Anja van de Stolpe

Prediction of drug-induced cardiotoxicity using human embryonic stem cell-derived cardiomyocytes

Recent withdrawals of prescription drugs from clinical use because of unexpected side effects on the heart have highlighted the need for more reliable cardiac safety pharmacology assays. Block of the human Ether-a-go go Related Gene (hERG) ion channel in particular is associated with life-threatening arrhythmias, such as Torsade de Pointes (TdP). Here we investigated human cardiomyocytes derived from pluripotent (embryonic) stem cells (hESC) as a renewable, scalable, and reproducible system on which to base cardiac safety pharmacology assays. Analyses of extracellular field potentials in hESC-derived cardiomyocytes (hESC-CM) and generation of derivative field potential duration (FPD) values showed dose-dependent responses for 12 cardiac and noncardiac drugs. Serum levels in patients of drugs with known effects on QT interval overlapped with prolonged FPD values derived from hESC-CM, as predicted. We thus propose hESC-CM FPD prolongation as a safety criterion for preclinical evaluation of new drugs in development. This is the first study in which dose responses of such a wide range of compounds on hESC-CM have been generated and shown to be predictive of clinical effects. We propose that assays based on hESC-CM could complement or potentially replace some of the preclinical cardiac toxicity screening tests currently used for lead optimization and further development of new drugs.

Workshop meeting report Organs-on-Chips : human disease models

The concept of “Organs-on-Chips” has recently evolved and has been described as 3D (mini-) organs or tissues consisting of multiple and different cell types interacting with each other under closely controlled conditions, grown in a microfluidic chip, and mimicking the complex structures and cellular interactions in and between different cell types and organs in vivo, enabling the real time monitoring of cellular processes. In combination with the emerging iPSC (induced pluripotent stem cell) field this development offers unprecedented opportunities to develop human in vitro models for healthy and diseased organ tissues, enabling the investigation of fundamental mechanisms in disease development, drug toxicity screening, drug target discovery and drug development, and the replacement of animal testing. Capturing the genetic background of the iPSC donor in the organ or disease model carries the promise to move towards “in vitro clinical trials”, reducing costs for drug development and furthering the concept of personalized medicine and companion diagnostics. During the Lorentz workshop (Leiden, September 2012) an international multidisciplinary group of experts discussed the current state of the art, available and emerging technologies, applications and how to proceed in the field. Organ-on-a-chip platform technologies are expected to revolutionize cell biology in general and drug development in particular.

Biology-inspired microphysiological system approaches to solve the prediction dilemma of substance testing

The recent advent of microphysiological systems - microfluidic biomimetic devices that aspire to emulate the biology of human tissues, organs and circulation in vitro - is envisaged to enable a global paradigm shift in drug development. An extraordinary US governmental initiative and various dedicated research programs in Europe and Asia have led recently to the first cutting-edge achievements of human single-organ and multi-organ engineering based on microphysiological systems. The expectation is that test systems established on this basis would model various disease stages, and predict toxicity, immunogenicity, ADME profiles and treatment efficacy prior to clinical testing. Consequently, this technology could significantly affect the way drug substances are developed in the future. Furthermore, microphysiological system-based assays may revolutionize our current global programs of prioritization of hazard characterization for any new substances to be used, for example, in agriculture, food, ecosystems or cosmetics, thus, replacing laboratory animal models used currently. Thirty-six experts from academia, industry and regulatory bodies present here the results of an intensive workshop (held in June 2015, Berlin, Germany). They review the status quo of microphysiological systems available today against industry needs, and assess the broad variety of approaches with fit-for-purpose potential in the drug development cycle. Feasible technical solutions to reach the next levels of human biology in vitro are proposed. Furthermore, key organ-on-a-chip case studies, as well as various national and international programs are highlighted. Finally, a roadmap into the future is outlined, to allow for more predictive and regulatory-accepted substance testing on a global scale.

Lessons from Toxicology: Developing a 21st-Century Paradigm for Medical Research

Summary Biomedical developments in the 21st century provide an unprecedented opportunity to gain a dynamic systems-level and human-specific understanding of the causes and pathophysiologies of disease. This understanding is a vital need, in view of continuing failures in health research, drug discovery, and clinical translation. The full potential of advanced approaches may not be achieved within a 20th-century conceptual framework dominated by animal models. Novel technologies are being integrated into environmental health research and are also applicable to disease research, but these advances need a new medical research and drug discovery paradigm to gain maximal benefits. We suggest a new conceptual framework that repurposes the 21st-century transition underway in toxicology. Human disease should be conceived as resulting from integrated extrinsic and intrinsic causes, with research focused on modern human-specific models to understand disease pathways at multiple biological levels that are analogous to adverse outcome pathways in toxicology. Systems biology tools should be used to integrate and interpret data about disease causation and pathophysiology. Such an approach promises progress in overcoming the current roadblocks to understanding human disease and successful drug discovery and translation. A discourse should begin now to identify and consider the many challenges and questions that need to be solved.

Towards a 21st-century roadmap for biomedical research and drug discovery: Consensus report and recommendations

Decades of costly failures in translating drug candidates from preclinical disease models to human therapeutic use warrant reconsideration of the priority placed on animal models in biomedical research. Following an international workshop attended by experts from academia, government institutions, research funding bodies, and the corporate and non-governmental organisation (NGO) sectors, in this consensus report, we analyse, as case studies, five disease areas with major unmet needs for new treatments. In view of the scientifically driven transition towards a human pathways-based paradigm in toxicology, a similar paradigm shift appears to be justified in biomedical research. There is a pressing need for an approach that strategically implements advanced, human biology-based models and tools to understand disease pathways at multiple biological scales. We present recommendations to help achieve this.

What Are Stem Cells

A stem cell is a cell that can divide to give rise to a new copy of itself and at least one other specialized (differentiated) cell type. Although this broad definition provides a useful framework, much still remains to be discovered about the different types of stem cells in the body, their common as well as unique properties, and how and for what applications they can be used in research, medical practice, biotechnology, and pharmacology. This chapter discusses stem cells and their key properties in detail. In addition to being able to divide and produce new copies of themselves (self-renew), stem cells can also differentiate, or specialize, into other cell types. If the stem cell is able to form all cell types of the embryo and adult, including germ cells (eggs and sperm) and the extra-embryonic structures such as placenta, it is considered totipotent. In order to be able to divide without losing the stem cell pool for later use, a stem cell is capable of multiplying by dividing in two, but after each cell division at least one of the two daughter cells retains the original stem cell properties. Stem cells can be divided into two types: embryonic stem cells and adult stem cells. Embryonic stem cells are derived from a very early embryo, and adult stem cells are found in postnatal tissues, not only of the body but also the umbilical cord. Although much controversy still exists about the potency of adult stem cells and their ability to form different cell types, adult stem cells are generally considered to be either multipotent or unipotent.

Cytostretch, an Organ-on-Chip Platform

Organ-on-Chips (OOCs) are micro-fabricated devices which are used to culture cells in order to mimic functional units of human organs. The devices are designed to simulate the physiological environment of tissues in vivo. Cells in some types of OOCs can be stimulated in situ by electrical and/or mechanical actuators. These actuations can mimic physiological conditions in real tissue and may include fluid or air flow, or cyclic stretch and strain as they occur in the lung and heart. These conditions similarly affect cultured cells and may influence their ability to respond appropriately to physiological or pathological stimuli. To date, most focus has been on devices specifically designed to culture just one functional unit of a specific organ: lung alveoli, kidney nephrons or blood vessels, for example. In contrast, the modular Cytostretch membrane platform described here allows OOCs to be customized to different OOC applications. The platform utilizes silicon-based micro-fabrication techniques that allow low-cost, high-volume manufacturing. We describe the platform concept and its modules developed to date. Membrane variants include membranes with (i) through-membrane pores that allow biological signaling molecules to pass between two different tissue compartments; (ii) a stretchable micro-electrode array for electrical monitoring and stimulation; (iii) micro-patterning to promote cell alignment; and (iv) strain gauges to measure changes in substrate stress. This paper presents the fabrication and the proof of functionality for each module of the Cytostretch membrane. The assessment of each additional module demonstrate that a wide range of OOCs can be achieved.

Innovative human-specific investigational approaches to autoimmune disease

Autoimmune diseases are exclusively human diseases with a complex genetic background and variable clinical presentation, of which the underlying pathophysiology is insufficiently understood. Current treatment is mainly empirical with limited efficacy and significant side effects. To develop more effective targeted therapy for personalized treatment, understanding of the human pathophysiology is crucial, implying a high need for human investigational disease models. Using the example of anti-neutrophil cytoplasmic antibody (ANCA) autoimmune vasculitis, the concept of building an in vitro organ-on-chip type human disease model, consisting of cultured organ-specific vascular tissue in interaction with relevant immune system components (e.g. lymph node and thymus tissue) is presented. This in vitro approach makes use of advances in engineering and human stem cell technologies, enabling derivation of pluripotent stem cell lines from patients, differentiation to required cell types, and incorporation in microfluidic chip-based culture systems to optimally mimic in vivo disease conditions. Knowledge-based computational disease modeling is introduced as a valuable complementary tool to generate an integral mechanistic picture of the disease. Combining these multidisciplinary developments promises breakthroughs in understanding autoimmune disease and targeted drug development, while simultaneously reducing use of animal models. Current state of the art and issues remaining to be solved are discussed.

The Biology of the Cell

This chapter introduces the most important molecular and cell biological principles needed to understand stem cells. Humans and animals, as well as plants and trees, contain many different functional organs and tissues. They are composed of a large variety of cells. Cells are therefore the basic building blocks, which make up the organism. All animal cells have a similar structure: an outer layer called the plasma membrane, made up of a double layer of lipid molecules, and an inner fluid known as cytoplasm. Most cell organelles are themselves separated from the cytoplasm by their own membranes. The form of the cell is determined and supported by the cytoskeleton, a flexible scaffolding composed of polymers of protein molecules which form a network that shapes the cell and allows it to move and walk. Inside the cell countless proteins—sometimes in cooperation with RNA molecules—facilitate the chemical and physical reactions and transport of other molecules required to carry out specific cellular functions. Although cells can have different shapes and functions, the DNA sequence in all cells of a given individual is in principle identical (with the exception of certain blood cells). Other prominent structures in the cell are the mitochondria. These organelles are present in large numbers and generate the energy required by the cell.

Wnt signaling in multiple myeloma: a central player in disease with therapeutic potential

Multiple myeloma is the second most frequent hematological malignancy in the western world and remains incurable, predominantly due to acquired drug resistance and disease relapse. The highly conserved Wnt signal transduction pathway, which plays a key role in regulating cellular processes of proliferation, differentiation, migration, and stem cell self-renewal, is associated with multiple aspects of disease. Bone homeostasis is severely disturbed by Wnt antagonists that are secreted by the malignant plasma cells in the bone marrow. In the vast majority of patients, this results in osteolytic bone disease, which is associated with bone pain and pathological fractures and was reported to facilitate disease progression. More recently, cumulative evidence also indicates the importance of intrinsic Wnt signaling in the survival of multiple myeloma cells. However, Wnt pathway-activating gene mutations could not be identified. The search for factors or processes responsible for Wnt pathway activation currently focuses on aberrant ligand levels in the bone marrow microenvironment, increased expression of Wnt transcriptional co-factors and associated micro-RNAs, and disturbed epigenetics and post-translational modification processes. Furthermore, Wnt pathway activation is associated with acquired cell adhesion-mediated resistance of multiple myeloma cells to conventional drug therapies, including doxorubicin and lenalidomide. In this review, we present an overview of the relevance of Wnt signaling in multiple myeloma and highlight the Wnt pathway as a potential therapeutic target for this disease.