3-D Co-culturing of Multiple Primary Cells for Drug Discovery, Diagnostics & Devices
Dawn Applegate, RegeneMed. Inc., USA
Response of 3-D liver co-cultures, obtained by growing all of the cells of native liver in a 3-D interconnecting porous scaffold to form tissue surrogates, to drug toxins over 90 days in culture demonstrates maintenance of liver-specific function including induction, inhibition, transporters and other factors important to drug discovery, diagnostics and medical devices. Development of other tissue types will also be discussed.

Human Embryonic Stem Cells - Applications for Drug Discovery & Medical Treatment
Veit Bergendahl, University of Wisconsin, USA

• Introduction; Human embryonic stem cells
• Discovery and current research
• Promises and Challenges
• Biomedical Applications
• Wisconsin Biotech with Human Embryonic Stem Cells

Human embryonic stem cells were first isolated only 10 years ago by James Thomson here in Madison and differ significantly in their biology and applications from mouse embryonic stem cells, which were discovered more than twenty years earlier. Much has been speculated about their use in research and biomedical applications, but it is clear now that a lot of research has to be done for some of these promises to come true. The talk will give an overview from the speaker’s personal point of view over the current status of human embryonic stem cell research and the biomedical applications that may have a promising chance to succeed in the near future. It will outline the challenges that will have to be overcome for some ambitious applications. It will also point out the benefits of human embryonic stem cell research that may sometimes be overseen like their ability to yield otherwise inaccessible amounts of biomaterial for basic research and their potential in toxicology. One example for a small molecule screen with human ES cells will be described and biomedical applications will be given by local companies specialized in this field.

Functional Tissue Engineering for Musculoskeletal Tissue Repair Using Autogenous Mesenchymal Stem Cells
David Butler, University of Cincinnati, USA
Mesenchymal stem cell (MSC) and drug therapies can accelerate repair of injured and diseased tissues, but benefits are usually assessed in monolayer. Functional tissue engineering will be presented whereby MSCs are introduced into 3-D scaffolds, stimulated with mechanical and chemical signals, and assessed for efficacy in culture and post surgery.

Rewiring GPCR Signaling in Stem Cells & Mice - Dissection, Design & Construction
Edward C. Hsiao, Gladstone Institutes, UCSF, USA
Our primary goal is to understand G-protein-coupled receptor (GPCR) signaling in key cell-fate decisions in cellular development. GPCRs are the largest class of cell-surface receptors and ideal therapeutic targets. We use human and mouse embryonic stem (ES) cells to test the role of specific GPCR pathways in tissue differentiation, computational methods (bioinformatics/GenMAPP) to identify/refine new pathways, and experimental molecular biology methods (RASSLs) to test these pathway circuits.

Primary Human Cell Systems for Drug Discovery: No More Black Box
Ellen L. Berg, BioSeek, Inc., USA
Assay systems that bridge the gap between molecular target discovery and clinical outcomes will improve our ability to identify safe and effective therapeutics. BioMAP Systems are an innovative platform that uses human cell-based disease models to assess the activities of drugs across a broad range of human tissue and disease biology and provide a better understanding of drug mechanisms. Key features of these assays include the utilization of primary human cells, often in co-cultures, and the use of selected combinations of input factors and readout measurements, optimized for therapeutic relevance. We have generated a large database of drug activity profiles in these assays and have developed analytical tools for the rapid deconvolution of active profiles to their target and pathway mechanisms. Applications of this technology for the selection of novel drug leads, characterization of drug function, including mechanism of action, identification of secondary or off-target activities, and clinical indication selection will be presented.

Heterotrimeric G proteins - How Your Cell Type Can Make a Difference in GPCR Signaling
Annette Gilchrist, Caden Bioscience, USA
Evidence for the concept that structurally diverse agonists for G protein coupled receptors (GPCRs) lead to distinct confirmations, and that these confirmations can differentially activate G protein signaling is mounting. We will review the topic for agonist-directed trafficking of GPCRs, and discuss how cell type selection can modulate preferential coupling and downstream signaling.

Incorporating Tissue Architecture and Gradients into Cellular Assays Using Existing HTS Infrastructure
Steven Hayes, Bellbrook Labs, USA
Cellular responses can be dramatically altered by the tissue microenvironment, including paracrine signals, which are often presented as gradients, and 3-dimensional matrices. Described is an approach for incorporating these aspects of tissue biology into cellular assays, using a highly miniaturized, and fully automatable format, compatible with existing liquid handling robotics. By combining tissue-like environment with primary cells, assays can be made to better resemble the in vivo condition.

Drug Discovery Targeting Chemokine Receptors: Applications for AIDS, Cancer and Stem Cell Therapies
Ziwei Huang, Burnham Institute for Medical Research; Runquan Zhang, Srinivas Duggineni, Xuejun Zhu, Jing An, Sameer Kawatkar, Harsukh Gevariya, Mi-Youn Lim, Pak-Nei Hon, Steve Eisold
Chemokines are small chemoattractant cytokines that regulate a wide array of biological and pathological processes. Chemokine receptors have become important and attractive targets for drug discovery. As members of the G protein-coupled receptor (GPCR) superfamily, chemokine receptors are also useful models to study ligand-receptor interactions and signaling mechanisms of GPCRs. Chemokine receptors such as CXCR4 and CCR5 play an important role in HIV-1 pathogenesis, including HIV-associated dementia in the brain. Furthermore, chemokine receptors such as CXCR4 and others mediate a wide variety of physiological and pathological functions including cancer metastasis, autoimmune diseases, transplant rejection, and stem cell migration. Particularly, stem cell migration mediated by chemokines and receptors offers an exciting avenue of developing new stem cell-based regenerative medicine. We have recently developed a powerful platform technique combining medicinal chemistry, protein structural modeling and structure-based ligand design to generate new synthetic molecules termed SMM (synthetically and modularly modified)-chemokines chemically engineered with very high receptor selectivity and affinity, and much reduced side effect and toxicity (ref. 1-3).   These molecules have been shown to be valuable probes of receptor binding and signaling mechanisms and new therapeutic leads for treating various human diseases mediated by chemokine receptors. In this presentation, we will discuss the latest discovery and application of these novel synthetic molecules for studying the molecular mechanisms of ligand binding and receptor signaling of GPCRs and developing new drugs for AIDS, cancer, autoimmune diseases, and stem cell-based therapies.

Using Primary Cells for HTS Follow-up in a Physiologically Relevant System
Eric Johnson, Merck, USA
The demands of high-throughput screening, including a small reaction volume and large signal-to noise ratio, necessitate the use of engineered systems to identify agonists and antagonists of many target proteins. Secondary studies of hits found in HTS campaigns using physiologically relevant cells should aid the projects teams' selection of leads to enhance the probability of identifying a small molecule that will prove successful in later stages of drug development including animal models and clinical trials.

Applications of Stem Cells to Target Validation in Drug Development
Dan Marshak, PerkinElmer, USA
Mammalian stem cells are categorized as "embryonic" if derived from the inner cell mass of the pre-implantation embryo, or as "somatic" if derived from tissues of the organism. Both sorts of stem cells are potentially useful for biomolecular analysis, in assays for toxicity or efficacy testing. In particular, stem cells allow the measurement of normal tissue differentiation which is often not possible with cell lines. Examples using human mesenchymal stem cells include bone, cartilage, and adipose lineage differentiation. These events are dependent upon key signaling pathways, including MAP/ERK kinases. Furthermore, measurement of the chromosomal stability of stem cells in long-term culture is a key parameter for quality assurance, and analysis can be done accurately and rapidly by comparative genomic hybridization on genomic arrays.

Human Primary Cell-Based In Vitro Experimental Systems for the Evaluation of Human Drug Safety
Albert Li, The ADMET Group LLC, USA
Accurate prediction of human drug toxicity is a major challenge for drug development. In vitro, human-based experimental systems can provide human-specific information that cannot be obtained with laboratory animals. This presentation reviews the application of in vitro, human-based, experimental systems in the evaluation of drug toxicity. A novel technology, the Integrated Discrete Multiple Organ Co-culture (IdMOC) system, which allows the co-culturing of cells from multiple organs as physically separated cultures interconnected by an overlying medium, will be described as a refined in vitro system for the evaluation of human drug toxicity. The IdMOC allows multiple organ interactions which is critical to drug toxicity and is a major deficiency for the routinely-used single cell type in vitro systems.

Stem Cells: Applications & Opportunities in Modern Drug Discovery (Plenary)
John McNeish, Pfizer, USA
Stem cells offer new opportunity to drug discoverer's in identifying new targets, screening for novel lead molecules, and assessing drug efficacy and safety pre-clinically. We have applied mouse ES and human adult stem cells to develop screens using RNAi and small molecules in primary and secondary cell-based assays. The advantages of stem cells in these applications, including a focus on the physiologic and pharmacologic relevance of stem cell derived cell types versus transformed or primary cells will be discussed.

Influence of Cell Phenotype on Pharmacology
Robert E. Pacifici, Ph.D. - Chief Scientific Officer
Huntington disease (HD) is a fatal, autosomal-dominant, neurodegenerative disorder for which there are currently no therapies. HD is caused by single known gene (htt) that is ubiquitously expressed, yet the preponderance of observable pathology is localized to the medium spiny neurons within the striatum (MSNs). Recent advances in stem cell biology suggest that soon, we may be able to orchestrate and validate the in vitro differentiation of either embryonic or neural stem cells into defined neuronal cell types. Assays developed using such cell populations should be more productive in elucidating the underlying susceptibility of MSNs to the htt insult than systems built around generic (non-neuronal) lines.

Nuclear Receptor Activation: Comparison Between Primary Hepatocytes & Engineered Cell Lines
Judy Raucy,  Puracyp,Inc., USA
A simplified, reproducible, and high-throughput tool for detecting the ability of candidate drugs to activate nuclear receptors was developed. When compared to human hepatocytes, results generated in the engineered cells were similar but were less costly, time consuming, and exhibited less variability. Thus, our cell lines can be used during drug discovery.

Embryonic Stem Cell Technologies: Applications in Drug Discovery & Development
Ralph Snodgrass, VistaGen Therapeutics, Inc.
Embryonic stem ("ES") cells provide a clinically relevant biological system that offers an unprecedented powerful discovery tool for understanding many significant diseases such as cancer, Alzheimer's, diabetes, stroke, heart disease, and various neurological diseases and conditions. The in vitro differentiation of ES cells offers the potential to develop powerful screens for drug effects, and ultimately developing novel therapies for these devastating diseases. This talk will give examples of four ES cell systems -- pancreas, cardiovascular, hepatic, and neuronal -- which VistaGen is using for R&D to identify and develop novel drugs, and for predictive pharmacology and toxicology assays. These systems will highlight and demonstrate the utility of embryonic stem cells as research tools for drug discovery, drug screening, and drug development.