High Content 2017 - 4th Annual SBI2 Conference
September 13th - 15th, 2017. San Diego Convention Center
O. Joseph Trask (PerkinElmer), &
Daniel R. Rines (Vala Sciences).
Session abstract: Coming Soon
Co-chair: Sharon Presell, Chief Scientific Officer, Organovo, Inc., & President at Samsara Sciences, Inc, San Diego, CA
Co-chair: David Egan, Co-founder, Core Life Analytics BV, Netherlands
- Jos Joore Co-founder & Managing Director, Mimetas, Netherlands
- Kapil Bharti, Stadtman Investigator, National Eye Institute, NIH
Co-chair: Kristen A. Johnson, Principal Investigator, California Institute for Biomedical Research, San Diego, CA
Co-chair: John Joslin, Senior Investigator, Assay Development & High Throughput Screening, The Genomics Institute of the Novartis Research Foundation, San Diego, CA
- Chris Hughes, University of California, Irvine
- Ron Davis, The Scriptts Research Institutes, FL
Co-chair: Anne Carpenter, Imaging Director, Broad Institute of Harvard and MIT, Cambridge, MA
Co-chair: Thierry Dorval, Servier Laboratories, Croissy-Sur-Seine, France
- William Godinez-Navarro, Novartis
- Yolanda Chong, Vice President of Biology at Recursion Pharmaceuticals
Abstract: Coming Soon
Co-chair: David Andrews, Director of Biological Sciences, Sunnybrook Research Institute, Professor, Dept. of Biochemistry, University of Toronto
Co-chair: Jeff Price, President and CEO at Vala Sciences, Inc., and Professor, Scintillon Institute, San Diego
- David Solecki, St. Jude Faculty, Department of Developmental Neurobiology, St. Jude Children's Research Hospital, TN
- Christian Tardif, Biophotonics Program Institute Nationale Optic INO, Quebec City, Canada
Scientific Program Abstracts
Session-1: Advanced and Complex Cell Models
Session Abstract: An increased focus on translational research in academia and the desire to reduce the attrition rate in industrial drug discovery programs has lead biologists to increasingly adopt more physiologically relevant cellular models. These include organoids, co-cultures, organ-on-a-chip technologies, iPSC-derived cultures and CRISPR-modified cell lines. These systems, while offering great opportunities, also bring attendant challenges, that need to be addressed with novel methods and technologies. Validation is also required to determine whether these more complex, and more expensive platforms, are worth the additional investment required. In this session speakers will describe a variety of advanced cellular systems and address the critical issues related to their adoption.
Jos Joore Co-founder & Managing Director, Mimetas (abstract submitted)
Title: The OrganoPlate: Human organ-on-a-chip tissue models for predictive drug testing in any throughput
Organ-on-a-chip has recently emerged as the new paradigm in enhanced, 3D tissue culture. The field builds on almost 26 years of developments in microfluidic and associated microfabrication techniques on the one hand and an urge towards ever more physiologically relevant cell and tissue culture approaches on the other hand. Application of microengineering techniques in cell culture enables structured co-culture, 3D culture, the use of flow and associated shear stress and application of controlled gradients. MIMETAS develops a commercially available platform based on a microtiter plate format that harbors up to 96 chips and enables perfused 3D co-culture in a membrane-free manner. The OrganoPlate® facilitates growth of tubules and blood vessels under continuous flow of medium, it allows engineering of organ complexity without usage of artificial membranes. The OrganoPlate® is fully compatible with liquid handling equipment and high-content readers and is easily adopted by end-users. Current flagship models in OrganoPlates® comprise the human kidney proximal tubule, central nervous system, colon, liver and blood vessels. These models are unsurpassed in terms of physiological relevance and throughput.
Kapil Bharti, Stadtman Investigator, National Eye Institute, NIH
Title: 3D bioprinting and iPS cells help generate patient-specific ocular tissue to model and treat age related macular degeneration
Pathological angiogenesis of capillaries located in the back of the eye (choroid) leads to an eye disease “wet” age-related macular degeneration (wet-AMD), one of the leading causes of blindness among elderly. In wet-AMD, choroidal capillaries grow and leak into the eye by breaching through the outer blood-retina-barrier that is formed by the tight junctions of the retinal pigment epithelium (RPE) cell layer located adjacent to these capillaries. Antibodies against Vascular Endothelial Growth Factor (VEGF) provide a temporary treatment by stopping capillary growth but do not cure the underlying disease. This is because there is no good model to identify mechanism of disease initiation. We have combined bioprinting and tissue engineering with induced pluripotent stem (iPS) cell technology to develop a 3D in vitro model of wet-AMD. Using a collagen-based gel for encapsulation of patient-specific iPS cell-derived endothelial cells, choroidal fibroblasts, and pericytes, we successfully bioprinted a microvascular network on one side of a ten-micron thick biodegradable scaffold. On the other side of the scaffold, we grow a RPE monolayer differentiated from the same patient’s iPS cells. The scaffold serves as a transient support for RPE and choroid to secrete extracellular matrix and forms a membrane similar to Bruch’s membrane in the back of the eye. This 3D tissue shows electrical properties that are reminiscent of the outer blood-retina-barrier of the eye. Furthermore, similar to VEGF induced vascular growth in wet-AMD, the in vitro microvascular network also responds to VEGF. The use of patient-specific iPS cells allows us to dissect genetic pathways associated with wet-AMD initiation. This work provides a platform to discover disease inducing pathways and the possibility of identifying potential therapeutic drugs for wet-AMD.
Session-2: Phenotypic Drug Discovery
Session Abstract: The challenge of phenotypic screening is to develop an assay amenable to high throughput automation but still preserve the connection to disease pathology. The combination of advanced automation and high content imaging have enabled many assays to be miniaturized to small plate formats, but still capture the relevant readouts required for many phenotypic assays. The field is continuing to push these boundaries using induced-pluripotent stem cells, microfluidics, complex 3D assays, and next generation sequencing. This session will highlight some of these efforts and provide insights into how phenotypic screening will continue to advance new discoveries for unmet medical needs.Chris Hughes, University of California, Irvine
Chris Hughes, University of California, Irvine
Title: Vascularized MicroTumors: A platform for phenotypic drug discovery and validation
The human body is a complex, 3-dimensional assembly of over 200 different cell types held together by a network of extracellular matrix (ECM) molecules. Sadly, our current laboratory models of human disease do not even begin to reflect this complexity, contributing to the >85% failure rate of FDA-approved trials of new drugs. Mouse studies, though helpful, are also fraught with difficulties, often including poor modeling of human drug metabolism. For these reasons, we have created a human Vascularized Micro-Organ (VMO) platform. These “organs-on-chips” contain multiple human cell types embedded in a 3D ECM that are supplied with nutrients (and drugs) through a living blood vessel network, just as they are in the body. This is a unique platform—no other system offers human micro-organs on a chip that are supported by true blood vessels—that represents an optimal balance between simplicity of use and physiological complexity. We have developed a version of the VMO—the Vascularized Micro-Tumor (VMT) platform—that provides unprecedented opportunities to study drug responses of tumors in a more natural environment than a plastic dish. In the VMT a vascular network forms in a central tissue chamber and anastomoses (connects) with outer channels that represent arterioles (high pressure) and venules (low pressure). A blood substitute flows from the arteriole, through the vascular network, where it nourishes the surrounding tumor, and then out through the venule. The basics of the platform have been developed and its utility has been established as we have incorporated cells from several tumor types within the VMT platform, including colon, breast, melanoma, prostate and glioma, and shown its effectiveness for drug screening. The VMT platform provides a new and superior way to screen drugs for efficacy and toxicity.
Ron Davis, Scripps Research Institute Florida, Department of Neuroscience, Jupter, FL
Title: High throughput and high content assays and screens for mitochondrial function and dynamics in neurons.
The effective treatment of mitochondrial diseases requires new therapeutics that target mitochondrial dynamics and function. Processes included under the rubric of “mitochondrial dynamics” that become dysfunctional in mitochondrial disease involve mitochondrial biogenesis (birth), fission (division), fusion, trafficking to subcellular compartments, and mitophagy (turnover). Major functions of individual mitochondria that can become dysfunctional include ATP generation and calcium buffering. Since the central nervous system is targeted in several mitochondrial diseases and neurons have their own unique physiology, we have established a high throughput, high content, and multiplexed assay for mitochondrial dynamics in primary neuronal cultures. The assay system employs mouse cortical neurons that conditionally expresses a mitochondrial-tagged fluorescent reporter; high content imaging of somatic, axonal and dendritic mitochondria; and an image analysis/bioinformatics pipeline that extracts critical features about the mitochondrial system in neurons with and without drug treatment or in the background of genetic disease. With this assay system, we are able to simultaneously quantify mitochondrial mass in the soma and neurites along with morphological features such as length, width, area, and circularity. These features allow us to determine the effects of manipulations on biogenesis, fission, fusion, and mitochondrial health. In parallel, we have established a high throughput assay of mitochondrial function that measures the potential gradient across the inner mitochondrial membrane as a surrogate measure of ATP synthesis. We have used these two assays to screen through several thousand small molecules and have identified compounds that alter mitochondrial biogenesis and health in neurons, and compounds that promote ATP synthesis. Since proper neuronal function is highly dependent on normal mitochondrial dynamics and mitochondrial generated energy, small molecules identified through these screens may improve neuronal health and stave off mitochondrial disease.
Session abstract: Several recent revolutions are increasing the amount and quality of information that can be extracted from large-scale imaging experiments. From 3D models of organs and tumors to complex cell and material systems, biologists are making ever more complex assay systems that push the limits of what can be accurately quantified. Advances in the world of computer science are pushing the field forward as well, from deep learning to high-dimensional data analysis techniques to methods that leverage the single-cell data inherent in images of cell populations.
William Godinez-Navarro, Novartis Institutes for Biomedical Research, Basel, Switzerland
Title: From pixels to phenotypes: Deep learning for analyzing high-content cellular images
Identifying phenotypes in high-content cellular images is challenging. We present a deep learning approach based on a multi-scale convolutional neural network (M-CNN) that, without segmentation and in one cohesive step, classifies cellular images into phenotypes by using directly and solely the images’ pixel intensity values. The only parameters in the approach are the weights of the neural network, which are automatically optimized based on a small number of training images annotated with phenotypic labels. The approach requires no object segmentation, no manual customization, and is applicable to single- or multi-channel images displaying single or multiple cells. We evaluated the classification performance of the approach on diverse cellular image datasets. The approach yielded overall a higher classification accuracy compared to state-of-the-art results, including those of other deep CNN architectures. In addition to using the network to simply obtain a yes-or-no prediction for a given phenotype, we use the probability outputs calculated by the network to quantitatively describe the phenotypes. Our study shows that these probability values correlate with chemical treatment concentrations, thus enabling chemical treatment potency estimation via CNNs. Our current supervised deep learning approach requires phenotypic labels to build the classification model. Future directions involving unsupervised deep learning methods that require no phenotypic labels for training will also be discussed.
Yolanda Chong, Vice President of Biology at Recursion Pharmaceuticals
Title: Identification of therapeutics for rare genetic diseases using phenotypic signatures.
There are approximately 6,000 rare diseases affecting an estimated 25 million people in the United States. Rare diseases disproportionately impact children, and many of these children do not live to see their 5th birthday. Development of therapeutic treatments for rare diseases has been slow, with less than 5% with an FDA-approved treatment. There is a clear unmet need for innovative approaches to rapidly develop new medicines for the millions of patients suffering from rare diseases.
Scientists at Recursion Pharmaceuticals have developed a highly efficient, broadly applicable, and readily scalable approach to drug discovery that simultaneously leverages automated biology and artificial intelligence. By using high-throughput microscopy to measure hundreds of sub-cellular structural changes, caused by pathogenic perturbations, we have been able to generate data-rich “biomarker-less” high-dimensional in vitro phenotypes. Our approach is amenable for use with complex in vitro disease models that are more translatable, increasing the potential for identifying relevant therapeutics.
Using our platform, high-throughput drug screens can be rapidly executed to uncover and repurpose promising drug candidates that rescue disease signatures. This unique approach allows us to efficiently model and find potential treatments for hundreds of traditionally refractory diseases, including Spinal Muscular Atrophy (SMA), Ataxia Telangiectasia (AT), and Neurofibromatosis Type 2 (NF2), making it ideally suited to tackle the urgent unmet medical need of patients with rare diseases.
Session-4: New Technologies
David Solecki, St. Jude Children's Research Hospital, Department of Developmental Neurobiology, TN
Title: Control of cell polarity, adhesion and germinal zone exit during neuronal progenitor differentiation
Cell polarity is a driving force that coordinates the choreography of neural development. How polarity signaling organizes the behavior of immature neurons and how polarity signaling cascades are regulated remain key questions facing the field of developmental neurobiology. These questions are critical to understand the pathology of neurodevelopmental diseases, where the production of neurons or their subsequent migration is defective. Studies combining necessity-sufficiency testing, cutting edge imaging technology and high throughput quantitative image analysis in the developing cerebellum show that a conserved polarity-signaling module, called the Pard complex, is essential for neuronal progenitor germinal zone exit by regulating cytoskeletal dynamics and cell-cell interactions needed for neuronal migration. I will present our progress identifying an upstream regulator of the Pard complex: an E3 ubiquitin ligase, Seven in Absentia, which mediates proteosomal degradation of Pard3; to control a shift from tangential to radial migration when cerebellar granule neurons leave their mitogenic niche, and drebrin; to control microtubule-actin crosslinking during CGN differentiation. While it is hypothesized that microtubule-actomyosin crosstalk is required for a neuron’s “two-stroke” nucleokinesis cycle, the molecular mechanisms controlling such crosstalk are not defined. By using the drebrin microtubule-actin crosslinking protein as an entry point into the cerebellar granule neuron system in combination with super resolution microscopy, we investigated how these cytoskeletal systems interface during migration. Lattice light-sheet and structured illumination microscopy revealed a proximal leading process nanoscale architecture wherein f-actin and drebrin intervene between microtubules and the plasma membrane. Functional perturbations of drebrin demonstrate that proximal leading process microtubule-actomyosin coupling steers the direction of centrosome and somal migration, as well as the switch from tangential to radial migration. Finally, drebrin function is antagonized by the Siah2 E3 ubiquitin ligase, suggesting a model for control of the microtubule-actomyosin interfaces during neuronal differentiation.
Christian Tardif, Jean-Pierre Bouchard, Pascal Gallant, Sebastien Roy, Ozzy Mermut, Biophotonics Program Institute Nationale Optic INO, Quebec City, Canada
Title: A new FLIM-FRET instrument with hyperspectral imaging for quantitative high content screening
Keywords—high content screening; high content analysis, FLIM, FRET, hyperspectral imaging
The combination of Fluorescence Lifetime Imaging Microscopy (FLIM) and hyperspectral imaging in drug screening research can provide information on the molecular specificity as well as the mechanism of action of candidate molecules. The Main drawbacks of such techniques are that they are typically time consuming, and are limited by the number of photons coming from the sample and the required spectral resolution. Conventional FLIM systems can record images of mCerulean3-Venus fluorescence protein pairs in live cells at a maximum rate of 1 image per 80 seconds for a 400x400 pixel image. Companion hyperspectral systems typically have 32 channels which either limit the spectral resolution or bandwidth of the system.
Given these limitations, we have developed a new FLIM and hyperspectral system for quantitative high content screening that images at a speed of greater than 0.1 frames per second, and without trade-off on the image quality or lifetime resolution, as encountered with commercial time correlated single photon counting. Our design and implementation of new FLIM-fluorescence resonance energy transfer (FRET) instrument is based on a parallelized detection scheme to overcome the pile-up limitation of conventional Time Correlated Single Photons Counting systems. Our instrument is complimented with an intelligent region of interest selection software and enhanced binning scheme to achieve better lifetime(s) accuracy. An interleaving method enables integrated hyperspectral detection with 64 spectral channels resulting in a spectral resolution of 8nm over a broadband range from 450nm to 850nm. A flexible supercontinuum source further provides flexibility for optimal tuning to any desired chromophore target.
In this presentation we explain lifetime measurement limitation, and how this new high speed confocal FLIM-FRET hyperspectral microscope, achieved eight times faster speed than the reference system without compromise to image resolution and accuracy in florescence decay lifetimes for quantitative high content screening. Recent results of benchmarking studies using live cells tested with this new apparatus will be shown.