Monday, 10 July 201708:00 | Conference Registration, Materials Pick-Up, Morning Coffee and Pastries | |
Session Title: Opening Plenary Session | Session Sponsors |
| | 09:00 |  | Keynote Presentation Integration of Systems Biology with Organs on Chips for Disease Modeling and Drug Development
Linda Griffith, Professor, Massachusetts Institute of Technology (MIT), United States of America
|
| 09:30 |  | Keynote Presentation Fitting iPSCs, 3D Cell Culture, Tissue Chips and Microphysiological Systems into the Grand Scheme of Biology, Medicine, Pharmacology, and Toxicology
John Wikswo, Gordon A. Cain University Professor, A.B. Learned Professor of Living State Physics; Founding Director, Vanderbilt Institute for Integrative Biosystems, Vanderbilt University, United States of America
As the engineering supporting body-on-chip (BoC) studies advances and
begins to penetrate both science and industry, we need to explore three
separate multidimensional spaces – one that spans BoC components, one
that covers the analytical techniques to characterize BoC performance
and drug response, and a third that spans the fields of application.
The component technologies being brought together include induced
pluripotent stem cells (iPSCs), 3D cell culture (which is beginning to
involve vascularization), tissue-chip bioreactors that enable the
recreation to tissue-like microenvironments, and the hardware required
to operate coupled microphysiological systems in a manner that
recapitulates human physiology and its response to drugs and toxins. The
second, analytical space is only now coming to the fore. To date, most
tissue-chip studies have reported morphological features, the expression
of small sets of genes, or the secretion of a few, organ-specific
compounds. A much more comprehensive battery of techniques is already in
regular use in the pharmaceutical industry, including genomics,
proteomics, and transcriptomics. Metabolomics is rapidly moving into
prominence as the instrumentation improves and the databases expand.
What is needed, though, are comprehensive comparisons between in vitro
and in vivo studies, as has been recently demonstrated with a weighted
gene coexpression network analysis that compare rat liver in vivo with
both mouse liver in vitro and rat primary hepatocytes growing in a dish,
which showed that a mouse liver was a better model of the rat liver
than the primary rat hepatocytes in a dish, which more closely resembled
a rat liver exposed to a significant toxic load. The BoC community
needs to compare, for example, a mouse with a mouse-on-a-chip to confirm
that the appropriate physiology is being recapitulated. The final space
spans biology, medicine, pharmacology, physiology and toxicology. BoCs
offer, for the first time, the ability to recreate in vitro and in
parallel, with an ever-dropping cost, the effects of organ-organ
interactions. Nowhere will this be more important than in studies of
absorption, distribution, metabolism, and excretion - toxicity
(ADME-Tox), where one may need skin, lung, or gut to absorb a drug or
toxin, liver and kidney to metabolize and excrete drug metabolites and
toxins, adipose and muscle tissue to store metabolites and toxins, and a
means to characterize in depth the underlying processes and how they
affect the chosen target organs. BoCs will thereby contribute not only
to toxicology, but our fundamental understanding of cellular biology and
systems physiology, thereby advancing both pharmacology and medicine.
Given that we will never create a perfect microHuman BoC, we can use
these three spaces to guide the compromises we make as we create useful
models, even toy models, of human physiology. |
| 10:00 |  | Keynote Presentation Engineered Living Systems: Current State and Future Potential
Roger Kamm, Cecil and Ida Green Distinguished Professor of Biological and Mechanical Engineering, Massachusetts Institute of Technology (MIT), United States of America
Following on recent advances in understanding single cell behavior (Carrera & Covert, Trends in Cell Biology, 2015), and in developing simple, proof-of-concept biological machines (Raman et al., PNAS, 2015, Park et al., Science, 2016), organoids (Fatehullah et al, Nature Cell Biology, 2016), and organ-on-chip technologies (Huh, et al., Science, 2010), efforts are underway to standardize manufacturing methods for engineered living systems (ELS). The approaches proposed, however, are widely divergent and often lack a sound basis due to the absence of a fundamental understanding of aspects unique to ELS – e.g., complexity, the central role of emergence – and fail to take advantage of their extraordinary capabilities – self-assembly, growth, self-repair, adaptation, learning.
We need to build on our current knowledgebase for the development and design of ELS, rethinking much of what we have learned from abiotic engineered systems. A major effort is therefore required to characterize, model and image the dynamical behavior of ELS, and thus establish the design principles needed for robust manufacture. While many ELS can survive merely by diffusion of gases and nutrients from their environment, most systems exceeding several hundred microns in lateral dimension require some means for convective transport, such as the circulatory system found in many living organisms. Several approaches have been employed to meet these needs, either by engineered conduits or induced network growth from seeded or suspended cells. In this talk, some of these methods will be described, focusing on networks that form by self-assembly, tend toward a stabilized perfusable network within 1-2 weeks, synthesize and organize their own matrix environment, and adapt to changing conditions. Both the successes and challenges of creating these networks will be discussed with the aim of developing reliable, vascularized ELS amenable to biomanufacture. |
| 10:30 | Coffee Break and Networking in the Exhibit Hall | 11:00 |  | Keynote Presentation Human “Body-on-a-Chip” Systems to Test Drug Efficacy and Toxicity
Michael Shuler, Samuel B. Eckert Professor of Engineering, Cornell University, President Hesperos, Inc., United States of America
Human microphysiological or “Body-on-a-Chip” systems are powerful tools to assess the potential efficacy and toxicity of drugs in pre-clinical studies. Having a human based, multiorgan system, that emulates key aspects of human physiology can provide important insights to complement animal studies in the decision about which drugs to move into clinical trials. Our human surrogates are constructed using a low cost, robust “pumpless” platform. We use this platform in conjunction with “functional” measurements of electrical and mechanical activity of tissue constructs (in collaboration with J. Hickman, University of Central Florida). Using a system with four or more organs we can predict the exchange of metabolites between organ compartments in response to various drugs and dose levels. We will provide examples of using the system to both predict the response of a target tissue as well as off-target responses in other tissues/organs. We believe such models will allow improved predictors of human clinical response from preclinical studies. |
| 11:30 |  | Keynote Presentation Human Emulation System: an Organs-on-Chips Platform for Advancing Drug Discovery and Development
Katia Karalis, Executive Vice President of Research, Emulate, Inc., United States of America
Micro-engineered Organs-on-Chips show physiological functions consistent with normal living human or animal cells in vivo. Each Organ-Chip is composed of a clear flexible polymer about the size of a AA battery that contains hollow channels lined by living human cells. The cells are cultured under continuous flow and mechanical forces thereby recreating key factors known to influence cell function in vivo. Cells cultured under continuously perfused, engineered 3D microenvironments go beyond conventional 3D in vitro models by recapitulating in vivo intercellular interactions, spatiotemporal gradients, vascular perfusion, and mechanical microenvironments. Integrating cells within Organs-on-Chips, enables the study of normal physiology and pathophysiology in an organ-specific context. Cellular/molecular level resolution is enhanced and demonstrates key insights into the mechanisms of action of drug induced toxicity. Numerous recent advances in applications of these systems are relevant in drug discovery/development for compound selection, and in de-risking mechanistic concerns using various organ systems. In this presentation we will highlight studies from collaborative efforts across our Human Emulation System with various academic and industry partners to demonstrate the utility of the system as a more predictive human-relevant alternative for efficacy and safety testing of new chemical entities in humans. |
| 12:00 |  | Keynote Presentation Novel Microphysiological Multi-Organ Systems for Studies of Human Metabolic Diseases in Drug Discovery
Tommy Andersson, Drug Metabolism and Pharmacokinetics, Cardiovascular and Metabolic Diseases, AstraZeneca, Gothenburg, Sweden
Currently used pre-clinical models often suffer from poor translation of
drug responses to the patient due to the limited knowledge gained in
the efficiency and mode of action of the drug candidate. This
contributes to high attrition rates in early clinical programs. Multi
organ-on-a-chip emulating human physiology have the possibility to
improve success rate by mimicking the human disease state and improve
selection of the right targets and compounds early in drug discovery.
Such models will not only improve translation to patients but also
reduce time spent in early clinical programs as well as reducing the
needs for animal models. We developed a human liver - pancreatic islets
chip model. The model allows cross talk between cells from both organs
in a fluidic system and responds in a physiological way to glucose load
by increased insulin secretion leading to increased glucose consumption
(figure). Initial studies indicate that the model can become insulin
resistant and thus can be used as a metabolic disease model. Ongoing
studies are investigating how insulin resistance in liver cells effects
islet function by using the insulin receptor antagonists. |
| 12:30 | Networking Lunch in the Exhibit Hall, Meet Exhibitors and View Posters | |
Session Title: The Deployment of iPSC-derived Cells for Drug Discovery |
| | 13:30 | .png) | Keynote Presentation Present (and Future) Uses for Stem Cells Derived Preparations in Drug Discovery and Toxicity Testing
Gary Gintant, Senior Research Fellow, Abbvie, United States of America
The advent of human pluripotent stem cells a decade ago sparked visions of novel screening approaches for drug safety and efficacy, narrowing the translational gap between traditional preclinical and clinical studies, and defining a new “proclinical space” where human derived preparations are used in preclinical assays. Not unlike other novel (and potentially disruptive) technologies, limitations learned along the way have provided new challenges for both the biology and technologies necessary for confident and efficient screening efforts. This presentation will highlight promising present and future applications for stem cell derived preparations, focusing on prospects for achieving translational success of new drug candidates. |
| 14:00 |  | Keynote Presentation What’s Next for Stem Cells?
Devyn Smith, Chief Operating Officer, Sigilon, United States of America
|
| 14:30 | Drug Testing on Patient-derived iPSC Cells
Bikash Pattnaik, M. D. Matthews Professor, McPherson Eye Research Institute, University of Wisconsin, Madison, United States of America
A presentation on disease modeling and drug testing using patient in a dish approach. | 15:00 | Complex Disease Genetics, Molecular Epidemiology, and the Power of iPSC-derived Cardiomyocytes
Ulrich Broeckel, Professor of Pediatrics, Medicine and Physiology - Chief, Section of Genomic Pediatrics, Medical College of Wisconsin, United States of America
| 15:30 | Development of a Novel Drug Discovery Platform for Progressive Multiple Sclerosis
Stephen Crocker, Associate Professor, Department of Neuroscience, University of Connecticut School of Medicine, United States of America
Primary progressive multiple sclerosis (PPMS) is a chronic demyelinating disease of the central nervous system (CNS) without any effective treatment. Patients with PPMS generally do not benefit from currently prescribed immuno-modulatory therapies which can be effective among relapsing-remitting MS (RRMS) patients. Hence, promoting endogenous brain repair by promoting the differentiation of myelin-forming oligodendrocytes (OLs) is viewed as a potential strategy to halt and possibly restore neurologic function in patients with PPMS. This presentation will provide an update on our current approach to develop a novel drug screening assay that can be used to identify compounds with potential to promote brain regeneration in PPMS patients. The basis for this assay is our recently developed and characterized induced pluripotent stem (iPS) cell lines from PPMS patient samples which we have determined can be used to model the lesion environment of the PPMS brain. When compared against iPS cells from age-matched, non-diseased control cell lines we recently reported that the cells from all PPMS patients tested have an inherent defect in their ability to protect or promote myelin forming OLs. This work provides an innovative approach with personalized medicine potential because it models a crucial aspect of the disease microenvironment. Outcomes from this assay may have important implications for understanding re-myelination failure in PPMS that may have therapeutic potential to benefit other forms of MS. | 16:00 | Leveraging Novel Technologies for Human iPSC-based Screening
Xianmin Zeng, Associate Professor, Buck Institute for Research on Aging, United States of America
Human induced pluripotent stem cell (iPSC) technology offers the benefits of a cell line coupled with the advantage of using human primary cells. We have developed a panel of iPSC lines for neurotoxicity assays and disease modeling. These include: 1) control lines, 2) patient-specific lines, 3) lineage-specific knock-in reporters, 4) isogenic controls of single and double knock-outs. We have also established scalable protocols for generating differentiated cells in an assay ready format. I will discuss the utility of these lines for neurotoxicity assays including assays to determine the specificity of different neural cell types for a small range of chemicals and drugs from the Tox21 library, as well as for neuroprotective assays with dopaminergic neurons. | 16:30 |  | Keynote Presentation 3D Cell-Material Micro-Tissue Devices in Application of ES/iPS Cells for Drug Screening/Toxicology and Cell Therapy
Norio Nakatsuji, Chief Advisor, Stem Cell & Device Laboratory, Inc. (SCAD); Professor Emeritus, Kyoto University, Japan
SCAD is a start-up company founded in 2014 based on the integration of stem cell technology and nanotechnology/micro-engineering developed at Kyoto University. We are currently focusing on the iPSC-derived cardiomyocytes, with possible addition of neurons and hepatocytes. Cardiomyocytes derived from human pluripotent stem cells (hPSC-CMs) are promising materials for drug screening/toxicology and cell therapy. However, there are two major issues: immaturity of hPSC-CMs and instability of cardiac cellular function. To overcome these issues, we use two technologies: the robust cardiac differentiation method and aligned nanofibers as a culture scaffold. Our cardiac differentiation method can efficiently generate cardiomyocytes from any hPSC lines under cytokine-free defined condition. The hPSC-CMs seeded on nanofiber scaffolds formed 3D multilayered structures (Micro-Tissues), which show upregulated expression of cardiac maturation marker genes and enhanced and stable cardiac cell functions. Our micro-tissue devices are easy to handle and highly useful for drug screening/toxicology assays. |
| 18:00 | Networking Cocktail Reception with Beer, Wine and Appetizers. Enjoy the Boston Skyline, Engage with Colleagues and Discuss Collaborations and Partnerships | 19:30 | Close of Day 1 of the Conference | 19:45 | Dinner Short Course on 3D-Culture [Separate Registration Required] |
Tuesday, 11 July 201707:00 | Morning Coffee, Breakfast Pastries, and Networking in the Exhibit Hall | |
Session Title: The Toxicity Screening Paradigm and Cell Types Deployed in Pharma/Biotech |
| | 08:00 | Electrophysiological Properties of Neurons Derived from Human Stem Cells In Vitro
Robert Halliwell, Professor of Neuroscience and Clinical Pharmacology, University of The Pacific, United States of America
Neurons derived from human stem cells promise great advantages for drug discovery and safety testing, especially in the development of agents for neurological and psychiatric disorders. This presentation will describe recent data showing that human stem cells from a variety of sources can develop the morphological features of neurons and glia, display immunocytochemical markers and express a complex array of functional voltage- and ligand-gated ion channels; I will also consider the advantages and challenges of using different types of human stem cells to derive such neurons. Finally, data will be presented showing the sensitivity of human stem cells and their neural phenotypes to a range of clinically important psychotropic agents. | 08:30 |  | Keynote Presentation Stem Cell Cardiomyocytes for Proarrhythmic Risk Assessment and Precision Medicine
Ksenia Blinova, Senior Technical Manager, Induced Pluripotent Stem Cell and Electrophysiology Core Facility, Center for Devices and Radiological Health (CDRH), US Food and Drug Administration (FDA), United States of America
|
| 09:00 | Using iPSC-derived Cardiomyocytes to Inform Kinase Inhibitor-induced Cardiotoxicity
Matthew White, ORISE Fellow, US FDA National Center for Toxicological Research, United States of America
Cardiotoxicity often mitigates the efficacy of anti-cancer therapeutics such as kinase inhibitors. Human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) offer an attractive model to study this toxicity in vitro. Here, I will discuss recent work from our laboratory investigating the effects of multiple FDA-approved kinase inhibitors on distinct iPSC-CM cell lines. | 09:30 | Stem Cell-Based Screening Platforms for the Efficient and Accurate Prediction of Nephrotoxicity in Humans
Daniele Zink, Principal Research Scientist and Team Leader, Institute of Bioengineering and Nanotechology, Agency for Science Technology and Research (A*STAR), Singapore
The presentation will summarize our work on stem cell-based screening platforms for the prediction of nephrotoxicity, and in particular renal proximal tubular toxicity, in humans. Our most advanced technology is based induced pluripotent stem cells (iPSC), which are differentiated by the currently most rapid and efficient protocol into renal proximal tubular-like cells ( Kandasamy et al., 2015). In combination with one of our recently established in vitro methods for nephrotoxicity prediction (Li et al., 2013; 2014) and machine learning (Su et al., 2014), an iPSC-based platform has been developed that predicts proximal tubular cell toxicity in humans with 87% test balanced accuracy (Kandasamy et al., 2015). Our iPSC-based technologies are compatible with our high-throughput platform for nephrotoxicity prediction (Su et al., 2015), and we are currently exploring predictive kidney-on-a-chip approaches. | 10:00 | Coffee Break and Networking in the Exhibit Hall | 10:30 |  Technology Spotlight:
Quality Control: Driving Standardization For Commercially-Available iPSC-derived Cell Types
Greg Luerman, Technical Director, Axiogenesis AG
Stem cell technologies have revolutionized our industry. In addition to humanizing drug discovery, these differentiated tissues now closely mirror the existing biology and physiology of an adult tissue system. This provides an ideal system for drug toxicity testing, disease modeling, and efficacy screening. For these cells to see further adoption into the industry, tight quality control metrics must be in place to guarantee reproducible results. Axiogenesis is helping to drive the establishment of industry-wide QC standards for iPS cell products.
| 11:00 | Kidney Organoids for Drug Discovery and Toxicity Screening
Ryuji Morizane, Associate Biologist, Renal Division, Brigham and Women’s Hospital Affiliated Faculty, Harvard Stem Cell Institute, United States of America
We have developed an efficient, chemically defined protocol for differentiating human embryonic stem (ES) cells and induced pluripotent stem cells (iPSCs) into multipotent nephron progenitor cells (NPCs) that can form nephron-like structures. By recapitulating metanephric kidney development in vitro we generate SIX2+SALL1+WT1+PAX2+ NPCs with 80-90% efficiency within 8-9 days of initiation of differentiation. The NPCs form PAX8+LHX1+ renal vesicles that self-organize into nephron structures. NPCs form kidney organoids containing epithelial nephron-like structures expressing markers of podocytes, proximal tubules, loops of Henle and distal nephrons in an organized, continuous arrangement that resembles the nephron in vivo. The organoids express genes reflecting many transporters seen in adult metanephric-derived kidney as well as important kidney endocrine genes such as the gene responsible for the production of erythropoietin. Stromal cells are also generated with the presence of PDGFRß+ (pericyte), endomucin+ (endothelial cell), or a-SMA+ (myofibroblast) interstitial cells. The entire procedure is performed with completely defined conditions without the need for embryonic spinal cord. This kidney differentiation system can be used to study mechanisms of human kidney development. Organoids can be used to evaluate nephrotoxicity of drugs as we have shown the expression of Kidney Injury Molecule-1 in structures that express markers of proximal tubules after exposure to nephrotoxicants. Glomerular toxins alter the cytoskeletal distribution of the glomerular structures. Epithelial toxins and TGFß can cause increased of stromal cells with characteristics of myofibroblasts. Hence the generated kidney organoids are effective tools to study genetic disorders of the kidney as well as mechanisms of toxicity. Generation of NPCs, when coupled with tissue engineering, may lead the way to generation of functional kidney replacement tissue in the future. | 11:30 | Toxicological Responses in Human iPSC-derived Neurons Using MEA System
Ikuro Suzuki, Associate Professor, Tohoku Institute of Technology, Japan
The functional network of human induced pluripotent stem cell (hiPSC)-derived neurons is a potentially powerful in vitro model for evaluating drug toxicity. Epileptiform activity is one of phenomena in neuronal toxicology. To evaluate the dynamics of epileptiform activities and the effect of anti-convulsant drug in cultured hiPSC-derived neurons, we used the multielectrode array (MEA) system, where we simultaneously record extracellular potentials for 384 channels on 24-well plates. We firstly confirmed the modulation of activity by typical glutamatergic and GABAergic receptor antagonists/agonists in spontaneous firings. Spontaneous activities and typical responses against synaptic related drugs were detected with high S/N ratio using high-throughput MEA system. Next, we examined chemically evoked epileptiform activity. Electrophysiological seizes were induced by pentylentetrazole (PTZ), 4-Aminopyridine (4-AP), and kainic acid (KA), the most widely used chemical convulsant in animal models to screen for new anti-epilepsy drugs. We also examined the anti-convulsant effects of common clinical anti-epilepsy drugs (AEDs), phenytoin. PTZ, 4-AP and KA induced an increase in synchronized burst firings (SBFs) in a concentration-dependent manner. Phenytoin suppressed induced epileptiform activity. However, the patterns of epileptiform activities and phenytoin effects were different with respect to each epilepsy drugs. From these results, we suggest that the electrophysiological assay in cultured human iPSC-derived neuron using high-throughput MEA system is a useful to investigate the neuronal toxicity in drug screening and pharmacological effects of human neurological disease. | 12:00 | Pluripotent Human Stem Cells as Models For Creating Placental Syncytiotrophoblast, The Major Cellular Barrier that Limits Fetal Exposure to Xenobiotics
Toshihiko Ezashi, Research Associate Professor, The University of Missouri, United States of America
The placenta provides the interface between the separate blood circulations of the mother and the fetus and has roles in controlling the movement of a variety of compounds, including dissolved nutrients and gases, between the two systems. It is also the barrier that limits direct exposure of the fetus to foreign chemicals circulating in maternal blood. The surface of the human placenta that makes direct contact with maternal blood, even in the earliest stages of pregnancy, is comprised of syncytiotrophoblast (STB), a multi-nucleated cell layer formed by fusion of underlying, proliferating cytotrophoblast. Pluripotent human stem cells can be differentiated efficiently towards placental trophoblast by exposure to BMP4 in presence of low molecular weight inhibitors of FGF2 and AVTIVIN/TGFB signaling. Within 48 h, the cells have lost their pluripotent phenotype and display marker features of trophoblast. By 5 days they begin to release hCG and progesterone and demonstrate the initiation of STB formation at discrete regions within the colonies. By day 7-8 it becomes possible to isolate sheets of syncytium. Transcriptome profiling reveals that these cells express a full complement of marker genes for STB, as well as an up-regulation of many others known to be involved in the metabolism, transport, and sequestering of xenobiotics, drugs and heavy metals. The model will provide a means of assessing how STB responds to exposure to xenobiotics and other foreign chemicals through changes in its normal developmental phenotype, including gene expression patterns. | 12:30 | Networking Lunch in the Exhibit Hall, Meet Exhibitors and View Posters | |
Session Title: Studying Biological Phenotypes and Dissecting Pathways of Relevance for Drug Discovery and Development |
| | 13:30 | Substrates for Generating Cancer Stem Cells and Highly Functional Hepatocytes
Mark Bradley, Professor of Therapeutic Innovation, Precision Healthcare University Research Institute, Queen Mary University of London, United Kingdom
In my talk I will introduce polymer microarray technology; including our inkjet mediated fabrication methodologies (which allows over 7000 different substrates to be made on a single glass slide) and describe how this approach has been used in a large number of stem cell based applications, notably:
(i). The use of polymer microarray technology to discover a novel thermo-responsive chemically-defined hydrogel for long term culture of human embryonic stem cells (Nature Communication, 2013) and mesenchymal adipose derived stem cells (Biomaterials, 2014)
(ii). Polymer discovery that were able to support highly functional hESC-derived hepatocyte like cells (as active as primary human hepatoctyes) (with David Hay see: Stem Cell Res, 2011 and WO2010106345)
(iii). The discovery and application of a substrate able to “lock-down” CSC’s in their CSC state
| 14:00 | Cardioprotectants: From Phenotype Screening to Pathway Targets
Siobhan Malany, Director, Translational Biology, Sanford Burnham Prebys Medical Discovery Institute, United States of America
In the United States alone, approximately one million heart attacks occur per year and only 37% of patients survive one year after suffering a heart attack. A major consequence of myocardial infarction is the loss of cardiomyocytes due to oxidative stress associated with reperfusion. Improving pharmacological therapies that provide protection during cardiac oxidative stress is the focus of significant research and exploratory medicine. We report a chemical biology phenotypic screening approach to identify and validate small molecules that protect human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) from oxidative stress. Cardioprotective activity of ‘hit’ compounds was confirmed using impedance-based detection of cardiomyocyte monolayer integrity and contractile function. Structure-activity relationship studies led to the identification of a potent class of compounds with 4-(pyridine-2-yl)thiazole scaffold. Examination of gene expression in hiPSC-CMs revealed that the hit compound, designated cardioprotectant 312 (CP-312) induces a marker of the antioxidant response network. CP-312 therefore represents a novel chemical scaffold identified by phenotypic high-throughput screening using hiPSC-CMs that activates the antioxidant defense response and may lead to improved pharmacological cardioprotective therapies.
| 14:30 | GlyR and GABAAR Targeted High-throughput Screening and Toxicity Testing Using Human Pluripotent Stem Cells
Katharina Kuenzel, Researcher, FAU Erlangen-Nuremberg, Institute of Medical Biotechnology, Germany
Pluripotent stem cells like the human embryonal carcinoma cell line NT2 provide an enormous potential for drug discovery and toxicity testing. NT2 cells differentiate upon exposure to retinoic acid into cells with properties of neurons of the central nervous system and have been shown to express ion channels including glycine receptors (GlyRs) and gamma-aminobutyric acid receptors (GABAARs) which are both increasingly considered as attractive drug targets for therapeutic intervention. By stable transfection of NT2 cells with YFP-I152L, a halide-sensitive YFP (yellow fluorescent protein) variant, we created a cell line for studying the pharmacological properties of GlyRs and GABAARs in native neurons. In concentration-response experiments with the receptor agonist glycine and GABA as well as with receptor-specific drugs we demonstrated the applicability of the established NT2-YFP-I152L cell line for in vitro-based drug and toxicity testing in high-throughput screening format. | 15:00 | Identification of Compounds that Restore FMR1 Gene Expression in Fragile X Syndrome
Daman Kumari, Staff Scientist, Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (NIH), United States of America
Fragile X syndrome (FXS) is the leading cause of inherited cognitive disability and autism spectrum disorder. The most common mutation in FXS patients is the expansion of a CGG-repeat sequence in the 5’-untranslated region of the Fragile X Mental Retardation-1 (FMR1) gene to >200 repeats that causes transcriptional silencing and loss of its protein product, FMRP. Most of the available therapeutic options for FXS target behavioral symptoms and currently there is no cure. Identification of FMRP targets and the elucidation of molecular mechanisms involved in the disease pathology have lead to the development of drugs that target the altered signaling pathways in the brains of FXS patients. However, these strategies are limited by the fact that loss of FMRP affects multiple pathways. Given that the FMR1 gene is silenced in FXS by aberrant epigenetic modifications which can be reversed, and the fact that expanded CGG-repeats are not a part of the open reading frame which is otherwise normal, strategies aimed at restoring FMRP production may be worthwhile. We have used two parallel approaches to identify small molecule compounds that are capable of restoring FMR1 expression in FXS patient cells. These include an unbiased high throughput screening (HTS) approach for identifying compounds that restore FMRP expression, and elucidating the mechanism of FMR1 gene silencing to target specific pathways with known small molecule inhibitors for gene reactivation. We developed a sensitive and quantitative assay for FMRP detection and optimized it for HTS. Using this assay in pilot screens with FXS neural stem cells, we identified a few compounds that were able to increase FMRP expression. This provides proof of principle that screening large compound libraries might yield better hits that could be developed into lead compounds. We have also identified a role for FMR1 mRNA in its own gene silencing. Our data suggest that the ability to interfere with the recruitment of repressive chromatin modifiers by FMR1 mRNA to the gene will allow the development of strategies that specifically target the FMR1 locus for restoring gene expression. | 15:30 | Coffee Break and Networking in the Exhibit Hall | 16:00 | Differentiating Arterial and Venous Endothelial Cells from Pluripotent Stem Cells
Guohao Dai, Associate Professor, Department of Bioengineering, Northeastern University, United States of America
In this talk, I will present the transcriptional regulation of arterial venous identity and our novel biomaterial approaches to control the vascular differentiation and functional specifications of pluripotent stem cells and their application in tissue regeneration and drug discovery. | 16:30 | Biomaterial-Guided Patient-Specific Cardiac Disease Modeling and Drug Toxicity Screening
Zhen Ma, Assistant Professor, Samuel and Carol Nappi Research Scholar, Syracuse Biomaterials Institute, Syracuse University, United States of America
Explosive progress in human induced stem cells (hiPSCs) has provided great opportunities for precision medicine and companion diagnostics on patient-derived cells with increasing ease. Our research integrates hiPSC biology, tissue engineering and gene editing technologies to lead the development of next generation hiPSC-based patient/disease-specific in vitro tissue models and organ-on-chip systems. These tissue models aim to accurately recapitulate the complexity of three-dimensional (3D) tissue architecture, the dynamics of in vivo-like biological response, and pathophysiological status of human tissue for better understanding human disease etiology and evaluating the efficacy of the personalized treatments to the individual patients. We have established the 3D mechanical-tunable cardiac model by populating synthetic organized filamentous matrices with wild-type and diseased cardiomyocytes derived from hiPSCs. This highly defined human 3D cardiac model will help us to better understand the disease mechanism and formulate better therapeutic strategies for this syndrome, moving drug discovery and development into the era of personalized medicine. | 17:00 | Acute and Chronic Molecular Markers Causally Linked with Tyrosine Kinase Inhibitor-induced Cardiotoxicity
Huan Wang, Research Fellow, Department of Systems Biology, Harvard Medical School, Harvard Therapeutic Science Program, United States of America
Tyrosine kinase inhibitors (TKIs), targeted at specific oncogenes, have significantly improved cancer patient survival, but many patients suffer from drug-induced cardiotoxicity. Toxic phenotypes range from congestive heart failure, hypertension, to cardiac arrhythmias, and onset of cardiotoxicity varies from weeks to months. This indicates that toxic effects are probably mediated by multiple biological processes and are likely determined by dose and duration of drug treatment and patients’ metabolism. We aim to illustrate underlying biological processes and molecular regulators that explain mechanisms of TKI-induced cardiotoxicity, and propose means to mitigate cardiotoxicity. To achieve this, we computationally model the relation between whole transcriptome and proteome with toxic phenotypes in human induced pluoripotent stem cells derived cardiomyocytes (iPS-CMs). Consistent with toxicity observed in clinic, Sunitinib and Sorafenib cause more significant cellular toxicity than Lapatinib and Erlotinib. Systematic profiling of gene and protein expression in iPS-CMs is highly reproducible between batches. Interestingly, gene regulation by all drugs, especially Sorafenib, is high correlated between acute (10 µM, 24h) and chronic (3.16 µM, 168h) treatment. Consistent biological functions are regulated by Sunitinib and Sorafenib, including mitochondrial respiration, metabolism, and contraction. Sorafenib induces mitochondrial uncoupling and increases glycolysis, leading to cellular toxicity. In summary, TKI-induced cardiotoxicity is mediated by numerous biological processes and molecular regulators in a dose and time dependent manner. We define a set of genes consistently regulated at RNA and protein levels as early indicators of chronic drug toxicity. | 17:30 | A Novel Optical Dynamic Clamp Platform For iPSC-derived Cardiomyocytes
Bonnie Quach, Researcher, Weill Cornell Graduate School, United States of America
iPSC-derived cardiomyocytes (iPSC-CMs) are a potentially viable platform for drug screening since they provide a renewable source of human cardiomyocytes that can be derived from a target patient population. However, one major obstacle to using iPSC-CMs for drug development is their fetal-like electrophysiology. Artificial addition of the missing inward rectifier potassium current, IK1, via dynamic clamp has been shown to produce an adult-like electrical phenotype. However, dynamic clamp is low throughput and limited to a single cell format because it requires a whole-cell patch. Optogenetic tools have been shown to stimulate neural and cardiac cells to fire action potentials using depolarizing opsins and inhibit electrical activity with hyperpolarizing opsins. We present a proof-of-concept of using optogenetics to dynamically generate a target current instead of an electrode. ArchT was used to mimic IK1, resulting in a more adult-like action potential morphology, similar to using traditional dynamic clamp methods. An in silico ArchT model was used to calculate the light intensity needed to activate ArchT in vivo and generate the target current (IK1). This is the first step towards the overarching goal of establishing a novel optical dynamic-clamp platform using iPSC-CMs for drug screening based on the principles of dynamic clamp. This method aims to be fully optically controlled, allowing for the use of iPSC-CM beating clusters and is more high throughput. | 18:00 | Close of Day 2 of the Conference |
|
