Exploring Ventricular Phenotypes in hiPSC-Derived Cardiomyocytes

In line with the FDA’s Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, high-quality cardiac ion channel services for the prediction of human arrhythmia risk is offered by Metrion.

  • High-quality in vitro human cardiac ion channel patch-clamp assays
  • Comprehensive in silico human action potential (AP) models
  • Predictive assays utilizing induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs)

iPSC-CMs that express a mature ventricular phenotype are required for the CiPA initiative. Electrophysiological profiling of Axol Human iPSC-Derived Ventricular Cardiomyocytes (hiPSC-vCMs) was carried out by Metrion Biosciences through the evaluation of their biophysical and pharmacological characteristics using:

  • The quantifications of inward Na+ and Ca2+ currents (INa and ICa), as well as outward and inward K+ currents (IK) through whole-cell voltage-clamp recordings.
  • Current clamp measurements of AP parameters and pharmacology, utilizing:
  • Compounds to distinguish between atrial and ventricular phenotypes
  • Core cardiac channel modulators from the CiPA validation toolbox

Ventricular phenotype, the functional expression and pharmacology of typical cardiac currents, including INa, ICa, and IKr were confirmed by this data.

Materials and Methods

Cell Culture: Human iPSC-Derived Ventricular Cardiomyocytes (ax2505, Axol Bioscience Ltd.) cultured in Cardiomyocyte Maintenance Medium (ax2530-500) at 37  ̊C (5% CO2). The initial 24 hours with 10% FBS, Pen/Strep, then going forward, serum-free.

Immunocytochemistry: Cells were fixed in 3% PFA, permeabilized with 0.2% Triton X-100 and blocked with BSA. Primary antibody was incubated overnight at voltage-clamp ̊C, and secondary antibody coupled to Alexa Fluor® dyes (Invitrogen) applied for 2 hours.

Western Blot: 30 μg protein ran on 10% SDS-PAGE gel for 70 min at 130 V and transferred to PVDF membrane. Membranes were incubated with primary antibody overnight at 4 ̊C, before being washed and incubated with secondary antibody for 1 hour — chemiluminescent imaging.

Manual Patch Clamp: For 4-5 days, Axol hiPSC-vCMs were seeded onto fibronectin-coated flasks and cultured at 37 °C (5% CO2) before being re-seeded onto fibronectin-coated coverslips for MP recordings. 7-10 days after cell seeding, AP were recorded at RT in current clamp mode using perforated patch (100 μg/ml gramicidin). Cells were typically paced at 1 Hz with a field stimulator for evoked AP. Single cells using the conventional whole-cell patch clamp configuration with protocols and solutions designed to isolate the ionic current of interest were used to obtain voltage clamp recordings.

Table 1: Manual patch clamp solutions. Composition (in mM) of external and internal solutions used for voltage and current (CC) manual patch clamp experiments. Source: Axol Bioscience

  External Internal
INa ICa IK CC INa ICa IK CC
NaCl 50 - 140 140 5 5 5 5
TEA-Cl 1 140 - - - - - -
KCl 5.4 5.4 5.4 5.4 - - 20 125
K-Asp - - - - - - 110 -
CaCl2 1.8 1.8 1.8 1.8 - - - -
MgCl2 1 1 1 1 - - 1 5
CsCl 90 - - - 130 130 - -
Glucose 10 10 10 10 - - - -
HEPES 10 10 10 10 10 10 10 10
MgATP - - - - 5 5 5 -
CdCl2 0.3 - 0.3 - - - - -
EGTA - - - - 10 10 10 5
4-AP - 2 - - - - - -
pH 7.4
CsOH
7.4
CsOH
7.4
NaOH
7.4
NaOH
7.2
CsOH
7.2
CsOH
7.2
KOH
7.2
KOH

 

EPC-10 amplifiers and PatchMaster software (HEKA Elektronik, Germany) were used to acquire data. Digitization 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. Figure 1 shows the AP parameters analyzed. Maximum Depolarisation Rate (MDR) could not be accurately estimated for evoked AP in this dataset. As such, Time To Peak (TTP) was used by measuring the time (ms) from the end of the stimulation artifact to the AP peak. Data are reported as mean ± SEM.

Action potential parameters Example action potential trace indicating the parameters which were quantified using HEKA FitMaster (evoked AP) and CAPA software (spontaneous AP) in this study.

Figure 1: Action potential parameters Example action potential trace indicating the parameters which were quantified using HEKA FitMaster (evoked AP) and CAPA software (spontaneous AP) in this study. Image Credit: Axol Bioscience

1. Characterisation of Axol ventricular iPSC-CM

Molecular and Physiological Characterisation of Axol hiPSC-vCMs

Figure 2: Molecular and Physiological Characterisation of Axol hiPSC-vCMs
A; Top Immunocytochemistry data showed the expression of ventricular cardiomyocyte markers (cardiac troponin-I; 93.5% expression). Data from Dr Christian Zuppinger, University of Bern and Prof Matt Daniels, University of Oxford. Bottom Western blot data confirmed that Axol hiPSC-vCMs express more cardiac troponin-T (cTnT) and α-Actinin than human skin fibroblasts (hSFs). Data from Abigail Robertson, University of Manchester. B; 87 % of Axol hiPSC-vCMs have a ventricular phenotype determined by MLC2v expression, compared to 13% expressing atrial MLC2a (n=1). Data from Dr Christian Zuppinger, University of Bern. C; Image showing Axol hiPSC-vCMs after 10 days in 2D culture. After 7 days in culture, cells treated with Fluo-4 chemical dye show regular beating. Data from Prof Matt Daniels, University of Oxford. Image Credit: Axol Bioscience

2. Voltage Clamp Snapshot of Native Ionic Currents

Voltage clamp snapshot of cardiac ionic currents in Axol hiPSC-vCMs

Figure 3: Voltage clamp snapshot of cardiac ionic currents in Axol hiPSC-vCMs
A: Representative traces of sodium (INa), L-type calcium (ICa,L), inward (IKin) and outward (IKout) potassium currents elicited by the voltage protocols shown. B: I-V relationships shown for INa (peak), ICa,L (peak), IKin (end of the pulse), transient IKout (peak current) and sustained IKout (end of the pulse). Image Credit: Axol Bioscience

3. Action Potential Characterisation

Characteristics of spontaneous and evoked action potentials

Figure 4: Characteristics of spontaneous and evoked action potentials
Representative traces of spontaneous (A) and evoked (1 Hz; B) AP recorded from Axol hiPSC-vCMs under control conditions. C; Average AP parameters for spontaneous (n = 18) and evoked (n = 31) AP in control conditions. Image Credit: Axol Bioscience

4. Atrial vs. Ventricular AP Phenotype

Pharmacological tools targeted against atrial specific currents show minimal effects

Figure 5: Pharmacological tools targeted against atrial specific currents show minimal effects
A; Representative traces of evoked AP recorded under control conditions (grey) and in the presence of 1 μM carbachol (orange), 100 nM tertiapin Q (pink) or 50 μM 4-AP (blue). B; Average effect of each compound on spontaneous AP parameters. Data presented as percent of control ± SEM, n ≥ 4. Significance calculated by a paired two-tailed Student’s t-test, * p<0.05, ** p<0.01. Image Credit: Axol Bioscience

5. Core Cardiac Channel Pharmacology

Core cardiac channel action potential pharmacology

Figure 6: Core cardiac channel action potential pharmacology
A; Representative traces of evoked AP recorded under control conditions (grey) and in the presence of 100 μM lidocaine (green), 100 nM nifedipine (blue) or 50 nM dofetilide (red). B; Average effect of each compound on spontaneous AP parameters. Data presented as percent of control ± SEM, n ≥ 4. Significance calculated by a paired two-tailed Student’s t-test, * p<0.05, ** p<0.01, *** p<0.001. Image Credit: Axol Bioscience

Conclusions

  • A range of ventricular cardiomyocyte markers are expressed by Axol hiPSC-vCMs and these function as a highly pure population of beating cells in culture.
  • Through extensive electrophysiological profiling of cardiac ion channel expression and function at Metrion, Axol hiPSC-vCMs was shown to:
  • Express the three major ionic currents INa, ICa,L, and IK (IKout and IKin)
  • Produce stable evoked AP when paced at 1 Hz
  • Predominately express a ventricular phenotype
  • Have the minimal effect of atrial-selective channel modulators
  • Exhibit appropriate core cardiac channel pharmacology
  • The presence of core cardiac currents INa, ICa,L, and IKr are confirmed with AP pharmacology

Acknowledgments

Produced from materials originally authored by Priyanka Dutta Passecker, George Gibbons, Ying Shao, Sarah Williams, Said El Haou, John Ridley, Louise Webdale, Kathy Sutton, and Marc Rogers from Axol Bioscience Ltd. And Metrion Biosciences Ltd.

This project received funding from the Eurostars-2 joint program with co-funding from the European Union Horizon 2020 research and innovation program.

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 11:34 AM

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