative spontaneous APs are shown from control  conditions (A. black) and in the presence of the compounds. Early after depolarisations (EADs), indicative of arrhythmic events, were observed following E-4031 application (arrow). G. Average effect of core cardiac ion inhibitors (% of control) on AP parameters (n ≥ 4). H. Average effect of atrial-specific current modulators (% of control) on AP parameters (n ≥ 5)

Figure 3: Functional confirmation of atrial phenotype of Axol’s hiPSC-ACMs
Manual patch clamp and pharmacological modulation was used to confirm the cardiac and atrial phenotype of Axol’s hiPSCs-ACMs. Key compounds were used to identify the three major cardiac currents: INa (B. Lidocaine, green), Ica,L (C. Nifedipine, blue) and IKr (D. E4031, red) and the atrial specific currents: IKur (E. 4-AP, blue) and IKACh (F. Carbachol, orange). IKur and IKACh are also targets for atrial fibrillation. Representative spontaneous APs are shown from control  conditions (A. black) and in the presence of the compounds. Early after depolarisations (EADs), indicative of arrhythmic events, were observed following E-4031 application (arrow). G. Average effect of core cardiac ion inhibitors (% of control) on AP parameters (n ≥ 4). H. Average effect of atrial-specific current modulators (% of control) on AP parameters (n ≥ 5). Image Credit: Axol Bioscience

Conclusion

An atrial phenotype is expressed with Axol’s hiPSC-derived atrial cardiomyocytes as confirmed by the molecular, pharmacological and electrophysiological data here. With no obvious arrhythmias, it is suitable for use on multiple electrophysiological platforms.

In the development of improved translational models of atrial fibrillation and cardiotoxicity, Axol hiPSC-ACMs represent a promising tool as they:

  • Express cardiac- and atrial-specific markers
  • Display stable, spontaneous and synchronized APs and fAPs within 4 days of culture, which are then maintained for 4 weeks or more in culture
  • Display no evidence of endogenous arrhythmias
  • Respond well to a core panel of cardiac ion channel inhibitors
  • Produce EADs in response to known pro-arrhythmic drugs
  • Exhibit atrial phenotypes to known selective modulators of atrial-specific currents and known targets of atrial fibrillation

Acknowledgements

Produced from materials originally authored by Broadbent, S., Clare, N., Dark N., Paonessa, M., El-Haou, S., Harper., S, Shi, Y from Axol Bioscience and Metrion Biosciences.

About AXOL Biosciences

Axol specializes in human cell culture.

Axol produces high quality human cell products and critical reagents such as media and growth supplements. We have a passion for great science, delivering epic support and innovating future products to help our customers advance faster in their research.

Our expertise includes reprogramming cells to iPSCs and then differentiating to various cell types. We supply differentiated cells derived from healthy donors and patients of specific disease backgrounds. As a service, we also take cells provided by customers (primary or iPSC) and then do the reprogramming (when necessary) and differentiation. Clearly, by offloading the burden of generating cells, your time is freed up to focus on the research. Axol holds the necessary licenses that are required to do iPSC work.

The package wouldn't be complete without optimized media, coating solutions and other reagents. Our in-house R&D team works hard to improve on existing media and reagents as well as innovate new products for human cell culture. We also supply a growing range of human primary cells; making Axol your first port of call for your human cell culture needs.


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Last updated: Feb 18, 2020 at 11:26 AM

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