axoCells™ human fibronectin, 1 vial

Phenotypic characterization: beating

Here you can see axoCells™ human iPSC-derived ventricular cardiomyocytes beating spontaneously in a smooth, synchronous monolayer. This demonstrates the high purity of the cell culture. This also demonstrates their optimization for electrophysiology assays, as you can perform spontaneous readings without the need for pacing.

axoCells™ human iPSC-derived ventricular cardiomyocytes beating spontaneously at 0.8Hz prior to cryopreservation. Video obtained using 10x magnification, 90fps.

Phenotypic characterization: ICC

Here you can see axoCells^TM ventricular cardiomyocytes expressing key markers of maturity and chamber-specificity. Expression of chamber-specific markers forms part of our QC for these cells. You can also appreciate the expected morphology with clear banding patterns.

1. Immunocytochemistry of axoCellsTM ventricular cardiomyocytes stained for myomesin (green) and actin (red). This image illustrates the presence of the M-line within the sarcomere, the contractile unit of cardiomyocytes. Magnification 63x oil objective. Scale bar = 20 µm
2. Immunocytochemistry of axoCellsTM cardiomyocytes stained for MLC2V (red) and MLC2A (green). Demonstrated is the mixture of ventricular (MLC2V) and atrial (MLC2A) Cardiomyocytes within the culture. Magnification 63x oil objective. Scale bar = 20 µm
3. Immunocytochemistry of axoCellsTM ventricular cardiomyocytes stained for cardiac alpha-actinin (green) and cardiac troponin T (red). This image illustrates the presence of the sarcomere, the contractile unit of cardiomyocytes. Magnification 40x
4. Immunocytochemistry of axoCellsTM ventricular cardiomyocytes stained for vimentin (red) and cardiac troponin T (green). Vimentin is a major filament protein in non-muscle cells whilst cardiac troponin T is a sarcomeric protein found in cardiomyocytes.

Phenotypic characterization: RNA sequencing

Here you can see distinct expression patterns between the iPSCs and the mature endpoint cells, demonstrating their functional maturity. You can also appreciate chamber-specific differences between the atrial and ventricular cardiomyocytes.

The utility of iPSC-derived cells over traditional hERG overexpression models can be further appreciated by the expression of multiple ion channels (allowing more physiologically-relevant models) and specific calcium handling machinery, which traditional models often lack.

Overall, this demonstrates the value of axoCells cardiomyocytes in chamber-specific, physiologically-relevant cardiac models.

RNASeq characterization of axoCells human iPSC-derived ventricular (ax2508) and atrial (ax2518) cardiomyocytes.  axoCells cardiomyocytes grown for 14 days compared to iPSCs from the same donor. Heatmap displays expression of several key cardiac markers in axoCells atrial and ventricular cardiomyocytes with respective Z-scores (White – high expression; Black – low expression)

Functional relevance: patch clamp

Here we show the functional validation of our axoCells ventricular cardiomyocytes against 3 major drug classes: Lidocaine (an Na+ channel blocker), Nifedipine (a Ca2+ channel blocker) and Dofetilide (a hERG channel blocker), measured by patch clamp, which is the “gold standard” electrophysiology method. There is expected response to the major drug classes, validating these cells for use in in vitro cardiac models.

axoCells human iPSC-derived ventricular cardiomyocytes express the core cardiac ion channels INa, ICa,L and IKr. (A) Representative traces of evoked action potentials recorded under control conditions (grey) and in the presence of 100 μM Lidocaine (green), 100 nM Nifedipine (blue) or 50 nM Dofetilide (red), which show expected effects on action potential amplitude and duration. B) Average effect of each compound on spontaneous action potential parameters, presented as percent of control ± SEM, N ≥ 4. Statistical significance calculated by a paired two-tailed Student’s t-test (* p<0.05, ** p<0.01, *** p<0.001).

Functional relevance: CiPA validation

axoCells ventricular cardiomyocytes have been externally validated against all 28 compounds in the CiPA panel, verifying their application for in vitro cardiac safety testing and detection of pro-arrhythmic risk of new compounds.

Here we present data from 2 of these compounds, Dofetilide and Verapamil.

Want to explore the full dataset? Take a look at our CiPA validation application note.

Dofetilide

Effect of increasing concentrations of Dofetilide on axoCells ventricular cardiomyocytes. There is strong hERG block even at the lowest concentrations, demonstrated by APD90 prolongation (well 1) and triangulation (well 7). Example raw trace demonstrates triangulation and APD shortening with eventual quiescence (Q, marker of arrhythmia).

Verapamil

Effect of increasing concentrations of verapamil on axoCells ventricular cardiomyocytes. At low concentrations, this produces hERG block, demonstrated by increased triangulation (well 1,7). At higher concentrations, Ca2+ channel block causes APD shortening (well 15). Example raw trace demonstrates increasing APD shortening with eventual quiescence (Q).

This demonstrates the utility of iPSC models over traditional hERG overexpression models, as verapamil would technically fail on a hERG overexpression model, but is actually perfectly safe: its multi-channel effects cancel out the pro-arrhythmic hERG blockade.

Take me to the full external CiPA validation data

Functional characterization: MEA

We’ve extensively characterized our axoCells ventricular cardiomyocytes using our industry-leading Axion Maestro Pro MEA system, which we also use for our axoServices electrophysiology assays.

Here you can see a physiological waveform at base, and then the expected electrophysiological response to Dofetilide (a commercially available hERG blocker), demonstrating the functional relevance of our axoCells ventricular cardiomyocytes.

Field action potential waveform of axoCells iPSC-derived ventricular cardiomyocytes, demonstrating the effect of Dofetilide (100nM, red) versus control (blue). Dofetilide, a hERG blocker, causes prolonged field potential duration (from 413ms to 514ms) and after-depolarization.

Functional validation in chronic cardiotoxicity model

axoCells ventricular cardiomyocytes have been validated for use in chronic cardiotoxicity models, via continuous 72 hours electrophysiological recordings of response to a commercially-available cardiotoxicity compound, doxorubicin.

72 hour continuous monitoring of axoCells ventricular cardiomyocytes on the Axion Maestro MEA system from day 10 post-thaw onwards. Upon addition of 3 µM doxorubicin (arrow) at 24 hours, chronic cardiotoxicity is observed with a reduction in beat rate, amplitude and beat width (not shown).