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In Vitro Modeling of Cardiac, Neuronal, and Metabolic Disorders with Human iPSC-derived Cell Types

Coby Carlson, Shannon Einhorn, Rachel Llanas, Steve Fiene, Arne Thompson, David Mann, Susan DeLaura, Blake Anson, Eugenia Jones, and Vanessa Ott

Abstract Introduction: To understand the onset and progression of a human disease, an effective model that combines known genetic elements with a predictive phenotypic readout is required. Recent advances in iPSC technology grant access to unique human samples and enable the generation of disease- and patient-specific cell lines. Terminally differentiated cell types – such as cardiomyocytes, neurons, and hepatocytes – derived from iPSC lines are extremely useful for understanding the patho-physiological mechanisms for cardiovascular, neurodegenerative, and metabolic disorders. Methods: We have used human iPSC-derived, cryopreserved cell types manufactured by CDI to create disease models for Cardiac Hypertrophy, Alzheimer’s Disease (AD), and metabolic diseases. Examples of phenotypic screening (using high content imaging and label-free systems), genetic analysis and modulation, and cell signalling analysis are highlighted. Results: First, we have developed a suite of hypertrophy assays with iPSC-derived cardio-myocytes that monitor endothelin-1 (ET-1)-induced cardiac hypertrophy, focused primarily on the expression of B-type natriuretic peptide (BNP), re-activation of fetal gene expression, and changes in cell size. Using a phenotypic screening approach, numerous compounds that modulate the hypertrophic response were evaluated. Secondly, using iPSC-derived neurons, we applied beta amyloid (1-42) peptide to mimic the undesired outcomes observed during AD. We measured the neurotoxicity and decrease in neurite outgrowth by cell viability and high content imaging assays. Additionally, we reversed some of the negative effects with small-molecules and RNAi. Finally, we have utilized iPSC-derived hepatocytes to begin to investigate the impact that insulin and glucagon have on glucose regulation and the associated cell signalling events. This same cell type also serves as a model system for understanding cholesterol homeostasis through LDLR and PCSK9 signal transduction. Discussion: The use of iPSC-derived cell types for modelling complex diseases is intensifying. Their use in high-throughput screening applications brings more physiologically relevant, human cell models into the drug discovery and development process sooner. Here we present a variety of cell-based approaches to test induced, infected, and inherited disease models with the hope that more efficient and reliable assessment of new chemical entities is possible.

Cardiac Hypertrophy Disease Model

Power of iPSC Technology

Normal Diseased

• iPS cells are somatic cells (eg. skin or blood) that have been genetically reprogrammed to a stem cell state through forced expression of pluripotent genes • iPSC technology provides a platform for generating human terminal cell types from donors with different disease genotypes or drug metabolism responses • CDI’s strengths are reprogramming, engineering, and differentiation at an industrial scale for drug discovery, toxicology, and disease modeling research

Neuronal Model for Alzheimer’s Disease Researchers at GSK used iCell Neurons to establish a cellular model of Alzheimer’s Disease. Neuronal loss was induced by exposure of the cells to an insult of Aβ1–42 aggregates. CDK2 was validated as an important signaling target for rescue of toxicity using known inhibitors and shRNA against CDK2. This model system was further utilized to identify novel modulators of this neurodegenerative disease in a focused drug screen.

GW8510

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• Development of assays and techniques to quantify and measure the phenotypic differences between cell types derived from disease-specific iPS cells and “normal” control lines is also a major focus at CDI • Cardiomyocytes from patient-specific iPSCs have been reported in the literature to study various arrhythmogenic heart diseases • In the figure above, samples derived from a Long QT Syndrome patient were analyzed in the cardiomyocyte beating assay on a FLIPR® Tetra • Irregular and prolonged beat rate due to the LQT2 mutation was detected (red trace) as compared to the rhythmic intracellular Ca2+ oscillations typically observed with iCell Cardiomyocytes (in blue)

• Cardiac hypertrophy is generally characterized by an increase in cardiomyocyte cell size and is manifested through numerous transcriptional, biochemical, and structural transformations

• Reversion back to the cardiac fetal gene program is a hallmark trait of the disease, and expression of B-type natriuretic peptide (BNP) is one of the most commonly used markers for the hypertrophic response

◄ Reducing the expression of fetal genes (eg. NPPB) post-thaw can be accomplished by either prolonged time in Maintenance Medium or by an alternative (SWE) culture medium

► NPPB expression is “turned off’” so that it can be re-activated through induction with endothelin-1 (ET-1)

Unstim + ET-1 (10 nM) ◄ Phenotypic screening assays look for modulation of a physiological readout of the hypertrophic response, such as increased BNP expression or changes in cell size

◄ Induction of iPSC-derived cardiomyocytes with ET-1 results in both robust expression of BNP over background levels and dramatic rearrangement of the actin cytoskeleton

◄ These assays can be miniaturized to 96-and 384-well format to detect both inducers and inhibitors of cardiac hypertrophy

qRT-PCR

High Content Imaging

Xu, X., et al. (2013) Stem Cell Res. 10: 213–227

▲ We have verified the Aβ-induced neuro-toxicity with this cell model in a cell viability assay and neurite outgrowth assay (data not shown) ▲ Additionally, iPSC-derived neurons secrete relevant biomarkers and the release can be modulated with small-molecules ◄ CDK2-specific knockdown or inhibition with GW8510 rescues the neurotoxicity induced by the peptide Aβ (1–42)

Carlson, C., et al. (2013) J Biomol Screen 18: 1203–1211

Metabolic Diseases Summary ◄ Using genetically normal iPSC-derived hepatocytes, we have developed cell-based assays to monitor levels of biologically relevant metabolic markers for future use with disease-specific lines

iCell CM control

LQT2 mutation

P1.57

Human iPSC-derived cell typess from both apparently normal and disease-specific donor backgrounds can be used to develop methods for “disease-in-a-dish” studies, thus enabling researchers to investigate mechanism of action and to evaluate new drug therapies.