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Cellular Dynamics at ISSCR 2015

Several posters at ISSCR 2015 featured iCell® and MyCell® products:

Differential Isoform Expression in an In Vitro Model of Cardiac Hypertrophy Using hIPSC-derived Cardiomyocytes

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Authors: Li W, Aggarwal P, Turner A, Matter A, Broeckel U

Abstract: Left ventricular hypertrophy (LVH) is accompanied by significant differential gene expression in cardiomyocytes (CMs). However, little is known about alternative splicing (AS) and the role of differentially expressed isoforms (DEIs). We performed Next-Generation RNA sequencing in an in vitro model of cardiac hypertrophy using hiPSC-derived CMs (hiPSC-CMs) to determine the prevalence, pattern and inter-individual variability of DEIs. Hypertrophy was induced by endothelin-1 stimulation in four hiPSC-CM lines from two patients with LVH. Transcripts were analyzed for relative abundance using Cufflinks2. We found that between 446 and 982 genes showed significant expression changes at isoform level in the four hiPSC-CM lines respectively. More than half of these genes have two or more annotated isoforms. A significant portion of the DEIs are unique to an individual CM line. Distinct functional motifs were found in these DEIs. Interestingly, DEIs are enriched in genes involved in hypertrophic cardiomyopathy and cardiac contraction. Taken together, we illustrated that changes in isoform expression were prevalent in an hiPSC-CM model of cardiac hypertrophy with a subset of DEIs unique to each CM line. Our study indicated that DEIs are an integral part of the overall disease phenotype and demonstrated the power of hiPSC-based disease modeling to dissect genetic, molecular, and environmental factors in complex disease.

Human iPS Cell-derived Cardiomyocytes Carrying MYH7-R403Q Exhibit Aspects of Hypertrophic Cardiomyopathy In Vitro

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Authors: Jones E, Aoyama N, McLachlan M, Hussey D, Burke T, Lange S,  Anson B

Abstract: Hypertrophic cardiomyopathy (HCM) is a common genetic heart condition affecting approximately 1:500 individuals, where the heart muscle becomes thick and blood flow is restricted. The condition is characterized by a thickening of the ventricular wall as a result of enlarged cardiac myocytes, changes in blood pressure due to restricted blood flow, and arrhythmias. The most prevalent form of familial HCM arises from a missense mutation in the gene encoding the beta-myosin heavy chain protein, resulting in a Arg-to-Gln change at a.a. 403, from (MYH7-R403Q). The study of cardiomyocyte diseases has been advanced by the advent of stem cell technology enabled production of stem cell-derived cardiomyocytes in sufficient quantities to facilitate large scale in vitro research. Further advances in stem cell technology enabled production of human induced pluripotent stem (iPS) cells from any individual, prompting development of large iPS cell collections. Cardiomyocytes (CM) can be produced from any iPS cell in a collection and used to gain a better understanding of mechanisms involved in heart disease. Here we describe the study of iPS cell-derived CM from normal and MYH7-R403Q. Hypertrophy is induced in CM cell-derived from normal human iPS with exposure to Endothelin-1 (ET-1). HCM-induced CMs exhibit hallmarks of cardiac hypertrophy including up-regulation of fetal genes, cytoskeletal rearrangements, and an increase in cardiomyocyte size. We show that induced and inherited HCM in iPS cell-derived CM have common features. CMs differentiated from MYH7-R403Q iPS cells exhibit cardiac morphology, and autonomous contractile activity similar to the WT iPS cell-derived CM. MYH7-R403Q CM and ET-1 induced HCM in normal CM have similar basal gene expression. ET-1 induction increases BNP expression in both control and MYH7-R403Q cardiomyocytes, but basal BNP levels are higher in MYH7-R403Q cardiomyocytes. These data show the progression of HCM characteristics in MYH7-R403Q cardiomyocytes and underscore the advantages of modeling cardiovascular disease with iPS cell technology.

Modeling Neurological Disease with Human iPS Cell-derived Neurons Containing a KCNT1 Mutation

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Authors: Jones E, Mangan K, McLachlan M, Burke T, Hussey D, Lange S

Abstract: The sodium-activated potassium channel, encoded by the gene KCNT1, is widely expressed in the CNS including cortical neurons, and mediates a sodium-sensitive potassium current (IKNa). This outward current regulates excitability and determines response to repeated high frequency stimulations, both of which are aspects of memory and learning. Mutations in KCNT1 and alterations to the IKNa current have pathophysiological consequences. Recent studies have described the emerging role of KCNT1 channels in cognitive deficits, and KCNT1 mutations in clinically distinct forms of severe early onset “childhood” epilepsies. The development of better therapies for neurological disorders has been hindered by limited access to clinically-meaningful cells for research and drug development. The advent of induced pluripotent stem (iPS) cell technology provides a platform to facilitate increased understanding of disease mechanisms in physiologically-relevant human cell types. We used this technology to generate human cortical neurons carrying the KCNT1 P924L mutation and assessed the functional effects on neural physiology. To introduce the P924L allele, we genetically engineered a “control” iPS cell line from an apparently healthy female donor and generated highly pure (>95% TUJ1-positive), terminally differentiated, cortical neurons from both the KCNT1 P924L and isogenic control iPS cell lines. Here, we present data from the functional comparison of wild-type vs. KCNT1 P924L mutant neurons, with a specific focus on the electrophysiological analysis.

The Utility of Induced Pluripotent Stem Cell (iPSC)-derived Cardiomyocytes and Hepatocytes as an In Vitro Human Model System for High Content Screening of Complex Chemical Substances

Authors: Sirenko O, Grimm F, Iwata Y, Crittenden C, Rusyn I, Cromwell E, Lange S

Abstract: In vitro model-based testing is part of routine safety evaluation of drugs and chemicals. Usually, screening is performed with pure compounds. In addition, chemical structure-based similarity “read across” is widely used for predictive safety assessments of non-pharmaceutical chemicals in regulatory submissions, especially in Europe. While either biological or chemical characterization of the potential human health hazard is sensible for chemically-characterized compounds, it is not applicable to assess the hazard of mixtures or complex substances (e.g., petroleum products). Thus, we suggest that safety evaluation centered on similarities in biological responses, i.e. a biological data-based read across, may represent a feasible alternative. In this work we tested a hypothesis that induced pluripotent stem cell (iPSC)-derived cardiomyocytes and hepatocytes represent a relevant in vitro model system that is applicable for high-content, multidimensional toxicity screening and biological read across of chemically complex substances. We selected 26 petroleum products from six distinct categories: SRGO (Straight Run Gas Oils), OGO (Other Gas Oils), VHGO (Vacuum & Hydrotreated Gas Oils), Bitumens, RAE (Residual Aromatic Extracts), and HFO (Heavy Fuel Oils). iPSC-derived cardiomyocytes and hepatocytes were exposed to a DMSO-based extract dilution series in logarithmical order over five logs for up to 48 hours. The cardiomyocyte-derived phenotypes included the measurement of effects on contractility, beating pattern and amplitude, as well as cell viability,morphology and mitochondria integrity measured through high-content live cell imaging. For hepatocytes we determined changes in cell viability, morphology, and mitochondria integrity by live and fixed cell imaging. Quantitative data were then used as high-dimensional “biological” data inputs for evaluation of the similarities and differences both within and across different substance categories. Our data clearly indicate cell- and substance group-specific effects. Collectively, our work demonstrates that iPSC- derived cardiomyocytes and hepatocytes are useful in vitro human model systems for high-content screening of complex chemical substances.