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Effects of transient hypoxia/ischemia on inducible human pluripotent stem cell (iPSC)-derived cardiomyocytes

Hypoxia/ischemia is a central elicitor of ischemia/reperfusion (I/R) injury in the human heart. However, there is an apparent lack of suitable in ­vitro models for the investigation of I/R ­induced cellular mechanisms in human cardiac cells. We investigate whether inducible human pluripotent stem cell (iPSC)-­derived cardiomyocytes are susceptible to physiologically defined transient hypoxic/ischemic conditions.

Spontaneously contracting iPSC-­derived cardiomyocytes were subjected to enzymatically induced hypoxic/ischemic conditions by using glucose oxidase (GO, 2U/ml) and catalase (CAT, 120U/ml). Morphological assessment and measurements of LDH activity released from damaged cells (Cytotoxicity Detection Kit; Roche, Mannheim, Germany) were used for evaluating and quantifying hypoxia/ischemia induced cytotoxicity (Figure 1).

Hypoxic/ischemic conditions were rapidly established after the addition of GO/CAT and resulted in pO2 levels <10mmHg after 30 minutes (lasting for at least 4 hours), a gradual decrease of glucose concentration (from 4g/l to <1g/l after 4 hours) and a decline of pH from 7.65 to 6.98. iPSC­derived cardiomyocytes showed spontaneous contractions after 10 days in culture with a beating frequency of 15­20 contractions/min under normoxia as well as under hypoxic/ischemic conditions (normoxia: 18.40±3.41 bpm; hypoxia/ischemia: 15.50±2.60bpm; low resolution video file). Directly after replacing the hypoxic/ischemic medium by normoxic culture medium, frequency of contractions increased by 2­fold in the hypoxia/ischemia group (normoxia: 16.72±0.93bpm; hypoxia/ischemia: 31.58±0.75bpm). 24 hours after the 4 hour hypoxia period, iPSC-­derived cardiomyocytes showed clear morphological signs of cell damage such as cell rounding, swelling and detachment from the growth surface. In cultures that were subjected to 4 hours of hypoxia, LDH release as a marker of cell damage was increased 3­fold while iPSC-­derived cardiomyocytes that were cultured under normoxic conditions did not reveal morphological changes or increased LDH release after 24 hours (normoxia: 0.10±0.00au; hypoxia/ischemia: 0.29±0.02au).

Inducible human pluripotent stem cell-­derived cardiomyocytes are susceptible to transient hypoxia/ischemia in ­vitro. The described culture system closely resembles hypoxic/ischemic conditions in­vivo and may help to elucidate cellular/molecular mechanisms of ischemia/reperfusion injury as well as cardioprotective strategies.


Serum-free human iPSC-derived cardiomyocytes for in vitro testing

Using defined factors OCT3/4, KLF4, SOX2 and c-MYC, adult cells from healthy and patient donors can be reprogrammed to generate induced pluripotent stem cells (iPSCs). Subsequently, these can be differentiated into a variety of cell types including cardiomyocytes. Human iPSC-derived cardiomyocytes (iPSC-CMs) can be cultured in vitro under serum-free conditions and as such, offer a platform investigate the effect of growth factors, cytokines and drugs on the development and functionality of human cardiomyocytes in vitro. Following differentiation, spontaneously beating iPS-CM’s were evaluated in 2D and 3D culture. Expression profiling by immunohistochemistry confirms the expression of cardiomyocyte selective markers including α-actinin, myosin heavy chain, atrial and ventricular myosin light chains, troponin-T and -I, β-catenin, vimentin, L-type calcium channels, connexin-40 and -43, telethonin and ankyrin repeat domain-1 (ANKRD1). Immunohistochemistry findings were validated by Western Blot for α-actinin and cardiac troponin-T expression. Additional analyses conducted, include bi-nucleate cell counts, cell form factor measurements and a comparison of plating efficiencies across a variety of substrates. The electrical activity of the iPSC-CMs was confirmed using a multi-electrode array (MEA), and the calcium dye Fluo4. One application of these cells is drug toxicity testing. To show proof of principle that this can be undertaken in a contactless manner using only genetically encoded tools, which offers several advantages compared to low throughput contact based methods with chemical dyes, we developed a simultaneous optical control/calcium imaging approach to replace the need for electrode stimulation and dyes. We are able to control the beat frequency of iPSC- CMs across the physiological range (0.3Hz – 2Hz) and can observe the anticipated effects of compounds such as Dofetilide, a known hERG inhibitor. Here, we have identified a range of characteristics in these human iPSC-CMs that confirms their ability to function as a highly-pure population of single beating human cardiomyocytes in vitro and presented evidence of a technically simple and scalable platform for cardiotoxicity screening assays.


In vitro assays and screening platforms for exploring ventricular and atrial phenotypes in iPSC-derived cardiomyocytes

Existing cardiac safety testing regimes have successfully prevented new drugs coming to market with pro-arrhythmic risk, but they are expensive (e.g. TQT clinical studies ICH-E14) and the reliance on preclinical hERG screening (ICH-S7B) is undermined by the fact that many new chemical scaffolds have been excluded from further drug development while older drugs that inhibit hERG are not associated with arrhythmia. This has prompted the FDA to implement the Comprehensive in vitro Pro Arrhythmia initiative (CiPA) which involves 3 parts: 1) High quality in vitro cardiac ion channel assays, 2) Comprehensive in silico action potential (AP) models and 3) Predictive assays using stem cell-derived cardiomyocytes.

Our presentation will focus on recent electrophysiological ion channel profiling undertaken at Metrion using human induced pluripotent stem (iPSC)-derived cardiomyocytes from Axol Bioscience. Action potentials and ionic currents were monitored using current- and voltage-clamp manual patch electrophysiology recordings, respectively. Pharmacology was assessed using representative compounds from the CiPA validation toolbox, as well as compounds selectively targeting ventricular and atrial ion channels.  Molecular and biophysical properties of these cells were assessed to explore the relative composition of functional and interdependent cardiac cell phenotypes. Phenotypic measurements of impedance (contraction) and field potential (excitability) were also undertaken using the CardioExcyte96 screening platform. Profiling of ion channel expression with a variety of pharmacological tools and techniques enabled us to ascertain the physiology of iPSC-derived cardiomyocytes and assess their potential utility in more efficacious drug screening and toxicology campaigns.


In vitro assessment of excitation-contraction coupling for predicting pro-arrhythmic risk in iPSC-derived ventricular cardiomyocytes

Human induced pluripotent stem cell-derived ventricular cardiomyocytes (hiPSC-vCMs) (Axol Bioscience) offer a physiologically relevant model for predictive toxicology screening in vitro. The CardioExcyte 96 (Nanion Technologies) is a hybrid screening instrument that simultaneously records cell contractility (impedance) and the extracellular electrical field potential (EFP) in a 96-well plate. Used in combination these tools could help predict the risk of human clinical pro-arrhythmias more accurately.

Here we present data on the optimization of hiPSC-vCMs on the CardioExcyte 96. We determined seeding parameters and identified the optimal time point for analysis. Excitation-contraction coupling was then assessed in response to three standard reference compounds from the Comprehensive in vitro Pro-arrythmia Assay (CiPA) guidelines. The three compounds tested were verapamil, a mixed ion channel blocker acting upon both L-type calcium channels (ICaV) and potassium channels (IKr); nifedipine, a selective calcium channel (ICaV) blocker; and dofetilide, a selective ion channel blocker for IKr. Both verapamil and nifedipine exhibit low pro-arrhythmic risk whereas dofetilide is classified as a high risk pro-arrhythmic compound by the Cardiac Safety Consortium. The addition of each of these compounds altered contractility and electrical excitation in the hiPSC-vCMs.

Here we have demonstrated that the CardioExcyte 96, a non-invasive, label-free, high temporal resolution tool may be used in conjunction with Axol hiPSC-vCMs to predict pro-arrythmic risk in vitro.


Are stem-cell cardiomyocytes a viable cellular reagent for automated patch-clamp?

ICH guidelines state that compounds in drug discovery must be tested for inhibition of hERG cardiac ion channel. It is often prudent to test compounds against a wider array of cardiac ion channels, e.g. hNaV1.5 and hCaV1.2 (Kramer et al., 2013). The CiPA initiative will demand testing and additional ion channel targets as well; namely hNaV1.5 late current, hKir2.1, hKvLQT1 and Kv4.3 (Gintant et al., 2016). These ion channel assays are all amenable to automated patch-clamp and have typically been run using recombinant cell lines over-expressing an individual ion channel. The aim of this research was to investigate whether human induced pluripotent stem cell-derived cardiomyocytes are a useful, affordable and predictive cellular reagent for use on the QPatch automated patch-clamp system.


Functional characterisation of human iPSC-derived atrial cardiomyocytes

Atrial fibrillation (AF) is the most common arrhythmia observed in the clinic, considerable effort has been made to identify the cellular mechanisms of AF and develop new safe and effective antiarrhythmic drugs. However, preclinical studies using non-cardiac cells and non-human animal models may not replicate the physiology of human atrial cardiomyocytes or predict patient efficacy and safety.

Here we outline our results from studies to validate human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-ACMs) generated by Axol Bioscience. The atrial phenotype of Axol hiPSC-ACMs was first characterised at the molecular level using immunocytochemistry with atrial specific markers before functional validation using manual patch clamp recordings of action potential (AP) parameters.

The atrial phenotype was further confirmed using modulators of the atrial specific acetylcholineactivated inward-rectifying potassium current (IKACh) and the ultrarapid delayed rectifier potassium current (Ikur) which are also targets in AF drug discovery.


In vitro validation of human iPSC-derived atrial cardiomyocytes for cell-based assays and drug discovery

The development of atrial cardiomyocytes from iPSCs offers the potential for disease models of atrial fibrillation to be established from patients which may provide information on therapeutics that modify the phenotypic markers of cardiovascular disease and atrial fibrillation.

Atrial fibrillation is one of the most common arrhythmias to affect the heart, as such there is a need to develop drugs to target atrial arrhythmia. However, current mouse models fail to translate in vitro due to fundamental differences in the electrophysiology of cardiac action potentials1.

Here we present data on the molecular and electrophysiological characterization of Axol’s Human iPSCderived Atrial Cardiomyocytes. We determined the protein and gene expression, beat rate and action potential parameters, along with identifying the functionality of the core cardiac and atrial-specific ion channels.

Molecular characterisation of Axol’s Human iPSC-derived Atrial Cardiomyocytes reveals 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.

Functionally, Axol’s Human iPSC-derived Atrial Cardiomyocytes elicit spontaneous action potentials, express functional core cardiac ion channels, INa, ICa,L and IKr and exhibit a steady beat rate.

Axol’s Human iPSC-derived Atrial Cardiomyocytes are shown to be a highly validated, physiologically relevant model that offers the opportunity to study atrial-specific disorders, such as atrial fibrillation, and develop cell-based assays to identify disease modifying treatments.