Thursday, January 17

7:30-8:00 Morning Coffee

SCREENING IN PRIMARY AND LIVE CELLS

8:00-8:25 Development of Cell-Based Assays for Study of Cardiac Disease and Drug Effi cacy/Toxicity Using Primary Cardiac Myocytes and Cardiac-Derived Cell Lines in a High-Content Format
Anthony Davies, Ph.D., High-Content Research Facility Director, Clinical Medicine, Trinity College Dublin
We are currently engaged in the development of a range of cell-based assays that utilize both primary adult cardiac myocytes and immortalized cell lines. To facilitate the development of these new assay tools, a detailed examination of both biochemical and structural changes in primary adult cardiac muscle cells has been conducted. These studies have yielded valuable information regarding the behavior of primary cardiac muscle cells in their quiescent and active states. Currently our work is focused on the use of muscle cells derived from a cardiac myoblast cell line as a basis for primary and secondary cardiac screens. Our ultimate goal is to develop a stable and biologically relevant assay that can be deployed and utilized in an automatable HCS format.

8:25-8:50 Title to Be Announced
Jeffrey Price, M.D., Ph.D., Associate Professor, Burnham Institute for Medical Research

8:50-9:15 High-Resolution Imaging of Living Cells in Flow Suspension Using Axial-Tomography: 3D Imaging Flow Cytometry
Spencer L. Shorte, Ph.D., Director Imagopole, Group Leader Plateforme d’Imagerie Dynamique (PFID), Institut Pasteur
Conventional flow cytometry methods report integrated averaged cellular fl uorescence signal intensities per cell analyzed, albeit with throughput rates of thousands of cells per second. Further, this is combined with the powerful utility to subsequently sort and/or retain the cells of interest. However, these methods mainly lack the fundamental ability to reveal spatial information on the sub-cellular compartmentalization of signals. This limitation has prompted efforts by some manufacturers to produce state-of-the art commercial flow cytometry systems allowing fl uorescence images to be recorded by an imaging detector (high-content imaging cytometry). Nonetheless, there remains an immediate and growing need for technologies facilitating three-dimensional (3D) fl uorescence analysis of cells in fl ow and/or suspension (“ultra”-high-content imaging
cytometry). Here, we report a novel methodological approach to this problem that combines micro-fluidic-fl ow, and microelectrode-dielectric-fi eld control to manipulate, image and finally sort individual cells in suspension. The method providing for 3D optical imaging of living intact cells in suspension also offers important advantages for imaging studies on cells in suspension. In particular, we report the system’s utility for confocal axial tomography using microrotation imaging that we demonstrate greatly enhances 3D optical resolution sampled from single intact living cells in suspension.

9:15-9:50 Networking Coffee Break

SCREENING IN MODEL ORGANISMS

9:50-10:15 High-Content Analysis of Zebrafish Angiogenesis by Cognition Network Technology
Andreas Vogt, Ph.D., Assistant Professor, Pharmacology, University of Pittsburgh
Advances in high-throughput screening and laboratory automation have substantially improved the speed of target-based drug discovery but it is unclear whether new drugs affecting single targets have improved therapeutic efficacy. An increasingly popular sentiment is that better models are needed to improve the quality of new drug candidates.  Whole organisms could provide such models but technical challenges limit their utility as drug screening tools. The zebrafish embryo may be amenable to high-throughput
screening methodology. We developed a fully automated analysis of angiogenesis in a transgenic zebrafish line. Images of fluorescent zebrafish embryos in 384 well plates were acquired on a high-content reader and analyzed by object-based image processing using Cognition Network Technology (CNT). The CNT algorithm detected embryos regardless of orientation, partitioned them into regions of biological relevance, and quantified the growth of intersegmental blood vessels (ISV). The assay delivered graded responses and documented antiangiogenic activity of a novel small molecule microtubule perturbing agent. The results demonstrate that it is feasible to adapt image-based high-content screening methodology to measure complex phenotypes in whole organisms.

11:00-1:00 Lunch on your Own

1:00-2:15 End-User/Vendor Panel Discussion

2:15-2:30 Break

SCREENING IN TISSUES

2:30-2:55 Challenges and Successes of Compound Screening in Brain Tissue
O. Joseph Trask, Jr., Ph.D., Head of Cellular Imaging Technologies, Center for Drug Discovery, Department of Neurobiology, Duke University
Neurodegenerative diseases are extremely diffi cult targets to investigate due to the complexity and
heterogeneity of the brain. For such reasons, simple non-neuronal cell linebased screens have proven to be limited in identifying promising new clinical candidates. We have thus developed an acute rat brain slice-based assay platform for Huntington disease (HD), a genetic neurodegenerative disease for which there is currently no effective treatment or cure. In this platform, novel imaging techniques are used to quantitate directly the neuroprotective properties of small molecule drug candidates against neurodegeneration induced by biolistically introduced mutant HD DNA constructs. In this talk I will discuss the challenges and successes of assay development, validation, and results of screening compounds in this model.

2:55-3:20 Application of High-Content Screening to Measure In Vivo Pharmacodynamic Response in Aurora Kinase Inhibitor Program
Douglas Bowman, Ph.D., Senior Manager, Imaging Sciences, Molecular and Cellular Oncology, Millennium Pharmaceuticals, Inc.
CS and related imaging technologies are widely used for in vitro assays throughout a drug discovery program including target identifi cation, lead optimization, and pre-clinical toxicology. These image-based assays provide us with an understanding of the biological effect of our compound, whether it is an upstream, direct, or downstream effect of target inhibition. We have extended HCS technologies to preclinical and clinical studies to characterize the mechanistic effects (pharmacodynamic response) of the drug. I will discuss these assays and the inherent challenges of adopting HCS techniques to tissue in our Aurora A Kinase inhibitor program.

3:20-3:40 Refreshment Break

SCREENING IN STEM CELLS

3:40-4:05 High-Content Screening for Molecules that Modulate Pancreatic Beta-Cell Growth and Differentiation
Fred Levine, M.D., Ph.D., Professor, UCSD Department of Pediatrics; Adjunct Professor, The Burnham Institute for Medical Research
Regeneration of pancreatic beta-cell by neogenesis from stem/progenitor cells or by enhancing beta-cell replication is an attractive approach to treating diabetes. Unfortunately, little is understood about the mechanisms that control regeneration. We have been using high-throughput, high-content screening of cell line models and human primary pancreatic cells to detect molecules that modulate beta-cell growth and differentiation.

4:05-4:30 A Chemical Approach to Stem Cell Biology
Sheng Ding, Ph.D., Associate Professor, Departments of Chemistry and Cell Biology, The Scripps Research Institute
Recent advances in stem cell biology may make possible new approaches for the treatment of a number of diseases. For better understanding of the molecular mechanisms that control stem cell fates as well as an improved ability to manipulate these cells, we have developed and implemented chemical and functional genomic tools, including highthroughput cell-based phenotypic screens of arrayed chemical, cDNA and RNAi libraries, and performed genomic and proteomic profi ling of homogenous undifferentiated/selfrenewing
or selectively differentiated cell populations under chemically defi ned conditions. In-depth biochemical and functional assays in vitro and in vivo, have been applied to identify and further characterize small molecules and genes that can control stem cell fate in various systems.

4:30-4:55 Non-Invasive Monitoring of Human Embryonic Stem Cell Growth, Death and Differentiation by Texture Analysis of Images
Paul Sammak, Ph.D., Research Associate Professor, Magee Women’s Research Institute, Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh
Quantitative monitoring of the expansion and fate of stem cells is important for routine culture and for drug screening. We have found that multiscalar texture analysis can be used to classify stem cell colonies into distinct groups based on overall morphology of phase contrast images and be used to monitor cell health and phenotype. This capacity will be especially important for reproductive toxicology using primitive human stem cells of various lineages. Varying densities of mouse embryonic fi broblasts were used to support human stem cell self-replication and neuronal differentiation. Statistically different categories of differentiation were determined based on non-destructive imaging and validated by molecular markers including Oct-4 (pluripotent cells) and nestin (neurectoderm and neural stem cells). Texture analysis can be used to measure amorphous biological samples where standard geometric measurements of shape and size are insuffi cient for quantitative, statistically signifi cant measures of cell morphology.

4:55-5:00 Closing Remarks

5:00 Close of Conference