Cellular Dynamics International Research Articles

A Bioprinted Heart-on-a-Chip with Human Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Evaluation.

In this work, we show that valve-based bioprinting induces no measurable detrimental effects on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The aim of the current study was three-fold: first, to assess the response of hiPSC-CMs to several hydrogel formulations by measuring electrophysiological function; second, to customise a new microvalve-based cell printing mechanism in order to deliver hiPSC-CMs suspensions, and third, to compare the traditional manual pipetting cell-culture method and cardiomyocytes dispensed with the bioprinter. To achieve the first and third objectives, iCell2 (Cellular Dynamics International) hiPSC-CMs were used. The effects of well-known drugs were tested on iCell2 cultured by manual pipetting and bioprinting. Despite the results showing that hydrogels and their cross-linkers significantly reduced the electrophysiological performance of the cells compared with those cultured on fibronectin, the bio-ink droplets containing a liquid suspension of live cardiomyocytes proved to be an alternative to standard manual handling and could reduce the number of cells required for drug testing, with no significant differences in drug-sensitivity between both approaches. These results provide a basis for the development of a novel bioprinter with nanolitre resolution to decrease the required number of cells and to automate the cell plating process.

Chronic toxicity assessment of BCR-ABL tyrosine kinase inhibitors using human-induced pluripotent stem cell-derived cardiomyocytes

Tyrosine kinase inhibitors (TKIs) have contributed to the improvement of the survival of patients with chronic myelogenous leukemia (CML). Growing evidence suggest that cancer therapy-related cardiac dysfunction has become important as the most undesirable side effects of chemotherapy. BCR-ABL TKIs, such as nilotinib and imatinib, has been reported to have a risk of QT prolongation associated with Torsades des Pointes and cardiac failure. We have previously reported that nilotinib caused QT prolongation and early afterdepolarization (EAD) using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). However, chronic cardiotoxicity of BCR-ABL TKIs has not been fully understood. In this study, we evaluated the chronic toxicity of imatinib and nilotinib using hiPSC-CMs. We used iCell Cardiomyocyte 2.0 (Cellular Dynamics International). To assess whether imatinib and nilotinib induced chronic cardiotoxicity, we investigated the QT interval recorded by multi-electrode array system (MED64, Alpha MED Scientific) and contractility velocity recorded by motion vector analysis (SI8000, Sony). We found that the imatinib decreased contraction velocity for long-term treatment. In addition, long-term treatment with nilotinib caused QT prolongation and decrease in contraction velocity in a concentration-dependent manner. These results suggest that hiPSC-CMs can be the useful tool for chronic cardiotoxicity assessment. We are planning to evaluate other types of BCR-ABL TKIs with hiPSC-CMs.

Human Induced Pluripotent Stem Cell-Derived TDP-43 Mutant Neurons Exhibit Consistent Functional Phenotypes Across Multiple Gene Edited Lines Despite Transcriptomic and Splicing Discrepancies.

Gene editing technologies hold great potential to enhance our ability to model inheritable neurodegenerative diseases. Specifically, engineering multiple amyotrophic lateral sclerosis (ALS) mutations into isogenic cell populations facilitates determination of whether different causal mutations cause pathology via shared mechanisms, and provides the capacity to separate these mechanisms from genotype-specific effects. As gene-edited, cell-based models of human disease become more commonplace, there is an urgent need to verify that these models constitute consistent and accurate representations of native biology. Here, commercially sourced, induced pluripotent stem cell-derived motor neurons from Cellular Dynamics International, edited to express the ALS-relevant mutations TDP-43M337V and TDP-43Q331K were compared with in-house derived lines engineered to express the TDP-43Q331K mutation within the WTC11 background. Our results highlight electrophysiological and mitochondrial deficits in these edited cells that correlate with patient-derived cells, suggesting a consistent cellular phenotype arising from TDP-43 mutation. However, significant differences in the transcriptomic profiles and splicing behavior of the edited cells underscores the need for careful comparison of multiple lines when attempting to use these cells as a means to better understand the onset and progression of ALS in humans.

Cardiovascular Effects of Polychlorinated Biphenyls and Their Major Metabolites.

Background:Xenobiotic metabolism is complex, and accounting for bioactivation and detoxification processes of chemicals remains among the most challenging aspects for decision making with in vitro new approach methods data.Objectives:Considering the physiological relevance of human organotypic culture models and their utility for high-throughput screening, we hypothesized that multidimensional chemical-biological profiling of chemicals and their major metabolites is a sensible alternative for the toxicological characterization of parent molecules vs. metabolites in vitro.Methods:In this study, we tested 25 polychlorinated biphenyls (PCBs) [PCB 3, 11, 52, 126, 136, and 153 and their relevant metabolites (hydroxylated, methoxylated, sulfated, and quinone)] in concentration–response () for effects in human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) and endothelial cells (ECs) (iPSC-derived and HUVECs). Functional phenotypic end points included effects on beating parameters and intracellular flux in CMs and inhibition of tubulogenesis in ECs. High-content imaging was used to evaluate cytotoxicity, mitochondrial integrity, and oxidative stress.Results:Data integration of a total of 19 physicochemical descriptors and 36 in vitro phenotypes revealed that chlorination status and metabolite class are strong predictors of the in vitro cardiovascular effects of PCBs. Oxidation of PCBs, especially to di-hydroxylated and quinone metabolites, was associated with the most pronounced effects, whereas sulfation and methoxylation of PCBs resulted in diminished bioactivity.Discussion:Risk characterization analysis showed that although in vitro derived effective concentrations exceeded the levels measured in the general population, risks cannot be ruled out due to the potential for population variability in susceptibility and the need to fill data gaps using read-across approaches. This study demonstrated a strategy for how in vitro data can be used to characterize human health risks from PCBs and their metabolites. https://doi.org/10.1289/EHP7030

Detection of Drug-Induced Torsades de Pointes Arrhythmia Mechanisms Using hiPSC-CM Syncytial Monolayers in a High-Throughput Screening Voltage Sensitive Dye Assay.

We validated 3 distinct hiPSC-CM cell lines-each of different purity and a voltage sensitive dye (VSD)-based high-throughput proarrhythmia screening assay as a noncore site in the recently completed CiPA Myocyte Phase II Validation Study. Blinded validation was performed using 12 drugs linked to low, intermediate, or high risk for causing Torsades de Pointes (TdP). Commercially sourced hiPSC-CMs were obtained either from Cellular Dynamics International (CDI, Madison, Wisconsin, iCell Cardiomyoyctes2) or Takara Bio (CLS, Cellartis Cardiomyocytes). A third hiPSC-CM cell line (MCH, Michigan) was generated in house. Each cell type had distinct baseline electrophysiological function (spontaneous beat rate, action potential duration, and conduction velocity) and drug responsiveness. Use of VSD and optical mapping enabled the detection of conduction slowing of sodium channel blockers (quinidine, disopyramide, and mexiletine) and drug-induced TdP-like activation patterns (rotors) for some high- and intermediate-risk compounds. Low-risk compounds did not induce rotors in any cell type tested. These results further validate the utility of hiPSC-CMs for predictive proarrhythmia screening and the utility of VSD technology to detect drug-induced APD prolongation, arrhythmias (rotors), and conduction slowing. Importantly, results indicate that different ratios of cardiomyocytes and noncardiomyocytes have important impact on drug response that may be considered during risk assessment of new drugs. Finally, we present the first blinded CiPA hiPSC-CM validation results to simultaneously detect drug-induced conduction slowing, action potential duration prolongation, action potential triangulation, and drug-induced rotors in a proarrhythmia assay.

Defining the Limits of Stem-Cell Derived Cardiomyocytes (SC-CMS) to Detect Cardiac Proarrhythmic Liabilities

Extracellular Field Potential (FP) and impedance recordings of SC-CMs are currently used to detect drug induced cardiac arrhythmia liabilities. Major endpoints include the field potential duration (FPD), the duration of the impedance twitch, and the presence of proarrhythmic markers including early and delayed afterdepolarizations and fibrillations. We used the cardioECR instrument (ACEA Biosciences) and iCell2 cardiomyocytes (Cellular Dynamics International) to 1) identify the FP parameter that best captures the intrinsic frequency-dependence of repolarization and, 2) assess the extent the system can detect the mitigating effect of drugs with multiple ion channels effects (MICE) on proarrhythmic activity. Cardiac repolarization in FP recordings is encapsulated by one positive (T3) and two negative (T1, T2) peaks. We detected a linear relationship with comparable slopes of 0.133 and 0.129 respectively (n=2) between T2 and T3 durations and beat period in the spontaneous frequency range. T1 shows a shallower slope (0.031) suggesting that the parameter does not follow closely the repolarization process and consistent with its limited response to drugs that prolong the FPD. MICE effects were investigated by assessing the effect of different combinations of pure hERG (E-4031) and hCav1.2 (nifedipine) channel blockers designed to simulate distinct IC50 hERG/IC50 hCav1.2 ratios (HCR). The functional relationship between the number of wells showing proarrhythmic markers and log (HCR) was fitted to a logistic function with an inflection point at −0.148 and a HCR of 0.71. Notably, this ratio is comparable to the 0.89 cutoff for torsadogenic drugs in the clinic (Kramer, Obejero-Paz et al., 2013). SC-CMs are useful tools to investigate the cardiac risks of drugs by using T2 and T3 to evaluate changes in cardiac repolarization and HCR values (≤ 0.71) to determine proarrhythmic activity.

Multielectrode array recordings of human iPSC-derived neurons reveal differences in network activity depending on differentiation protocol and genome modification

Event Abstract Back to Event Multielectrode array recordings of human iPSC-derived neurons reveal differences in network activity depending on differentiation protocol and genome modification Sandra K. Fischer1* and Frank Gillardon1 1 Boehringer Ingelheim (Germany), Germany With increasing prevalence of neuropsychiatric disorders, there is an ever-growing need for suitable model systems to understand the underlying pathological mechanisms. Currently most data are derived from animal models, although correlations between animal and human are often weak. However, acquisition of human neuronal material is rarely possible and afflicted with ethic concerns. Human induced pluripotent stem cell (hiPSC) derived neuronal cultures offer a possibility to overcome these limitations. Synaptic activity and neurotransmission deficits have been identified as core pathophysiologies of neuropsychiatric disorders. The multi electrode array (MEA) technology allows for repeated measurements of electrical activity of functional neuronal networks over an extended period of time. We cultivated iPSC-derived neuronal cultures on the Multichannel Systems MEA multiwell platform and assessed synaptic activity over time and in response to treatment with pharmacological agents. The hiPSC-derived neurons developed electrical activity from single spikes to bursts over time indicating maturation. Dot-seeded GlutaNeurons (iCell Neurons, Cellular Dynamics International) showed their peak activity after 2-3 weeks in culture with the highest burst activity in week 3. Ngn2-induced Neurons (iCell Neurons, Cellular Dynamics International) displayed a weaker total activity with lower spike rate, less burst activity and a maximum activity after 6 weeks in culture. Treatment with the GABA antagonist Bicuculline evoked synchronized burst firing in GlutaNeuron cultures containing approx. 20% GABAergic neurons, whereas the spontaneous activity of the purely glutamatergic culture of Induced Neurons was unchanged as expected. Loss-of-function mutations in ErbB receptor tyrosine kinases have been identified as genetic risk factors for neuropsychiatric diseases. Upon pharmacological inhibition of ErbB receptor tyrosine kinases, neuronal network activity was inhibited, consistent with our MEA recordings from primary rat cortical neuron cultures. Moreover, we generated a loss-of-function mutation in a high risk gene for schizophrenia in hiPSC from a healthy donor utilizing the CRISPR/Cas9 gene editing system. Following neuronal differentiation into the forebrain lineage electrophysiological activity was monitored on the MEA multiwell system during network formation and maturation. Preliminary data indicate altered spontaneous network activity in mutant iPSC-derived neurons compared to isogenic controls. In summary, our findings demonstrate that hiPSC-derived neurons form functional neuronal networks on MEA plates which can serve as suitable system for modeling of neuropsychiatric diseases. Acknowledgements The authors thank Dr. Sabine Lange (Cellular Dynamics International) for helpful discussions. Keywords: IPSC-derived neurons, neuropsychiatric disorder, electrical network activity, multielectrode array plate, CRISPR/Cas9 gene editing Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018. Presentation Type: Poster Presentation Topic: Stem cell-derived applications Citation: Fischer SK and Gillardon F (2019). Multielectrode array recordings of human iPSC-derived neurons reveal differences in network activity depending on differentiation protocol and genome modification. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00015 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Mar 2018; Published Online: 17 Jan 2019. * Correspondence: Mrs. Sandra K Fischer, Boehringer Ingelheim (Germany), Hannover, Germany, sandra.fischer@boehringer-ingelheim.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Sandra K Fischer Frank Gillardon Google Sandra K Fischer Frank Gillardon Google Scholar Sandra K Fischer Frank Gillardon PubMed Sandra K Fischer Frank Gillardon Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

ELECTROPHYSIOLOGICAL PHENOTYPE CHARACTERIZATION OF HUMAN IPSC-DERIVED DOPAMINERGIC NEURONAL LINES BY MEANS OF HIGH-RESOLUTION MICROELECTRODE ARRAYS

Event Abstract Back to Event ELECTROPHYSIOLOGICAL PHENOTYPE CHARACTERIZATION OF HUMAN IPSC-DERIVED DOPAMINERGIC NEURONAL LINES BY MEANS OF HIGH-RESOLUTION MICROELECTRODE ARRAYS Michele Fiscella1, 2*, Noelle Leary1, Ebru Acun1, Silvia Ronchi1 and Andreas Hierlemann1 1 ETH Zürich, Switzerland 2 MaxWell Biosystems, Switzerland High-resolution-microelectrode-array (MEA) technology enables to study neuronal dynamics at different scales, ranging from axonal physiology to network connectivity (Müller et. al, Lab on a Chip, 2015). We have used this MEA technology to characterize and compare the electrical phenotypes of commercially available human dopaminergic neurons (iCell DopaNeurons, MyCell DopaNeurons A53T α-synuclein, Cellular Dynamics International, Madison, WI, US). Furthermore, we have studied the effect of human astrocytes (iCell Astrocytes, Cellular Dynamics International, Madison, WI, US) on neural culture development. Astrocyte/neuron co-cultures showed higher signal amplitudes and higher firing rates than neural cultures without astrocytes. Adding astrocytes to neural cultures changed the whole culture morphology by promoting cell clustering. Interestingly, astrocyte/neuron co-cultures showed a lower sample-to-sample variability across multiple MEA recording sessions compared to neural cultures without astrocytes. We compared velocities of action potential propagation along axons between dopaminergic A53T α-synuclein neurons and the wild-type isogenic control cell line. We found that in both, wild-type and disease-model neurons, axonal action potential propagation velocities were lower than, for example, in rat primary cortical neurons (Bakkum et. al, Nature Communications, 2013). Furthermore, we found different axonal action-potential-velocity development profiles of A53T α-synuclein dopaminergic neurons and the wild-typecell line. Finally, we were able to precisely and reproducibly evoke action potentials in individual single human IPSC-derived neurons through subcellular-resolution electrical stimulation. High-resolution MEA systems enable to access novel electrophysiological parameters of iPSC-derived neurons, which can be potentially used as biomarkers for phenotype screening and drug testing. Acknowledgements EU, ERC Advanced Grant “neuroXscales” contract number 694829 EU, ERC-PoC “MwHresEP" contract number 755383 CH, Project CTI-No. 25933. 2 PFLS-LS “Multi-well electrophysiology platform for high-throughput cell-based assays” References J. Müller, M. Ballini, P. Livi, Y. Chen, M. Radivojevic, A. Shadmani, V. Viswam, I. L. Jones, M. Fiscella, R. Diggelmann, A. Stettler, U. Frey, D. J. Bakkum, A. Hierlemann, "High-resolution CMOS MEA platform to study neurons at subcellular, cellular, and network levels", Lab Chip, 2015, 15, pp. 2767-2780 (DOI: 10.1039/C5LC00133A) Douglas J. Bakkum, Urs Frey, Milos Radivojevic, Thomas L. Russell, Jan Müller, Michele Fiscella, Hirokazu Takahashi, Andreas Hierlemann, "Tracking Axonal Action Potential Propagation on a High-density Microelectrode Array across Hundreds of Sites", Nature Communications, 2013, 4:2181 (DOI: 10.1038/ncomms3181). Keywords: CMOS-MEAs, IPSC-derived neurons, Parkinson Disease, microelectrode arrays, phenotyping Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018. Presentation Type: Poster Presentation Topic: Stem cell-derived applications Citation: Fiscella M, Leary N, Acun E, Ronchi S and Hierlemann A (2019). ELECTROPHYSIOLOGICAL PHENOTYPE CHARACTERIZATION OF HUMAN IPSC-DERIVED DOPAMINERGIC NEURONAL LINES BY MEANS OF HIGH-RESOLUTION MICROELECTRODE ARRAYS. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00014 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 05 Mar 2018; Published Online: 17 Jan 2019. * Correspondence: Dr. Michele Fiscella, ETH Zürich, Zurich, Switzerland, michele.fiscella@mxwbio.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Michele Fiscella Noelle Leary Ebru Acun Silvia Ronchi Andreas Hierlemann Google Michele Fiscella Noelle Leary Ebru Acun Silvia Ronchi Andreas Hierlemann Google Scholar Michele Fiscella Noelle Leary Ebru Acun Silvia Ronchi Andreas Hierlemann PubMed Michele Fiscella Noelle Leary Ebru Acun Silvia Ronchi Andreas Hierlemann Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Induced Pluripotent Stem Cells: Inventors turning into competitors

Induced Pluripotent Stem Cells (IPSCs) are a kind of adult cells that have been genetically reprogrammed to become different cell types. Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with embryonic stem cells. IPSCs technology was pioneered by Shinya Yamanaka from Kyoto University. This breakthrough has inspired researchers to start working around IPSC technology. James Thomson from University of California has developed IPS cell lines derived from Human Somatic Cells. Subsequently, he also established a large scale human IPSC manufacturing company named Cellular Dynamics International. Thus, increasing interest in the commercial exploitation of IPSCs patents has lead us to look into the patent portfolios of top three patent assignees in IPSC technology. In this study, we have discussed technological patent trends and multiple factors which reflect the competitive scenario between the top assignees of IPSC technology. Our conclusions suggest that Kyoto University led by inventor Shinya Yamanaka is the leader of IPSC technology. However, patent-product linkage analysis suggests that Cellular Dynamics International led by inventor James Thomson may surpass Kyoto University in near future.

Characterization of GABAA receptor ligands with automated patch-clamp using human neurons derived from pluripotent stem cells

IntroductionAutomated patch clamp is a recent but widely used technology to assess pre-clinical drug safety. With the availability of human neurons derived from pluripotent stem cells, this technology can be extended to determine CNS effects of drug candidates, especially those acting on the GABAA receptor. MethodsiCell Neurons (Cellular Dynamics International, A Fujifilm Company) were cultured for ten days and analyzed by patch clamp in the presence of agonist GABA or in combination with positive allosteric GABAA receptor modulators. Both efficacy and affinity were determined. In addition, mRNA of GABAA receptor subunits were quantified by qRT-PCR. ResultsWe have shown that iCell Neurons are compatible with the IonFlux microfluidic system of the automated patch clamp instrument. Resistance ranging from 15 to 25MΩ was achieved for each trap channel of patch clamped cells in a 96-well plate format. GABA induced a robust change of current with an EC50 of 0.43μM. Positive GABAA receptor modulators diazepam, HZ-166, and CW-04-020 exhibited EC50 values of 0.42μM, 1.56μM, and 0.23μM, respectively. The α2/α3/α5 selective compound HZ-166-induced the highest potentiation (efficacy) of 810% of the current induced by 100nM GABA. Quantification of GABAA receptor mRNA in iCell Neurons revealed high levels of α5 and β3 subunits and low levels of α1, which is similar to the configuration in human neonatal brain. DiscussioniCell Neurons represent a new cellular model to characterize GABAergic compounds using automated patch clamp. These cells have excellent representation of cellular GABAA receptor distribution that enable determination of total small molecule efficacy and affinity as measured by cell membrane current change.

Abstract 29: Porcine Decellularized Heart Tissue Enhance the Expression of Contractile Proteins in Human Cardiomyocytes and Differentiated Cardiac Progenitor Cells

Tissue engineering is an evolving tool for repair of cardiovascular damage, in particular after myocardial infarction. Currently, most studies are focused on providing gel or thin synthetic matrices to provide structural support for injured valves, or ventricle. But little is known about how cardiac cells (eg, endothelial cells [ECs], cardiomyocytes [CMs] and cardiac progenitor cells [CPCs]) are modified by an exposure to a scaffold much less how a xenogeneic scaffold may affect cell physiology or function. Our study was designed to examine the effect of rat or pig collagen matrix and decellularized left ventricle extracellular matrix (dECM) tissues on the phenotype of human cardiac cells. Human ECs, CMs and CPCs derived from IPs cells (Cellular Dynamics International) were cultured singly or together on rat or pig collagen matrices or on dECM. Protein and gene expression was investigated by immunohistochemistry, histology and real time PCR. All 3 cell types survived on both types of support when delivered singly or together. Endothelial cell PECAM-1 expression was enhanced on decellularized tissues particularly on rat matrix. Cardiomyocyte contractile protein expression did not differ with support type, whereas CPC contractile protein gene expression increased on both decellularized tissues more so on pig matrix. Additionally, when the cells were co-cultured, single cell type gene expression was modified. Our study showed that human cardiac cells are influenced by the structural support on which they are cultivated. Cardiac ECM enhanced the expression of contractile proteins and endothelial markers but matrix species had minimal influence on this effect. Furthermore, cell co-culture altered gene expression suggesting cell communication is intact on the matrix. This study shows that cell phenotype and behavior is impacted by the tissue matrix to a greater degree than occurs with a simple one protein support and suggests complex decellularized matrix may be superior to successfully engineer cardiac constructs with intact physiology. Additionally, the successful differentiation of IPs cells into cardiomyocytes on decellularized matrix opens avenue to build tissue engineer constructs from patients with heart disease as a screening tool.

Abstract 425: Induced Pluripotent Stem Cell Derived Cardiomyocyte Tissue Engineered Scaffold Improves Left Ventricular Function and Electro-Mechanical Coupling in Rats with Heart Failure

Background: Chronic Heart Failure (CHF) is the leading cause of hospital readmissions in the United States. It may result from systolic or diastolic dysfunction, which often coexists. Here we report the effects of delivering human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) via a bioengineered patch on left ventricular function in rats with CHF. Methods: Adult male Sprague-Dawley rats underwent left coronary artery ligation and were randomized to Sham, CHF, and hiPSC-CM patch. High purity human hiPSC-CMs were obtained from Cellular Dynamics International, seeded and co-cultured onto a vicryl matrix embedded with human dermal fibroblasts. Echocardiography was performed at 3 and 6 weeks post-randomization. Hemodynamic pressure measurements were performed at 6 weeks post-ligation with Millar solid state micromanometer catheters. Open chest Electrophysiologic (EP) mapping was performed at 6 weeks post ligation. Results: Patches constructed with hiPSC-CMs displayed synchronized and spontaneous contractions within 48hrs of culture which developed in robustness over time. At maximal robustness, contractions were visualized across the full thickness of the construct. Contractions were recorded at 36+5 beats BPM. Three weeks after implantation, the hiPSC-CM patch decreased LV EDP (45%), Tau (29%), E/e’ (23%) and increased, EF (14%), e’ (20%), and e’/a’ (36%) versus CHF. EP studies show electro-mechanical coupling between the patch and the native myocardium with normal activation through the patch and increases (P<0.05) voltage amplitude in CHF versus hiPSC-CM patch treated rats (1±0.5 mV vs 6±1.5mV). Conclusion: Cardiac patch implantation with human iPSC derived cardiomyocytes is an effective and feasible method of treating CHF with improvements in systolic function, diastolic function, and electro-mechanical coupling in rats with CHF.

Characterization of Neurons Derived from a Human Induced Pluripotent Stem Cell Line Using Patch‐Clamp Electrophysiology

Neuropharmacological studies using animal‐derived tissues are not always directly translatable to humans, and the occurrence of discrepancies can be difficult to predict. Sources of human neurons have been limited in the past, but developments in stem cell science are now providing new opportunities to generate human neurons from stem cells. iCell neurons are derived from a human induced pluripotent stem cell (hIPSC) line and may have the potential to replace animal neurons in neuropharmacological studies. Here, we have explored the neurophysiological and neuropharmacological properties of iCell neurons using immunocytochemistry and single‐cell patch‐clamp techniques.Whole cell recordings were obtained from iCell neurons cultured for up to 4 months in vitro. Functional expression of voltage‐gated sodium and potassium ion channels, as well as ligand‐gated GABAA, glutamate, NMDA, AMPA, kainate and glycine receptors was determined. Spontaneous and evoked action potentials were inhibited by the sodium channel blocker tetrodotoxin, and spontaneous activity levels increased over time in culture. Immunocytochemical analysis revealed a corresponding increase in neurite density over time. iCell neurons therefore appear to have several important functional properties consistent with human neurons and might be a viable alternative to animal neurons in drug studies.Research support was provided by Cellular Dynamics International and the Department of Physiology and Pharmacology at the University of the Pacific.

Efficient and cost-effective batch analysis of multi-electrode array (MEA) data from human iPS cardiomyocytes using Intelligent Waveform Service (IWS)

• Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) with multi-electrode array (MEA) technology could be used to develop assays for predictive electrophysiological safety screening. • Large volumes of MEA data are generated and analysis using existing tools is time consuming. There is a need to improve timeliness and cost-effectiveness of data reporting. • Neural ID’s Intelligent Waveform Service (IWS) is propriety enterprise software that uses pattern-based learning of waveforms. • IWS was used to analyze MEA data generated from hiPSC-CMs treated with Terfenadine and results were compared to existing analysis tools. • We show that analysis and reporting times using IWS can be reduced by up to five times and employing pattern recognition technology will improve accuracy and facilitate greater use of MEAs in compound screening. Methods Results • The non-sedating anti-histamine Terfenadine was chosen to validate the IWS software. • Data was recorded from spontaneously beating hiPSCCMs (Cellular Dynamics International) using the MEA1060 system (Multi Channel Systems). Data was filtered (1Hz high pass, 3kHz low pass) and sampled at 10kHz. • After baseline recordings, Terfenadine was cumulatively added to the bath. The last minute of recording at baseline and each Terfenadine concentration was used for analysis. • IWS was used to analyze the following parameters: Field potential duration (FPD), total spike amplitude, beat rate and conduction time. • Data analysis is conducted on the server using templates which are batch processed for efficient resource utilization. • Gradient markers are defined to determine spike interval and context of targeted patterns. • Based on the marker locations, patterns are defined graphically (Fig. 2) to detect repolarization wave location and measure its peak value. • IWS creates an empirical data model and does not rely on a predetermined library of patterns, leading to higher recognition rates than industry standards and the ability to analyze a broader range of data. • By simply clicking and dragging, additional pattern examples can be added to the knowledge database (Fig. 3). • The same process is used to detect other types of patterns, such as spikes, with amplitude (maximum/minimum) measurements applied (Fig. 4). Intelligent Waveform Service (IWS) IWS is proprietary enterprise software (Fig. 1) that: • Leverages machine learning to provide waveform analytics of biosignals. • Automates and accelerates discovery & preclinical research, providing more complete results at a fraction of the time and cost. • Learns example patterns, then runs batch jobs against multiple file sets and data formats, making analytics shareable among researchers and groups. Figure 1 – IWS Architecture Figure 4 – Detecting Spike Patterns and Measuring Amplitude Figure 3 – Adding Pattern Examples to the Template Figure 2 – Pattern Creation and Metrology Set-Up

Abstract 2801: The use of induced pluripotent stem (iPS) cells for the safety testing of enhanced affinity TCR-transduced T cells

Abstract Engineering T cells with enhanced affinity T cell receptors (TCRs) is a promising therapeutic strategy for treating cancer. Engineered TCRs overcome the limitations of naturally occurring cancer-antigen specific TCRs which are typically of extremely low affinity and fail to efficiently recognize tumor cells. However, the use TCRs with enhanced affinity for adoptive therapy carries with it the risk that they might recognize normal tissue, either as on-target or off-target reactivity. Two fatalities in a clinical trial of an affinity-enhanced MAGE-A3-specific TCR have highlighted the need for more biologically relevant cellular testing systems to minimize cross-reactivity-related toxicity in future adoptive immunotherapy trials. Cellular Dynamics International Inc. supply a range of iCell products that are highly pure induced pluripotent stem (iPS) cell-derived cells that reproduce normal human biology in an in vitro setting. The advantage of iCell cultures over standard cultures of primary cells is that they better represent human organs with respect to their antigen presentation by HLA. In this study, a panel of iCell sub-types, representing major organs of the human body, were used to test the reactivity of T cells transduced with an enhanced affinity TCR for HLA-A2-restricted peptide antigen NY-ESO-1/LAGE-1157-165. IncuCyte FLR technology, which enables direct visualization of caspase-3/7-dependent apoptosis at 37°C in real time, was used to assess the killing of the iCells by NY-ESO TCR-transduced T cells over a 4 day time course. While potent killing by the NY-ESO TCR-transduced T cells was observed against an HLA-A2+, NY-ESO-1+/LAGE-1+ tumor cell line, A375, no reactivity was observed against any of the tested iCells sub-types unless the iCells were pulsed with 1 µM NY-ESO peptide. The screening of TCR-transduced T cells against biologically appropriate cell cultures, such as iCells, further advances preclinical safety testing of new TCRs for use in adoptive immunotherapy. Citation Format: Katherine J. Adams, Andrew B. Gerry, Joanna E. Brewer, Nicholas J. Pumphrey, Alan D. Bennett, Bent K. Jakobsen. The use of induced pluripotent stem (iPS) cells for the safety testing of enhanced affinity TCR-transduced T cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2801. doi:10.1158/1538-7445.AM2014-2801

Minimized Cell Usage for Stem Cell-Derived and Primary Cells on an Automated Patch Clamp System

Abstract Introduction Chip-based automated patch clamp systems are widely used in drug development and safety pharmacology, allowing for high quality, high throughput screening at standardized experimental conditions. The merits of automation generally come at the cost of large amounts of cells needed, since cells are not targeted individually, but randomly positioned onto the chip aperture from cells in suspension. While cell usage is of little concern when using standard cell lines such as CHO or HEK cells, it becomes a crucial constraint with cells of limited availability, such as primary or otherwise rare and expensive cells, like induced pluripotent stem (IPS) cell-derived cardiomyocytes or neurons. Methods We established application protocols for CHO cells, IPS cell-derived neurons (iCell® Neurons, Cellular Dynamics International), cardiomyocytes (Cor.4U®, Axiogenesis) and pancreatic islet cells, minimizing cell usage for automated patch clamp recordings on Nanion's Patchliner. Use of 5 μl cell suspension per well for densities between 55,000 cells/ml and 400,000 cells/ml depending on cell type resulted in good cell capture. Results We present a new cell application procedure optimized for the Patchliner achieving > 80% success rates for using as little as 300 to 2000 cells per well depending on cell type. We demonstrate that this protocol works for standard cell lines, as well as for stem cell-derived neurons and cardiomyocytes, and for primary pancreatic islet cells. We present recordings for these cell types, demonstrating that high data quality is not compromised by altered cell application. Discussion Our new cell application procedure achieves high success rates with unprecedentedly low cell numbers. Compared to other standard automated patch clamp systems we reduced the average amount of cells needed by more than 150 times. Reduced cell usage crucially improves cost efficiency for expensive cells and opens up automated patch clamp for primary cells of limited availability.

Minimized cell usage for stem cell-derived and primary cells on an automated patch clamp system

IntroductionChip-based automated patch clamp systems are widely used in drug development and safety pharmacology, allowing for high quality, high throughput screening at standardized experimental conditions. The merits of automation generally come at the cost of large amounts of cells needed, since cells are not targeted individually, but randomly positioned onto the chip aperture from cells in suspension. While cell usage is of little concern when using standard cell lines such as CHO or HEK cells, it becomes a crucial constraint with cells of limited availability, such as primary or otherwise rare and expensive cells, like induced pluripotent stem (IPS) cell-derived cardiomyocytes or neurons. MethodsWe established application protocols for CHO cells, IPS cell-derived neurons (iCell® Neurons, Cellular Dynamics International), cardiomyocytes (Cor.4U®, Axiogenesis) and pancreatic islet cells, minimizing cell usage for automated patch clamp recordings on Nanion's Patchliner. Use of 5μl cell suspension per well for densities between 55,000cells/ml and 400,000cells/ml depending on cell type resulted in good cell capture. ResultsWe present a new cell application procedure optimized for the Patchliner achieving>80% success rates for using as little as 300 to 2000 cells per well depending on cell type. We demonstrate that this protocol works for standard cell lines, as well as for stem cell-derived neurons and cardiomyocytes, and for primary pancreatic islet cells. We present recordings for these cell types, demonstrating that high data quality is not compromised by altered cell application. DiscussionOur new cell application procedure achieves high success rates with unprecedentedly low cell numbers. Compared to other standard automated patch clamp systems we reduced the average amount of cells needed by more than 150 times. Reduced cell usage crucially improves cost efficiency for expensive cells and opens up automated patch clamp for primary cells of limited availability.

Evaluation of Contractile Activity of Stem Cell-Derived Human Cardiomyocytes using Impedance Analysis

Single stem cell-derived human cardiomyocyte (SC-hCM) electrophysiolo-gy is a valuable tool to assess cardiac risk associated with drugs (Peng 2010, Ma 2011). However, impedance-based measurement of SC-hCM contractile activity (Roche xCELLigence RTCA Cardio system) adds a new dimension to cardiac risk assessment as events downstream of excitation-contraction coupling can now be investigated in a higher throughput assay. SC-hCMs show spontaneous contractile activity resulting in transient changes in impedance (impedance twitches). We performed a series of pharmacological studies to investigate the mechanisms underlying this transient behavior in iCELL cardiomyocytes (Cellular Dynamics International). Here we show that impedance twitches are dependent on calcium entry since calcium channel blockers (verapamil, nifedipine) and agonists (FPL64176) decrease and increase twitch amplitude, respectively. Twitch frequency is slowed by the HCN channel blocker ZD7288 (33±8% @ 300 nM), a phenomenon associated with prolongation of relaxation by 26±7% (n=5, p<0.05), but independent of changes in amplitude. β-adrenergic stimulation increases twitch frequency and amplitude, and shortens the time to peak and twitch relaxation time, consistent with results from other cardiac preparations. Impedance twitch activity reflects contractile activity since it is completely obliterated by the myosin II inhibitor blebbistatin at concentrations that do not affect the shape or frequency of the spontaneous action potentials. These results suggest that several aspects of the excitation-contraction coupling mechanism observed in adult cardiomyocytes are intact in SC-hCMs, including the dependence of the twitch contraction on Ca2+ entry and the regulation of contraction by frequency and β-adrenergic stimulation.

Understanding hepatotoxicity – from patients to mice to computer

Drug Induced Liver Injury (DILI) remains the major adverse drug event that leads to termination of clinical development programs and regulatory actions including failure to approve for marketing, restricted indications, and withdrawal from the marketplace. The type of DILI that is most problematic is “idiosyncratic” meaning that only a very small fraction of treated patients are susceptible to the DILI. Current preclinical models, even “humanized” ones, do not reliably identify molecules that have this liability and, conversely predict liabilities in molecule that are in fact quite safe for the liver. For example, current preclinical testing does not detect the DILI potential of xymelagatran, a drug withdrawn from worldwide markets due to idiosyncratic hepatotoxicity but would predict a high liver risk for acetaminophen (paracetamol) even when taken as directed. Reliable preclinical testing will probably not be developed until there is greater understanding of the mechanisms underlying DILI. Reasoning that the best models to study DILI are the people who have actually experienced it, we are capitalizing on the resources of the US Drug-Induced Liver Injury Network (DILIN-supported by the National Institutes of Health) that is collecting genomic DNA and other biospecimens from patients who have experienced DILI. In addition to genetic studies, we are partnering with Cellular Dynamics International to reprogram induced pleuripotent stem cells from DILIN subjects in whom we have complete exomic DNA sequence with the goal of producing patient-specific liver cultures, and ultimately, humanized mice. We are also partnering with the Shanghai Centers for Disease Control to prospectively collect a variety of biospecimens, including whole blood for transcriptome analysis, from patients treated for active tuberculosis as part of a larger biomarker discovery initiative. We are using panels of inbred mice to mimic patient population genetic diversity and to identify genes and pathways that may underlie DILI susceptibility in patients. Finally, we have established the DILIsim Initiative which is a public-private partnership that is building a computer model to synthesize the rapidly accumulating data with the goal of predicting DILI liability in drug candidates. DILIsim involves 10 pharmaceutical companies and the Food and Drug Administration.

Novel Application of Human Neurons Derived from Induced Pluripotent Stem Cells for Highly Sensitive Botulinum Neurotoxin Detection

Human induced pluripotent stem cells (hiPSC) hold great promise for providing various differentiated cell models for in vitro toxigenicity testing. For Clostridium botulinum neurotoxin (BoNT) detection and mechanistic studies, several cell models currently exist, but none examine toxin function with species-specific relevance while exhibiting high sensitivity. The most sensitive cell models to date are mouse or rat primary cells and neurons derived from mouse embryonic stem cells, both of which require significant technical expertise for culture preparation. This study describes for the first time the use of hiPSC-derived neurons for BoNT detection. The neurons used in this study were differentiated and cryopreserved by Cellular Dynamics International (Madison, WI) and consist of an almost pure pan-neuronal population of predominantly gamma aminoisobutyric acidergic and glutamatergic neurons. Western blot and quantitative PCR data show that these neurons express all the necessary receptors and substrates for BoNT intoxication. BoNT/A intoxication studies demonstrate that the hiPSC-derived neurons reproducibly and quantitatively detect biologically active BoNT/A with high sensitivity (EC(50) ∼0.3 U). Additionally, the quantitative detection of BoNT serotypes B, C, E, and BoNT/A complex was demonstrated, and BoNT/A specificity was confirmed through antibody protection studies. A direct comparison of BoNT detection using primary rat spinal cord cells and hiPSC-derived neurons showed equal or increased sensitivity, a steeper dose-response curve and a more complete SNARE protein target cleavage for hiPSC-derived neurons. In summary, these data suggest that neurons derived from hiPSCs provide an ideal and highly sensitive platform for BoNT potency determination, neutralizing antibody detection and for mechanistic studies.