Track 1: Safety Toxicology
10:45 am |
Holistic View on State-of-the-art Cardiac Safety Screening
Elena Dragicevic, Nanion Technologies
Elena Dragicevic, PhD, PhD
Senior Scientist / Sales Manager
Electrophysiology techniques, such as high-throughput automated patch-clamp and extracellular field potential recordings, coupled with optogenetic tools, have become a standard in cardiac safety pharmacology. Here, we present the development and optimization of various systems and their applications not only for safety pharmacology, such as the Comprehensive In-vitro Proarrhythmia Assay (CiPA) initiative, but also for benefits in toxicity screens of adherent, proliferating or non-proliferating cells. We focus on assays which yield reproducible concentration-dependent effects of compounds using impedance measurements in combination with extracellular field potential (EFP) recordings. The changes in the impedance signal indicate effects on cell contractility, cell morphology and proliferation over prolonged periods of time, giving a crucial advantage of this technique over standard cytotoxicity assays as it allows continual monitoring of the development of cytotoxicity. We will show data obtained from testing the concentration dependent effects of chemotherapeutic drugs on cancer cells and their proliferation patterns. Furthermore, hiPS-derived hepatocytes or monocyte-derived hepatocytes were investigated and outcomes were compared to standard hepatotoxicity assays. Our data and technical innovations strengthen the importance of testing compounds in assays complementary to patch clamp electrophysiology, to provide an all-inclusive safety and toxicity compound profile.
11:15 am |
Evaluation of Potential TCR-mediated Neurotoxicity Using iPS Cells
Maja Bürdek, Medigene Immunotherapies
Maja Bürdek, PhD
Senior Scientist / Sales Manager
Medigene Immunotherapies, GmbH
Clinical studies using TCR-transgenic T cells for adoptive T cell therapy revealed the efficacy of this therapeutic approach but also uncovered possible toxic effects against healthy tissues due to on-target/off-tumor or off-target toxicity (Morgan et al., J Immunother. 2013; Linette et al., Blood 2013). Therefore, relevant models for prediction of potential TCR-mediated toxicity against healthy tissues need to be developed.
Here we describe the use of neurons and astrocytes derived from iPS cells for evaluation of potential TCR-mediated neurotoxicity in vitro. iPS-derived cells served as target cells in functional co-culture assays with CD8+ T cells transduced with an HLA-A2-restricted, PRAME-specific TCR. To show general susceptibility to recognition and killing via T cells, the iPS-derived cells were exogenously loaded with PRAME peptide or co-cultured with T cells transduced with an allo-HLA-A2-reactive TCR. Recognition of target cells via T cells was measured via IFN-γ release of T cells and detection of apoptotic target cells in a real-time killing assay. Interestingly, iPS-derived neurons only showed cell surface expression of HLA-A2 after IFN-γ treatment and their peptide loading did not seem to work as efficiently as for iPS-derived astrocytes. Importantly, neither iPS-derived cell type triggered activation of PRAME TCR-transduced T cells.
In summary, we showed that functional assays of astrocytes and neurons derived from iPS cells represent an elegant approach to assess the potential risk of TCR-mediated neurotoxicity in vitro.
11:45 am |
Lunch (provided), Poster Session
1:15 pm |
Excitation-Contraction Coupling Plasticity in Pluripotent Stem Cell-Derived Cardiac Myocytes
Cesare Terracciano, Imperial College of London
Cesare Terracciano, MD, PhD
Professor of Cardiac Electrophysiology
Imperial College of London
Calcium regulation by the sarcoplasmic reticulum (SR) is a fundamental property of heart muscle that ensures efficient excitation-contraction (EC) coupling. SR calcium cycling is fully functional in the adult healthy myocardium but poorly utilised during development and disrupted during cardiac disease, indicating that SR contribution is a highly regulated and plastic function. Leveraging on the naïve and plastic properties of human induced pluripotent stem cell-derived cardiac myocytes (iPSC-CMs), we studied the effects of multicellular patterns and extracellular matrix (ECM) on the development of SR calcium cycling. We found that culture with human fibroblasts affects the EC coupling machinery in iPSC-CMs. Our data suggest the importance of heterocellularity and the ECM in the development of specialised features, particularly EC coupling, of adult cardiac muscle in naïve cardiac cells. This is not only relevant for the applications of iPSC-CMs in translational medicine and cardiovascular research but also to understand and target the plasticity of the EC coupling machinery in physiological conditions and during cardiac disease.
1:45 pm |
Functional In Vitro Assessments of Drug-induced Neuronal Modulation: Comparing and Contrasting Human and Rodent Models by Multi-variate Artificial Intelligence-based Analyses.
Benjamin Bader, NeuroProof GmbH
Benjamin Bader, PhD
Business Director - Customer Projects
Using neuronal cell cultures on micro electrode arrays (MEAs) for phenotypic screening is an elegant and efficient and widely used way to explore neuro-active compounds or toxins in preclinical development. Dissociated rodent primary neuronal cell cultures derived from specific areas of the brain offer a reliable tool for analyzing brain region specific effects. Despite lacking a genetically defined or layered topology, primary neuronal cultures share many features with their source tissues, including receptors and ion channel composition, intrinsic electrical membrane properties, synaptic development and plasticity. As a result of their phenotypic receptor and cellular composition, primary rodent neuronal cell cultures show very specific and complex activity pattern after four weeks in vitro. This complexity results from a high level of organization e.g. synaptic connectivity of different neuronal types in network cultures, which is present in primary cultures and what is aimed for in human iPSC-derived neurons.
The technology of generating human induced pluripotent stem cell-derived (hiPSC) neuronal cultures is widely used to generate different phenotypes and numerous differentiation protocols exist. In order to establish physiologically relevant human in vitro culture models for drug testing and disease models, we apply artificial intelligence and machine learning methods to describe the differences and similarities of electrical functional activity patterns from primary murine neuronal cell cultures and hiPSC neuronal networks.
1) We show that primary cell cultures show brain region-specific activity pattern which can be clearly distinguished by pattern recognition methods. We describe and analyze these activity patterns with hundreds of spike train-derived parameters and show that the pattern complexity from hiPSC neuron cultures is unique and reproducible. Comparing the functional phenotypes of hiPSC neuronal networks with those of primary mouse neurons we show that iPSC neuronal networks are able to resemble certain brain-specific activity patterns depending on their differentiation protocol.
2) We show that hiPSC neurons allow compound screening for phenotypic profiling as well as for toxicity and side effect prediction (e.g. seizure) and discuss differences between mouse and human compound responses.
3) We show examples for brain-associated monogenetic disorders which can be modeled in vitro using functional phenotypic MEA readouts and that discuss exampled for rescuing these phenotypes.
In conclusion, we provide a full assay panel for drug screening, safety and disease modeling in our functional phenotypic tool box allowing using primary mouse brain cultures and hiPSC cultures to improve preclinical research and translation using mouse and human in vitro data.
2:15 pm |
Myotoxicity Evaluation Using High-content Analysis (HCA)
Anthony Perrier, Servier Laboratories
Anthony Perrier, PhD
Preclinical Project Leader, Pharmacology & Toxicology
Abstract: Hepatotoxicity and cardiotoxicity remain major causes of attrition during drug development. Therefore, research has been focused in elucidating such toxicities at early stages of drug discovery, helping decision making process, reducing late attrition, animal use and drug development costs. Depending on the candidate, other targets, such as the skeletal muscle, may show limitating toxicity and no or few specific screening tests are available for early selection. For that reason, we developed in vitro tests for muscular toxicity, focusing on mitochondrial toxicity and phospholipidosis.Fifteen reference compounds with known toxicities and various mechanisms on human skeletal muscle (global toxicity, mitotoxicity, phospholipidosis) and negative controls were tested on human iPSC myoblasts. Global toxicity, mitotoxicity and phospholipidosis were evaluated with appropriate stainings and quantified using High Content Analysis after 24 h or 72 h of exposure to 8 concentrations of each compound. The different tests were then qualified.Under the conditions of this first data set, the data obtained were considered as suitable for detecting the three evaluated end-points. Therefore, HCA on human iPSC myoblasts has been used as a screening tool for the selection of different candidates. HCA could also be used as a mechanistic tool to document a muscular toxicity emerging during drug development.
2:45 pm |
Get Ready to LEAP: Advances in Automated Arrhythmia Detection for Next-generation hiPSC-CM MEA Assays
Jim Ross, Axion Biosystems
Jim Ross, PhD.
Chief Technology Officer
Abstract: Trends in cardiac safety testing, as exemplified by CiPA and JiCSA, emphasize a move beyond surrogate measures of Proarrhythmia. The cardiomyocyte and multiwell microelectrode array (CM-MEA) assay offers an ideal approach to evaluate pre-arrhythmic indicators in vitro. Join Axion BioSystems to discuss advances in automated arrhythmia detection using Local Extracellular Action Potential (LEAP) technology to support next-generation CM-MEA assays.
Track 2: Drug Discovery
10:45 am |
Phenotypic Screening in iPSC-derived Neurons - A New Paradigm in Drug Discovery
Sandra Lubitz, Evotec A. G.
Sandra Lubitz, PhD
VP Stem Cell Biology
Evotec A. G.
Abstract: One of the major obstacles in studying neurodegenerative diseases is the difficulty in obtaining relevant cell types for analyses. The differentiation of neural cell populations from pluripotent stem cells presents an exciting opportunity to obtain large numbers of human neuronal cell types for disease modelling and drug screening. However, it is of prime importance that quality standards to the cells and assays are being defined. To realise disease modelling and drug discovery based on in vitro differentiated, patient-specific iPSC-derived cells it is necessary to develop robust, reproducible, and relevant assays with reasonable throughput. While these characteristics are essential to all small molecule screening assays, the use of patient-specific, stem cell-derived cells presents unique challenges: In addition to iPSC validation, further optimisation of in vitro differentiation protocols is required to achieve similar robustness and reproducibility as with traditional cell lines for screening campaigns. This includes use of defined media conditions, as well as adaptation and scale up to HTS assay formats. High throughput screening in iPSC-derived neurons with the intent of identifying novel therapeutic compounds has the potential to transform the way drug discovery is performed. Evotec is systematically using iPSC-derived neurons to screen for new mechanisms, targets and compounds for a number of neurodegenerative disorders, such as ALS, PD and HD.
11:15 am |
Development and Application of Cardiac Microtissues to Structural Cardiotoxicity
Caroline Archer, AstraZeneca
Caroline Archer, PhD
Senior Scientist/Cardiovascular in Vitro Specialist
Cardiotoxicity remains a major cause of attrition during drug discovery and development. There is little known about the mechanisms underlying structural cardiotoxicity, which limits the potential for developing predictive assays for early safety screening. In addition, current 2D in vitro approaches utilise monolayer stem cell derived cardiomyocytes, are limited in terms of the physiological phenotype. We have developed a 3D cardiac microtissue model (hiPS-CMs, cardiac fibroblasts and endothelial cells) that offers increased physiological relevance in a format amenable to drug discovery. These cardiac microtissues have been morphologically characterised and validated against a panel of tool structural cardiotoxins. Our presentation will describe the utility of these cardiac microtissues and the development of a high content biology assay to predict structural cardiotoxicity in a high-throughput manner.
11:45 am |
Lunch (provided), Poster Session
1:15 pm |
High-resolution Microelectrode Array Platform for Biomarker Discovery and Novel Functional Assays with Human iPSC-derived Neurons
Michele Fiscella, ETH Zurich/MaxWell Biosystems
Michele Fiscella, PhD
VP Scientific Affairs
MaxWell Biosystems AG
Microelectrode-array (MEA) technology enables label-free and high-throughput recording of cellular electrical signals. MEAs are currently used for in vitro phenotype characterization and drug toxicity/efficacy testing with iPSC-derived cells. Here we present a high-resolution microelectronics-based MEA featuring 26’400 microelectrodes within a sensing area of 3.85 mm × 2.10 mm. The electrical activity of whole cell networks can be monitored and studied at high spatio-temporal resolution. The electrical activity of single cells can be isolated and studied, together with subcellular details, such as the propagation of action potentials along single axons. The high-resolution MEA system can extract novel parameters from iPSC-derived cells, which can be used as potential biomarkers for phenotype screening and drug testing.
We performed novel functional assays in iPSC-derived midbrain floorplate dopaminergic neurons. We analyzed the electrical phenotype of an A53T α-synuclein dopaminergic neural line, modeling Parkinson's disease. We found differences in physiological activity between the A53T α-synuclein cell line and the isogenic control cell line. Phenotype differences were detected at different scales, ranging from network activity to subcellular structures as axons.
1:45 pm |
Influence of Cell Density on the Evaluation of Pharmacological Test Compounds Using MEA Technology
Alejandra Baerfuss, NMI TT GmbH
Alejandra Baerfuss, PhD Student
Electrophysiologist in Marketing and Sales
NMI TT Pharmaservices and Ruprecht-Karls-Universität Heidelberg
The contraction of cardiomyocytes within the heart is preceded by an accurately tuned electrical action potential (AP). It requires a finely timed orchestration of the different underlying ion channels. Of special interest are ion channels responsible for the precise termination of the action potential. Disturbances, e.g. due to drug induced modulation of the channels, can lead to life-threatening arrhythmia of the heart (Sanguinetti and Tristani-Firouzi, 2006). Therefore regulatory authorities such as the Food and Drug Administration (FDA) have very strict regulations regarding the approval of new drugs which are continuously updated (Food and Drug Administration, HHS, 2005).
The electrical activity of cardiomyocytes can be investigated using several technologies from the gold standard patch clamp up to microelectrode arrays (MEA). The latter consists of substrate-integrated electrodes in a chip allowing the cultivation and the non-invasive recording and evaluation of extracellular field action potentials (fAP) of electrically active cells including cardiomyocytes (Stett et al., 2003; Kraushaar et al., 2009, 2011).
The usability of human stem cell-derived cardiomyocytes (hiPS-CM) for cardiac safety pharmacology and drug approval is currently investigated by a large consortium of industrial and academic laboratories (Comprehensive in vitro proarrhythmia assessment, CiPA), led by the ILSI Health and Environmental Sciences Institute (HESI). Here, a major focus is set not only on drug-induced prolongation of the cardiac AP but furthermore on the occurrence of small irregular depolarization events like early (EAD) and delayed afterdepolarizations (DAD), respectively, which are significant risk indicators of arrhythmia induction (Sager et al., 2014).
The health of cardiomyocytes cultivated on MEA is not only determined by the correct plating and culture medium but also depends on the cell density on the surface area (Uesugi et al., 2013). As commercially available hiPS-CM are comparably cost-intensive, one tends to plate cells at the lowest possible density, especially as optimization of a MEA based test assay also includes cost optimization.
We investigated the influence of different cell densities of cultivated cardiomyocytes (Cellular Dynamics iCell cardiomyocytes2, hiPS-CM from other sources and primary cardiomyocytes) on the electrophysiological properties and sensitivity to cardioactive drugs. fAP duration was highly dependent on the plating density. iCell cardiomyocytes2 plated at densities of 9k and 18k cells/µl, respectively, displayed fAP durations of 473 ± 12 and 387± 9 ms, respectively, resulting in a difference of approximately 22%. Other commercially available hiPS-CM showed similar values, pointing towards the fact that high cell densities result in a decreased cardiac action potential duration. When plating at different densities a linear relationship between cell density and fAP duration was observed. Interestingly primary cardiomyocytes reacted inversely; here duplication of the cell density resulted in an increase of the fAP duration (~20%). Also drug sensitivity was a function of cell density. For iCell cardiomyocytes2 e.g. application of the specific hERG inhibitor E4031 at 30 nM resulted in a prolongation of 124 ± 15% at 6k and 44 ± 9% at 12k cells/µl, in line with findings for other hiPS-CMs. Furthermore, the occurrence of proarrhythmic markers was increased at lower cell densities.
Taken together these findings demonstrate the importance of highly standardized plating and cultivation of cardiomyocytes on MEA chips, especially when data needs to be compared between different labs (like in the running CiPA study). With iCell cardiomyocytes2 plated at the correct plating density, the overlap between drug induced effects and predictions was well overlapping when challenged with a drug test library, making Cellular Dynamics iCell cardiomyocytes2 a predictive and easy-to-use hiPS-CM test system.
2:15 pm |
Human iPSC-derived Organ-on-a-Chip Models for Better Therapies
Sebastiaan Trietsch, Mimentas
Sebastiaan Trietsch, PhD
Head of Research
Drug development and prediction of neurotoxicity remains challenging due to the lack of relevant (disease) models of the human brain. Current neurotoxicity assessments rely heavily on expensive and time-consuming ex vivo and in vivo animal testing, and are limited in disease modelling. Here, we describe the development of a human in vitro 3D neuronal model consisting of human iPSCs-derived neurons and astrocytes in the OrganoPlate®. This microfluidic-based platform enables high-throughput culturing and screening of predictive organ and tissue models.
In the 3D OrganoPlate® neuronal cultures, we show proper network formation by immunofluorescent staining. The neuronal electrophysiology was shown based on a calcium sensitive dye indicating spontaneous neuronal firing. Compound exposures modulated the neuronal activity, neurite outgrowth, viability and mitochondrial membrane potential showing the ability of the model to predict neurotoxicity. Next, we are developing the neurovascular unit by combining the blood-brain barrier model with neurons, allowing us to study diseases such as Alzheimer's.
Overall, the studies display the feasibility of using our 3D human iPSC-derived models as a high-throughput screening platform for assessment of pharmaceutical neurotoxicity and application to patient- and disease-specific drug discovery and development.
2:45 pm |
The Effect of Different Oxygen Tensions on Modulating the Early Differentiation Potentials of Human Induced Pluripotent Stem Cells
Reema Mohammed, UCL
Reema Mohammed, PhD
University College London
Early development of mammalian embryos occurs in a relatively low oxygen microenvironment in the reproductive tract (1.5 - 5.3% O2). Yet, ESCs and iPSCs are routinely cultured in atmospheric conditions. In this study, our aim was to investigate the effect of different oxygen tensions on the short-term culture of human iPSCs and on stem cell-fate determination during early differentiation. We performed gene-profiling analysis of human iPSCs maintained under normoxic (20% O2) and a range of hypoxic (0% to 12% O2) conditions. The expression of genes associated with pluripotency, embryonic germ layers and hypoxia were studied using qualitative RT- PCR, Immunostaining and Flow Cytometry. Preliminary results revealed that after four days of culturing human iPSCs at different hypoxic levels, morphological changes were observed. Additionally, hypoxia down-regulated the expression of pluripotency markers. Hypoxic conditions also promoted the expression of genes associated with the three germ layers and genes that are involved in the hypoxia-signalling pathway. Interestingly, mild hypoxia (8% O2) increased the number of cells expressing Brachyury (Mesodermal marker), while acute hypoxia (2% O2) caused 95% of human iPSCs to differentiate into ectodermal lineage indicated by Nestin up-regulation. Thus, our results suggest that hypoxia is an important component of in vitro differentiation for the generation of clinically relevant progenitors.
3:15 pm |
Refreshments (provided), Poster Session
4:00 pm |
hPSCreg - A Central Data Resource for Human Pluripotent Stem Cells
Andreas Kurtz, Charite - Universitätsmedizin Berlin
Andreas Kurtz, PhD
Charité - Universitätsmedizin Berlin
Berlin-Brandenburg Center for
Human pluripotent stem cells are increasingly deposited in large biobanks but also established in numerous individual labs and institutional facilities. The continuing establishment of ever new induced PSC lines is partially due to the lack of common quality standards and subsequent uncertainty regarding the quality of available lines, but also because of difficulties to access, search and find already established lines. The human pluripotent stem cell registry (hPSCreg) has been established in 2007 to collect information of pluripotent stem cells in a central open platform to facilityte access to information of available human PSC-lines. The Registry enforces the deposition of data in standardized formats. All data and lines are manually validated and certified for use in EU-funded research. In addition, a unique identifier, or reference, is provided for each line to avoid the risk of erroneous identification and referencing. Data models, standards and validation processes for human PSC related information will be discussed and utility of the Registry for global access and harmonization of assays and mandatory information addressed.
4:30 pm |
Human Pluripotent Stem Cells: Paving the Way for Clinical Applications
Elsa Abranches, UK Stem Cell Bank, NIBSC
Senior Stem Cell Scientist
UK Stem Cell Bank, NIBSC
The high cost of delivering new products to clinical trial presents a significant hurdle for the development of cell therapies.In particular, the raw and starting material(e.g culture media components and cell lines) require careful consideration in order to enable a reliable and safe final product. As many regulators will repine, it is not possible to test safety into a product, therefore increasingly early and robust risk assessment and traceability of these materials is crucial to minimise risk to patients and investors.The UK Stem Cell Bank is a key component of the UK regenerative medicine infrastructure, charged with procuring, processing and distributing seed stocks of human pluripotent stem cell (hPSC) lines for research and human application and which is equipped with laboratories designed to meet EU GMP. In its latest phase of development, the UKSCB is focused on 37 hPSC lines established in the UK, under appropriate conditions and traceability (i.e. Human Tissues Authority (HTA) licensed) and under principles established by the International Stem Cell Banking Initiative. All cell lines are subject to ethical scrutiny by a national Steering Committee, after which the UKSCB starts its process.Key elements of this include ethics documentation review, “due diligence” for compliance with European regulation, banking and testing, storage and distribution, which assure suitability of each cell line for clinical application and the requirements of the UKSCBs HTA license.
5:00 pm |
Developing the World’s First iPSC-derived MSC Therapeutic Product
Ross MacDonald, Cynata Therapeutics
Ross MacDonald, PhD
Managing Director and Chief Executive Officer
The therapeutic use of allogeneic mesenchymal stem cells (MSCs) is receiving much worldwide attention. There are over 650 clinical trials underway investigating the therapeutic use of these versatile cells in a wide range of diseases including such major clinical and economic problems as stroke, cardiovascular disease, osteoarthritis and autoimmune disease. Manufacture of the finished MSC product poses many regulatory, biological, logistic and cost challenges given the reliance of first-generation manufacturing processes on deriving the starting material from multiple donors and massively expanding the isolated MSCs to produce sufficient quantities for clinical use. Recent developments in differentiation methodologies based on induced pluripotent stem cells (iPSCs) have facilitated many new approaches to production of cell based therapeutics. This presentation describes Cymerus™, a unique and patented process for manufacture of MSCs for clinical use using iPSCs as starting material which eliminates many of the challenges posed by first-generation production methods. The presentation also describes recent progress in bringing the world’s first allogeneic iPSC-derived MSC product into the clinic.