iCell® Cardiomyocytes, derived from human induced pluripotent stem cells (iPSCs), are suitable for in vitro toxicity screening and drug development. Functionality and relevant responses in pharmacological applications have been recently demonstrated for human iPSC-derived cardiomyocytes (1, 2, 3). Currently used preclinical cardiomyocyte models, such as in vivo animal testing, explanted hearts, cardiac tissue preparations, cardiomyocyte-like cell lines, or primary cardiomyocytes, are plagued by supply limitations, questionable relevance, stability issues, and inconsistency with respect to disease state and genetic background (4, 5).
Cellular Dynamics’ iCell Cardiomyocytes overcome the limitations of current models. They are manufactured with high purity in industrial quantities, exhibit properties of native cardiomyocytes, are of human origin, and are amenable to long-term culture. These human iPSC-derived cells are manufactured through reproducible differentiation protocols and have a uniform genetic background to improve consistency across experiments. In addition, iPSC technology holds significant promise for creating cardiomyocyte panels from ethnically diverse populations or simulating cardiac diseases in vitro.
In addition to displaying typical cardiac phenotypes, iCell Cardiomyocytes express cardiac specific transcription factors and structural genes. In addition, functional analysis has shown that iCell Cardiomyocytes have the ionic currents present in adult cardiomyocytes. Together, these findings demonstrate that iCell Cardiomyocytes are more physiologically relevant than in vitro models currently used for non-clinical cardiac safety studies.
Read more about applications of iCell Cardiomyocytes in cell viability, cytotoxicity, caspase activity and apoptosis, and mitochondrial membrane potential.
Cell viability assays are commonly used in academic, biotech and pharmaceutical research to obtain information on cell health, proliferation and toxicity. The viability of iCell Cardiomyocytes after compound exposure can be assessed by using the Promega CellTiter-Glo® Luminescent Cell Viability, a viability assay based on the quantitation of ATP in metabolically active cells (6). Promega’s proprietary luciferase enzyme catalyzes the mono-oxogenation of the luciferin substrate in the presence of Mg2+, ATP and molecular oxygen resulting in a light signal that is proportional to the number of viable cells in a cell population.
96-well plates (Corning #3603) were precoated with gelatin (0.1% solution, Sigma #G1890). iCell Cardiomyocytes (99% purity) were seeded in iCell Cardiomyocytes Plating Medium to provide 15,000 plated cells/well in a final volume of 100 µL. 48 hours after plating, wells were washed and cells were fed with 90 µL iCell Cardiomyocytes Maintenance Medium. Nine point drug compound titrations of staurosporine (AG Scientific #S-1016), imatinib mesylate (gift from Roche Pharmaceuticals), emetine dihydrochloride hydrate (Sigma #E2375), amoxapine (Sigma #A129) and doxazosin mesylate (Sigma #D9815) were prepared at 10X in iCell Cardiomyocytes Maintenance Medium containing 10% of dimethyl sulfoxide (DMSO). 10 µl of each compound dilution were added to triplicate wells bringing the final volume in each well to 100 µl and the final DMSO concentration to 1%. After 24 hours of drug exposure, cellular ATP concentrations were assessed using the CellTiter-Glo Luminescent Cell Viability Assay as per the manufacturer's instructions. Luminescence readings were taken on the Tecan GENios Pro microplate reader (1 second integration time/sample).
All compounds assayed significantly reduced cell viability with various potencies while the negative controls did not have a significant effect (Figure 1). The EC50 values calculated were 2.27 μM for staurosporine, 2.5 μM for amoxapine, 2.62 μM for doxazosin mesylate, 3.02 μM for imatinib mesylate, and 9.07 μM for emetine dihydrochloride hydrate respectively. The Z’ values for all assays were > 0.5 indicating highly robust assays.
iCell Cardiomyocytes are sensitive to known cardiotoxic compounds, as the cell viability assay results measured by the CellTiter-Glo Luminescent Cell Viability Assay demonstrates. Drug compound-induced general cytotoxic effects can be reliably assessed on iCell Cardiomyocytes using the CellTiter-Glo Luminescent Cell Viability Assay.
Cell viability assays are commonly used in academic, biotech and pharmaceutical research to obtain information on cell health, proliferation and toxicity. The viability and cytotoxicity of iCell Cardiomyocytes after compound exposure can be assessed by using the Promega MultiTox-Fluor Multiplex Cytotoxicity Assay, a ratiometric fluorescent assay that simultaneously measures the relative number of live and dead cells in cell populations by detecting two distinct proteases. A fluorogenic, cell-permeant, peptide substrate glycyl-phenylalanyl-amino-fluorocoumarin (GF-AFC) is used to measure live-cell protease activity which is restricted to viable cells. After entering a viable cell, the GF-AFC substrate is cleaved by the live-cell protease activity resulting in a fluorescent signal proportional to the number of viable cells. Loss of cell membrane integrity renders the live-cell protease inactive. To measure dead cell protease activity, a fluorogenic cell-impermeant peptide substrate called bis-alanyl- alanyl-phenylalanyl-rhodamine 110 (bis-AAF-R110) is used. Upon loss of membrane integrity, protease is released and cleaves the bis-AAF-R110 substrate to generate the fluorescent signal. The impermeable nature of bis-AAF-R110 ensures specificity to dead cells. The products generated by the live and dead-cell protease have different excitation and emission spectra and can therefore be measured simultaneously. The live-cell protease produces the AFC product and the dead-cell protease results in the R110 product which can be measured at 400Ex/505Em and 485Ex/520Em respectively (7).
96-well plates (Corning #3603) were precoated with gelatin (0.1% solution, Sigma #G1890). iCell Cardiomyocytes (99% purity) were seeded in iCell Cardiomyocytes Plating Medium to provide 15,000 plated cells/well in a final volume of 100 µL. 48 hours after plating, wells were washed and cells were fed with 90 µL iCell Cardiomyocytes Maintenance Medium. Nine point drug compound titrations of staurosporine (AG Scientific #S-1016), imatinib Mesylate (gift from Roche Pharmaceuticals), emetine dihydrochloride hydrate (Sigma #E2375), amoxapine (Sigma #A129) and doxazosin mesylate (Sigma #D9815) were prepared at 10X in iCell Cardiomyocytes Maintenance Medium containing 10% of dimethyl sulfoxide (DMSO). 10 µl of each compound dilution were added to triplicate wells bringing the final volume in each well to 100 µl and the final DMSO concentration to 1%. After 24 hours of drug exposure, cell viability and cytotoxicity were assessed using the MultiTox-Fluor Multiplex Assay per the manufacturer's instructions. Live-cell and dead-cell fluorescence levels were measured at 390Ex/485Em and 485Ex/520Em respectively using a Tecan GENios Pro microplate reader.
All compounds assayed (staurosporine, imatinib mesylate, emetine, amoxapine and doxazosin) significantly reduced cell viability while the negative control did not (Figure 2). The EC50 values calculated were 613 nM for staurosporine, 10.2 μM for imatinib mesylate, 17.3 μM for emetine dihydrochloride, 19.3 μM for amoxapine and 20.6 μM for doxazosin mesylate, respectively. The Z’ values for all assays were > 0.5 indicating highly robust assays.
The cell viability and cytotoxicity assay results as measured using the Promega MultiTox-Fluor Multiplex Cytotoxicity Assay demonstrate that iCell Cardiomyocytes are sensitive to known cardiotoxic compounds. Overall, the Promega MultiTox-Fluor Multiplex Cytotoxicity Assay can be used to reliably assess drug compound induced general cytotoxic effects on iCell Cardiomyocytes independent of cell number.
Cysteine aspartic acid-specific proteases (caspases) are critical effector molecules of programmed cell death and are one of the most commonly used markers in the field of mammalian apoptosis (8). Both intrinsic and extrinsic pathways of apoptosis can activate the caspase signaling cascade. The intrinsic pathway of apoptosis is initiated through the release of cytochrome c from the mitochondria and its subsequent association with Apaf-1, dATP, and caspase 9 to form the complex known as the apoptosome (9). Formation of the apoptosome initiates caspase 9 to cleave and activate effector caspases 3 and 7. Extrinsic apoptosis signaling, mediated by cell surface receptors, can directly activate caspases 3 and 7 via caspases 8 and 10 without involvement of the mitochondria (10). Caspases 3 and 7 are cellular executioners that cause the destruction of the cell. Cleavage of their substrates lead to the morphological changes associated with apoptosis, including DNA fragmentation, cell shrinkage, chromatin condensation, and membrane blebbing (11).
The Caspase-Glo® 3/7 Assay (Promega) is used to detect effector caspase activity in apoptotic cells (12). Active caspase 3 and 7 cleave a protease-specific DEVD tetrapeptide substrate to release aminoluciferin. Luciferase cleavage of aminoluciferin produces a luminescent signal that is directly proportional to the amount of caspase 3 and 7 activity present. Staurosporine, amoxapine, emetine, and imatinib mesylate (Gleevec) were selected to investigate caspase activation in iCell Cardiomyocytes.
96-well plates (Corning #3603) were precoated with gelatin (0.1% solution, Sigma #G1890). iCell Cardiomyocytes (99% purity) were seeded in iCell Cardiomyocytes Plating Medium to provide 15,000 plated cells/well in a final volume of 100 µL. 48 hours after plating, wells were washed and cells were fed with 90 µL iCell Cardiomyocytes Maintenance Medium. The following compounds were administered:
Compound dilutions were performed in iCell Cardiomyocytes Maintenance Medium with 10% DMSO for a final concentration of 1% DMSO during treatment. Apoptosis was measured using the Caspase-Glo®3/7 Assay (Promega #G8091) after 6 hours (staurosporine) or 24 hours (amoxapine, emetine, imatinib mesylate) of compound treatment with a Tecan GENios Pro microplate reader (1 second integration time). Relative luminescence units (RFU) were background-corrected to control wells with cells and vehicle only.
Caspase activity can be detected in iCell Cardiomyocytes in response to staurosporine, amoxapine, and emetine using the Caspase-Glo®3/7 Assay (Figure 3). The EC50 value for 6 hour treatment with staurosporine was 1.5 µM. The EC50 values for 24 hour treatment with amoxapine and emetine were 67 µM and 6.5 µM, respectively. No caspase 3 or 7 activity was detected at 24 hours in the range of imatinib mesylate tested despite widespread cell death at the highest concentrations.
Monitoring caspase activity is critical to detecting and understanding drug toxicity. The use of iCell Cardiomyocytes in preclinical safety testing could accelerate the detection of toxic compounds and provide a mechanism of action for cardiac toxicity. iCell Cardiomyocytes are an amenable cellular tool for in vitro apoptosis assays investigating the activation of caspase pathways.
Mitochondrial function is critical for cardiomyocyte viability through ATP synthesis, ion homeostasis, and the regulation of apoptosis and necrosis (9). The maintenance of the mitochondrial inner membrane potential (ΔΨm) is a critical component of cell health, and its integrity is often used as a measure of cellular viability (13). Loss of ΔΨm results in decreased ATP production and can promote a cascade of pro-apoptotic factors. Mitochondrial toxicity is linked to many of the drugs receiving Black Box Warnings from the FDA, and at least three drugs have been pulled from the market because of organ toxicity directly related to the collapse of ΔΨm (14).
The Cell Meter™ JC-10 Mitochondrial Membrane Potential Assay Kit is used to detect the loss of ΔΨm (15). In healthy cells, JC-10 selectively accumulates in mitochondria as orange “J-aggregates.” As the inner membrane potential is lost in apoptotic or necrotic cells, the monomeric form of JC-10 is released into the cytoplasm and the cells fluoresce green. The shift of fluorescence in cells from orange to green indicates apoptosis or necrosis. Valinomycin and staurosporine, two compounds known to disrupt ΔΨm in other cell types (16, 17), were selected to investigate their response in iCell Cardiomyocytes.
96-well plates (Corning #3603) were precoated with gelatin (0.1% solution, Sigma #G1890). iCell Cardiomyocytes (99% purity) were seeded in iCell Cardiomyocytes Plating Medium to provide 15,000 plated cells/well in a final volume of 100 µL. 48 hours after plating, wells were washed and cells were fed with 90 µL iCell Cardiomyocytes Maintenance Medium. The following compounds were administered:
Compound dilutions were performed in iCell Cardiomyocytes Maintenance Medium with 10% DMSO for a final concentration of 1% DMSO during the treatment. Mitochondrial membrane potential was measured using the Cell Meter™ JC-10 Mitochondrial Membrane Potential Assay Kit (ABD Bioquest #22800) following six hours of compound treatment using a Tecan GENios Pro microplate reader (ex490em535 and ex490em580). Relative fluorescence units (RFU) were background-corrected to the levels in control wells containing media only. Data was reported as a ratio of em535 to em580 after background correction.
The loss of mitochondrial membrane potential can be detected in iCell Cardiomyocytes in response to valinomycin and staurosporine using the Cell Meter™ JC-10 Mitochondrial Membrane Potential Assay Kit. The EC50 values for a six hour treatment with staurosporine and valinomycin were 9.1 µM and 1.2 nM respectively (Figures 4 and 5).
Figure 4
