Cellular Dynamics at the Biophysical Society Annual Meeting 2015
Check out these posters that detail the utility of CDI’s iPS cell technology.
T Knott1, M Chevalier2, B Amuzescu1, J Eisfeld1, O Scheel1, H Abriel2
- Cytocentrics Bioscience
- University of Bern
Abstract: The cardiac sodium current plays a central role in excitability and cardiac impulse propagation. This current is mainly generated by the voltage-gated sodium channel Nav1.5 encoded by the gene SCN5A. Nav1.5 channels lead to the macroscopic transient “peak” that causes the phase 0 of the cardiac action potential (AP). It is also well known that this fast inward current is followed by a much smaller late current (INaL). An increase of INaL prolongs the AP duration and may cause a long QT syndrome (LQTS). Therefore, drug-induced increase of INaL represents a new concern for safety pharmacology, and assessing the influence of new pharmaceutical compounds on INaL should be part of the routine safety screening as discussed in the current CiPA initiative redefining the cardiac risk assessment paradigm. In order to increase INaL amplitudes for pharmacological studies, other groups have used toxins such as ATX-II or H2O2 preexposure, but these protocols may induce shifts in dose-response curves. Pharmacology assays at physiological temperature (35°C) are prone to yield highly relevant data.
E Jones, N Aoyama, J Wang, M McLachlan, R Learish, T Burke, C Carlson, B Anson, Cellular Dynamics International
Abstract: Hypertrophic cardiomyopathy (HCM) is a common genetic heart condition affecting approximately 1 in 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 change of amino acid 403, from Arg-to-Gln (MYH7-R403Q). The study of diseases affecting cardiomyocytes has been advanced by the advent of stem cell technology which has enabled the production of stem cell-derived cardiomyocytes in sufficient quantities to facilitate large scale in vitro research. Further advances in stem cell technology enabled the production of human induced pluripotent stem (iPS) cells from any individual, apparently healthy normal as well as affected individuals, prompting production of large collections of iPS cells. Cardiomyocytes (CM) can be produced from any iPS cell in a collection and used to gain a better understanding of mechanisms involved in complex heart disease. Here we describe the study of iPS cell-derived CM from normal and MYH7-R403Q.
Hypertrophy can be induced in normal human donor iPS cell-derived CM with exposure to Endothelin-1 (ET-1). HCM-induced CMs exhibit classic 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 showed autonomous contractile activity similar to the control 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.
E Jones, K Mangan, M McLachlan, T Burke, B Meline, C McMahon, C Carlson, S DeLaura, Cellular Dynamics International
Abstract: The sodium-activated potassium channel Slack, encoded by the gene KCNT1, is expressed in neurons throughout the brain, including the frontal cortex, and mediates a sodium-sensitive potassium current (IKNa). This outward current regulates neuronal excitability and determines how neurons respond to repeated high frequency stimulations, both of which are aspects of memory and learning. Mutations in KCNT1 and alterations to the IKNa current have patho-physiological consequences. Recent studies have described the emerging role of KCNT1 channels in cognitive deficits, and role of 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 cell 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 a physiologically-relevant human cell type. We have leveraged this technology to generate human neurons cells carrying the KCNT1 P924L mutation and assessed the function effect of the mutation on physiology. To introduce the P934L allele, we genetically engineered a “control” iPS cell line from an apparently healthy female donor with no family history of neurological disorders and generated highly pure (>95% TUJ1-positive), and terminally differentiated cortical neurons from the KCNT1 P924L and isogenic control iPS cell lines.
Here, we present data from the functional comparison of these human neurons (wild-type vs. KCNT1 P924L mutant), with a specific focus on the electrophysiological analysis using multi-electrode array (MEA). The ability to engineer isogenic wild type and disease associated alleles by genome editing human iPS cells gives researchers unprecedented access to models for neurological disorders. Our ability to produce pure populations of sub-type specific human neurons is revolutionizing our approach to studying diseases in vitro, and is opening new avenues to develop treatment for central nervous system diseases.