April 15, 2012 (Vol. 32, No. 8)

Predictive in vitro Model Useful for HTS, Cardiac Toxicity, and Disease Modeling

The high cost associated with developing new chemical entities (NCEs) creates a clear demand for better prediction of toxicity and efficacy to thus enable more effective SAR early in the discovery process. The primary causes of compound attrition are poor pharmacokinetics, lack of efficacy, and off-target toxicity.

Compound performance in these areas is often not adequately addressed until pre-clinical development, at which point failures are quite costly. The implementation of more physiologically relevant models and assays earlier in the discovery and development pipeline could reduce the overall cost of drug development and potentially diminish market withdrawal.

Several platforms exist for precise, high-throughput interrogation of biological responses in cellular models. However, current in vitro models used on these platforms typically include immortalized cell lines, human cadaveric tissue, or primary cultures of nonhuman animal origin.

Each of these cellular models presents limitations that contribute to the overall low predictability of in vivo effects, including limited biological relevance of immortalized cell lines, functional variability of primary cell cultures, and/or the low throughput of assays based on isolated tissues.

Terminal cell types differentiated from human embryonic or induced pluripotent stem cells (ESCs or iPSCs, respectively) have emerged as a valuable alterative to current cell-based models. The indefinite self-renewing capacity of iPSCs provides an inexhaustible supply of consistent starting material.

Highly structured differentiation protocols followed by rigorous quality control procedures ensure a highly reproducible cellular model. Additionally, the industrial-scale production of cellular models and their intrinsic relevant human biology make stem cell-derived cell types suitable for high-throughput screening in drug discovery.

Human iPSCs also offer the opportunity of generating disease- or genotypic-specific cell lines for use as more clinically relevant models to identify new targets and populations susceptible to adverse side effects.

iCell® Cardiomyocytes from Cellular Dynamics International (CDI) are human iPSC-derived cardiomyocytes that exhibit the genomic, biochemical, electrophysiological, and mechanical functionality expected of human cardiac myocytes. These cardiomyocytes are manufactured at an industrial scale in quantities suitable for use with many of the platforms and standard assay techniques already employed by pharmaceutical and academic settings.

Their availability in high quality, quantity, and purity; relevant biology; and ease of use make human iPSC-derived cardiomyocytes an excellent cellular model for use in basic research, drug discovery, and toxicity investigations.

Figure 1. (A) iCell Cardiomyocytes mitochondrial toxicity before (left) and after (right) treatment with Antimycin A. (B) Mitochondrial membrane potential was quantified through application of an analytical mask.

High-Content Imaging Techniques in Assessing Cardiac Toxicity

Cardiotoxicity is a primary cause of compound attrition in the development pipeline and recall of marketed pharmaceuticals. The inherent normal cell biology of iCell Cardiomyocytes and relevant responses to pathological stimuli can be coupled with the granular investigative capabilities of high-content imaging to provide a system suitable for providing detailed and specific assessments of compound action on cellular function.

Mitochondrial membrane integrity is one of the early markers for cardiac toxicity or tissue ischemia, and the ability to easily detect and quantify mitochondrial toxicity in cultured iCell Cardiomyocytes with high-content imaging (ImageXpress® Micro Automated Imager, Molecular Devices) exemplifies the advantages of coupling advanced cellular and platform technologies.

Figure 1C. The dose-response relation for the effects of Valinomycin

In this example, cardiomyocytes were cultured on 96-well assay plates, mitochondrial toxicity was induced with either Antimycin A or valinomycin, and the effect of compound addition on mitochondrial membrane potential was monitored with the JC-10 voltage-sensitive indicator.

Images of cardiomyocytes in the absence and presence of the toxicant were obtained (Figure 1A), the drug-induced effects were quantified through vendor-supplied routines whereby a standard analytical mask was applied (Figure 1B), and the data was expressed as a dose response relation (Figure 1C).

Figure 2A. Fluorescence intensity versus time for a single control well of beating iCell Cardiomyocytes loaded with Fluo-4 dye on the FLIPR Tetra System before and after addition of epinepherine.

High-Throughput Phenotypic Screening

Rhythmic electrical oscillation at the cardiomyocyte plasma membrane drives cyclical changes in intracellular Ca2+ levels that in turn initiate myocyte contraction. Modulating this behavior can be an intended endpoint in a subset of therapeutic applications and a toxic outcome in others. The biochemical pathways underlying electrical-contractile (EC) coupling are complex and intertwined with many of the molecular players existing across multiple pathways. Early predictive assessment for intentional or inadvertent modulation of these pathways is a critical component of many development programs.

In culture, hiPSC-derived cardiomyocytes form an electrically and mechanically active syncytium that recapitulates the functional behavior of native myocytes. The FLIPR® Tetra system (Molecular Devices) is a widely used high-throughput cellular assay system for monitoring real-time changes in fluorescence. When coupled with fast-acting calcium-sensitive dyes and iCell Cardiomyocytes, the system provides a powerful platform for assessing modulations in Ca2+ signaling and beat rate (chronotropy) in an in vivo-like human cellular model.

Figure 2B. Dose-response relations for positive and negative chronotropes on iCell Cardiomyocytes loaded with Fluo-4 dye on the FLIPR Tetra System. IC50 values: isoproterenol 7nM, epinephrine 15 nM, propranolol 2 uM

As an example, chronotropic modulation of iCell Cardiomyocytes’ activity before and after treatment with β-adrenergic receptor agonists and antagonists was measured in a 384-well format and quantified using the FLIPR Tetra system (Figures 2A and 2B). iCell Cardiomyocytes reacted to the chronotropic agents at physiologically relevant concentrations with expected alterations in beating frequency: increased beat rate with the β-adrenergic receptor agonists epinephrine and isoproterenol, and decreased beat rate with the β-adrenergic receptor antagonist propranolol.

The positive and negative alteration of electrical activity through GPCR modulation exemplifies the ability of hiPSC-derived cardiomyocytes to act as a suitable model across both drug discovery and drug toxicity applications.

Figure 3A. Perinuclear staining of ET-1 induced BNP expression in iCell Cardiomyocytes (red; BNP labeling, blue; nuclear staining)

Disease Modeling

Cardiac hypertrophy is another cellular phenomenon that can exist as either a therapeutic target or a potential toxic outcome. Endothelin-1 (ET-1) is a potent, vasoconstricting peptide that acts through an endogenous GPCR receptor signal cascade leading to a hypertrophic response whereby fetal gene expression, such as A and B-type natriuretic peptide (ANP and BNP, respectively), is re-activated. The response of iCell Cardiomyocytes in this disease model is shown in Figures 3A, 3B, and 3C.

Figure 3B. qPCR quantification of ET-1 induced increases in ANP mRNA

iCell Cardiomyocytes were cultured according to the user’s guide, exposed to ET-1, and analyzed with commonly available techniques. ET-1 induced BNP expression is shown qualitatively with immunocytochemical staining (Figure 3A), while quantitative measurements of ANP mRNA and BNP protein responses are illustrated with QPCR (Figure 3B) and flow cytometry (Figure 3C).

Both methods gave a reproducible EC50 of ~100 pM, attesting to the robustness of the assay. The relevant induction of the ANP and BNP biomarkers in iCell Cardiomyocytes demonstrates the utility of the system for use as a hypertrophic disease model as well as a sentinel able to detect adverse hypertrophic effects.

The applications illustrated in this tutorial highlight the promise of human iPSC-derived cardiomyocytes to provide a human in vitro test system with cross-platform functionality in a biologically relevant cellular environment. Implementation of this unique and clinically relevant model system across both toxicity and discovery studies presents a significant advantage by avoiding complications that can arise when translating data from models across different species and biological characteristics.

Ultimately, by providing access to relevant human biology early in the discovery process, iPSC technology holds the potential to increase efficiency of the drug development pipeline and thereby reduce overall costs.

Figure 3C. Flow cytometry quantification of ET-1 induced increases in BNP protein

Giorgia Salvagiotto, Ph.D. ([email protected]), is technical applications scientist III, and Steven Kattman, Ph.D., is R&D, cell biology group leader at Cellular Dynamics International. Oksana Sirenko, Ph.D., is research scientist R&D and Jayne Hesley is applications scientist, cellular imaging at Molecular Devices.

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