Industrialized Production of Human iPSC-Derived Cardiomyocytes for Use in Drug Discovery and Toxicity Testing - The authors describe an industrialized process for the manufacture of iPSC-derived human

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Industrialized Production of Human iPSC-Derived Cardiomyocytes for Use in Drug Discovery and Toxicity Testing
The authors describe an industrialized process for the manufacture of iPSC-derived human cardiomyocytes.


BioPharm International
Volume 24, Issue 3, pp. 58-67

HUMAN iPSC-DERIVED CARDIOMYOCYTE CHARACTERIZATION

Although purified iPSC-derived cardiomyocytes have the physical appearance of cardiomyocytes and aggregations of the cells exhibit synchronous contractile activity (i.e., the cells beat), we tested the cells' biochemical and electrophysiological properties to determine their utility for drug development and toxicity testing.


Figure 3. Gene expression of iCell Cardiomyocyte-specific mRNAs during differentiation.
Cardiomyocytes manufactured using industrial-scale culture and our cell differentiation process expressed several genes found specifically in cardiomyocytes (see Figure 3). Gene expression of human cardiomyocyte mRNAs was tested using real-time PCR between 0 and 32 days post initiation of differentiation. Gene expression was quantified using Taqman (Applied Biosystems, Carlsbad, CA) gene expression assays for a number of transcription factors, cytoskeletal components, and ion channels. Levels of the stem cell transcription factor Oct-4 decreased during differentiation of iPSC-derived cardiomyocytes, while all cardiomyocyte-specific mRNAs expression levels increased.


Figure 4. Differentiated cardiomyocytes express cardiac protein markers: (I) Bright field image; (II) Nkx 2.5, RND Systems #AF2444; (III) Bright field image; (IV) Sarcomeric Alpha Actinin, Abcam #ab9465; (V) Bright field image; (VI) Troponin T, Abcam #ab8295. Images taken at 10X (I-II) and 64X (III–VI) magnification.
The presence of cardiac-specific protein markers was also investigated. iPSC-derived cardiomyocytes were shown to express the cardiomyocyte-specific proteins sarcomeric alpha actinin and troponin I (see Figure 4).


Figure 5. Differentiated cardiomyocytes generate cardiac action potentials. Action potential tracings recorded from a spontaneously beating single iCell Cardiomyocyte using the perforated (gramicidin) patch clamp methodology. Atrial (left), nodal (center), and ventricular-like (right) action potentials were recorded from the cell population. The horizontal line over each tracing represents 0 mV.
Cardiomyocyte subtypes of the heart have distinctive electrophysiological profiles that can be characterized by, among other items, early depolarization events (Phase 4 depolarization) and the action potential duration. As shown in Figure 5, action potentials produced by individual iPSC-derived cardiomyocytes recapitulate qualities of action potentials of native nodal, atrial, and ventricular cardiomyocytes.

HUMAN iPSC-DERIVED CARDIOMYOCYTE PHARMACOLOGY


Figure 6. Response of iPSC-derived cardiomyocytes to (1) hERG channel blocker, E-4031, (II) calcium channel blocker, nifedipine, and (III) the field potential response to E-4031 measured via a microelectrode array.
iPSC-derived cardiomyocytes should also mimic responses to chemical perturbations seen in normal human cardiomyocytes. Electrophysiological responses of cardiomyocytes were tested against exposure to E-4031, a blocker of the human Ether--go-go Related Gene (hERG) potassium channels that are primarily expressed in the heart, and to nifedipine, a blocker of the voltage-dependent L-type calcium channel also expressed in the heart. In both cases, the change in shape of the cardiac action potential from single cells recorded with the patch clamp or the shape of the field potential of a population of beating cardiomyocytes recorded with a microelectrode array exhibit the expected prolongation or shortening of the signal as expected based on work with native cells and tissue (see Figure 6).


Figure 7. Cytotoxic responses of cardiomyocytes following exposure to staurosporine.
iPSC-derived cardiomyocytes showed sensitivity to known cardiotoxic compounds (see Figure 7) affecting biochemical processes. Cardiomyocytes exposed to staurosporine for 16 hours showed half maximum effective values (EC50 values) of 575 nM for the live assay and 482 nM for the dead assay. Caspase activity was measured after six hours of drug exposure and demonstrated an EC50 of 585 nM, once again demonstrating the expected responses of native cardiomyocytes.


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