Abstract Book: Abstracts Plenary Lecturers 1

Abstract Book: Abstracts Plenary Lecturers 1 Embryonic development of hematopoietic stem cells: implications for clinical use C. Robin Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands Hematopoietic Stem Cells (HSCs) are responsible for constant replenishment of all blood cells during the entire life of an organism. Because of their properties, HSCs are the pivotal cells in regenerative therapies of the blood system and in some cancer treatments. Several stem cell sources are currently applied clinically but the low number of HSCs available, particularly in cord blood, is a limiting factor. One of the major goals in the field of regenerative medicine is to get clinically relevant amount of transplantable HSCs by in vitro expansion of already existing HSCs or by reprogramming somatic cells into transplantable HSCs, notably by expression of transcription factors. So far, success has been limited. HSCs are initially generated during embryonic development. First detected in the aorta, they subsequently transit through different anatomical niches where they will expand before colonizing the bone marrow. Although the existence of HSCs has been known for more than 50 years and despite extensive research, there is still uncertainty regarding the mechanisms that underlie HSC production, self-renewal and differentiation. Over the past years, we demonstrated by using live imaging technology that hematopoietic cells, organized in clusters, are de novo generated from specialized hemogenic endothelial cells in the aorta of the mouse embryo. These clusters (named IntraAortic Hematopoietic Clusters or IAHCs) contain the first HSCs produced during development. We recently demonstrated that IAHCs mainly contain pre-HSCs that will progressively mature into HSCs. The analysis of pre-HSCs at the molecular level by RNA-sequencing reveals important successive steps of maturation, occurring in vivo at different developmental time points. The novel insights in pre-HSC to HSC transition represent an important source of information (molecular pathways and cellular events) needed to pave the way for successful in vitro production of transplantable HSCs.

2 Metabolism and cardiac regeneration T. Doenst Department of Cardiothoracic Surgery, University of Jena, Jena, Germany Regeneration in cardiovascular medicine is generally considered replacement of injured or irreversibly damaged cardiac cells with new ones. These new cells can come from exogenous sources by the injection of stem cells, by stimulating proliferation of viable cardiomyocytes, or by reprogramming of fibroblasts. In the broader biology perspective, regeneration is the process of renewal, restoration, and growth, that makes genomes, cells, organisms, and ecosystems resilient to natural fluctuations or events that cause disturbance or damage. Regeneration here mainly refers to the morphogenic processes that characterize the phenotypic plasticity of traits allowing multi-cellular organisms to repair and maintain the integrity of

their physiological and morphological states. Thus, regeneration may also encompass repair processes for injured or dysfunctioning cells, as occurs during ischemia/reperfusion, induction of apoptosis or pressure overload and other causes of heart failure. However different definitions and perspectives may be, all the above addressed processes share one common feature. They require a well-functioning ATP producing machinery because they all require vast amounts of energy. The main producing site of ATP in the cell is the mitochondrion. It is therefore no surprise to find mitochondria involved in critical steps of all of the above mechanisms. This presentation will review the role of mitochondria and energy metabolism in the broader sense of cardiac regeneration and will highlight metabolic steps that are critical for successful regeneration under different pathological conditions. The presentation will conclude that energy metabolism is not only an important contributor to regeneration but also a potential therapeutic target.

3 A high throughput toolbox for tissue engineering C.A. van Blitterswijk, N. Rivron, R. Truckenmueller & J. De Boer Department Tissue Regeneration, University of Twente, Enschede, The Netherlands A need for new technology platforms: Since the introduction of the term Tissue Engineering the field has grown impressively both as regards the number of involved researchers, disciplines and output in the form of papers, patents etc. In spite of this, the actual successes in the form of e.g. clinical and commercial applications are still lagging behind expectations. There may be many reasons underlying this phenomenon and among those we certainly find an initial underestimation of the complexity of the field. However, even as we currently better realize the fundamental challenges of our field the mere acceptance of these challenges will not solve the issue. In order to overcome the fundamental hurdles that stand at the basis of our field it will be essential to develop novel technologies and/or strategies upon which we can build future successes and at a faster rate as done previously. After roughly a decade of efforts to generate effective tissue engineering therapies in the field of musculoskeletal regeneration/repair our group concluded that conventional tissue engineering methodologies would not suffice to achieve the results that we had set as a goal for ourselves. Largely, these conventional methodologies were based on an assumption that success would automatically follow from a fundamental understanding of the associated elements like for instance cell behavior and material aspects and, when these were lacking, by adapting protocols previously developed for similar applications. In reality it turns out that this approach, although it follows a conventional scientific way of thinking, might not be the most optimal approach to move forward in our field. Time and time again we and others would discover that what looked promising in vitro would either not work in vivo or through completely different mechanisms than originally foreseen. As it turns out the complex interactions between intracellular pathways, between cells, tissues and organs and our scaffolds at the macro, meso, micro

© 2014 The Authors. European Journal of Clinical Investigation © 2014 Stichting European Society for Clinical Investigation Journal Foundation European Journal of Clinical Investigation, 44 (Suppl. 1), 1–3

2 Abstract Book: Abstracts Plenary Lecturers and nano level seem fundamentally unpredictable, with only few exceptions. To this we may add that the scientific methods that we apply are inherently slow. Typically one will test a few formulations or protocols in vitro, will then select the best and move on to further in vivo screening in small experimental animals followed by functionality testing in large animals in order to end in human trials. This method easily takes a decade, if successful. If anywhere in this trajectory the system fails we start all over again. We are of the opinion that this conventional system is inefficient in several ways. First of all it underestimates the severe variation in response to our hybrid constructs that we encounter between the various species that we use, both from purely physicochemical properties (like size) and genetic interspecies differences but also related to the frequently underestimated distinct variation between individuals. Although this latter aspect is less prominent in, our frequently inbred, experimental animals, it is hard reality in our human recipients. Secondly, as we start the process with few experimental variations in combination with an incomplete, at best, understanding of the systems we apply or work with we are bound to end up with suboptimal technologies, even when successful. Currently, we are trying to deal with this in our lab while developing a new technology toolbox that might, at least partially overcome, the above issues. The platform is based on four pilars. First we use high throughput assessment of biologically active agents for our cells as is common in drug discovery. As big pharma, with a frequently much better biological fundamental knowledge base, is forced in this direction it is bound to be useful in our much smaller field with technologically more complex applications. Although, the hit compounds may still fail in the following process, at least we will now be testing the most powerful among many instead of the best of few. Second, as our systems are so complex we will frequently have to use multiple compounds simultaneously. As these tend to interact, routine lab protocols will not suffice to obtain the optimal combination of factors which is why we now invest in lab on a chip platforms to allow the testing of multifactorial combinations in the shape of multiplex arrays. Third, as we see the rise of instructive, smart, scaffolds and/or materials and also here lack most of the fundamental knowledge on the driving mechanism(s) we have implemented array technology to assess the instructive abilities of libraries with millions of surface topographies in an approach referred to as materiomics. Finally in order to overcome the uncertainties and limitations of animal experimentation we have embarked on developing platforms that allow us to generate complex tissues in the meso to macro range in high throughput version. In addition this approach will create possibilities to assemble such microtissues into larger structure which may mimic the complex structure of organs or parts thereof. The combination of the above technologies will now offer us a toolbox that is better fit for dealing with tissue engeering complexity than most conventional techniques applied today.

4 Lgr5 stem cells in self-renewal and cancer H. Clevers Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences & University Medical Centre Utrecht, Utrecht, The Netherlands

get gene, transcribed in colon cancer cells. Two knock-in alleles revealed exclusive expression of Lgr5 in cycling, columnar cells at the crypt base. Using lineage tracing experiments in adult mice, we found that these Lgr5+ve crypt base columnar cells (CBC) generated all epithelial lineages throughout life, implying that they represent the stem cell of the small intestine and colon. Lgr5 was subsequently found to represent an exquisitely specific and almost ‘generic’ marker for stem cells, including in hair follicles, kidney, liver, mammary gland, inner ear tongue and stomach epithelium. Single sorted Lgr5+ve stem cells can initiate ever-expanding crypt-villus organoids, or so called ‘mini-guts’ in 3D culture. The technology is based on the observation that Lgr5 is the receptor for a potent stem cell growth factor, R-spondin. Similar 3D cultures systems have been developed for the Lgr5+ve stem cells of stomach, liver, pancreas and kidney. Intestinal cancer is initiated by Wnt pathway-activating mutations in genes such as APC. As in most cancers, the cell of origin has remained elusive. Deletion of APC in stem cells, but not in other crypt cells results in progressively growing neoplasia, identifying the stem cell as the cell-of-origin of adenomas. Moreover, a stem cell/progenitor cell hierarchy is maintained in early stem cell-derived adenomas, lending support to the “cancer stem cell”-concept.

5 Regenerative medicine: ethics from bench to bedside A.L. Bredenoord Julius Center, Department of Medical Humanities, University Medical Center Utrecht, Utrecht, The Netherlands Background: Regenerative medicine based interventions such as stem cell transplantation, gene transfer and tissue engineering provide innovative treatment prospects for a wide array of disorders. The amount of (early) clinical studies is expected to rapidly expand in the near future. Regenerative medicine however faces several layers of cross-cutting complexity, not only technological but also with respect to the introduction into clinical trials, patient care and society. Here we identify the main ethical challenges of translating regenerative medicine from bench to bedside to society – and back. Materials and methods: Literature review, qualitative research and ethical analysis. Results: Regenerative medicine is different from traditional pharmaceuticals due to the novelty and complexity of the intervention (such as different kind of stem cells), the invasiveness of the procedure, and the novel aim of regeneration. These characteristics have an impact on the science and ethics of clinical trials, for example with regard to the risk-benefit balance, designing a trial in terms of outcome measures and control group, participant selection and informed consent. In addition, regenerative medicine will also influence society, encompassing both hard impacts (such as costs and risks) and soft impacts (such as changing perceptions on health, disease, aging and justice). Conclusion: Regenerative medicine is promising for many disease conditions but also faces challenges that simultaneously involve widely divergent fields of expertise. In order to guarantee ethically sound innovation, constructive interdisciplinary collaborations and dialogue are necessary to proactively identify and evaluate the ethics of translational regenerative medicine.

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We originally defined Lgr5 as a Wnt tar-

© 2014 The Authors. European Journal of Clinical Investigation © 2014 Stichting European Society for Clinical Investigation Journal Foundation European Journal of Clinical Investigation, 44 (Suppl. 1), 1–3

Abstract Book: Abstracts Plenary Lecturers 3

6 Radiographic progression in ankylosing spondylitis – how can we explain it and how can we modify it? X. Baraliakos Rheumazentrum Ruhrgebiet Herne, Ruhr-University Bochum, Herne, Germany Inflammatory and chronic/structural changes of the spine are pathognomonic in patients with ankylosing spondylitis (AS). In the last years, in the evaluation of the natural course of AS has been in the focus of research, especially with respect to the relationship between inflammation or postinflammatory, structural changes detected by magnetic resonance imaging (MRI) and bone formation detected by conventional radiographs. Based on the analysis of MRI data for both inflammatory and fatty (postinflammatory) lesions, development of de novo syndesmophytes is directly associated with the development of fatty changes on the affected edges of the vertebral bodies, independently of whether inflammation was present on the same edges or not, at the time of first presentation of the patient. On the other hand, vertebral edges that show only inflammation but no transformation into fatty (postinflammatory) changes, show a decreased relative risk for development of new bone formation over time. In a recent publication we could show for the first time that patients with AS benefit from treatment with TNF-blockers in the long term since development of new bone formation can be decelerated. However, this seems to happen only in those edges that are inflamed without any signs of fatty degeneration prior or during treatment. According to these results, It can now be understood that the failure of these compounds to show a benefit on radiographic progression in the first 2- or 4-years of treatment, as reported in other, previous publications, was based on the fact that this progression was the consequence of reparative process (via the pathway of fatty degeneration) where TNFblockade could not interrupt this ongoing process. These data are crucial in the understanding of the long-term clinical course of patients with AS in daily practice. According to these results, it becomes obvious that the initiation of antiinflammatory treatment, especially by using TNF-blockers, has the most benefit not only for the clinical but also for the radiographic outcomes when it is initiated in early phases of the disease, where inflammation is the driving force of disease activity and structural, postinflammatory changes have not yet occurred. These findings argue against the previously proposed ‘TNFbrake hypothesis’, which had indicated that treatment with TNF-blockers in AS might show a beneficial clinical effect but, in the same time, might accelerate the rates of radiographic progression in the same patients. Modern imaging techniques, such as Positrone Emission Tomography (PET) combined with MRI may even improve our knowledge about the pathophysiology of radiographic progression in rheumatic diseases. First data of patients with AS are available and will be demonstrated at the presentation.

We are using mammalian stem cell models to understand the interplay between gene regulation, chromatin structure and DNA methylation. Towards this goal we combine molecular biology and functional genomic approaches. This enables us to monitor the epigenome and its dynamics in an unbiased way and to generate regulatory models from genome-wide datasets, which we test in cellular models by genetic perturbation and genome editing approaches. In order to create functional genomic binding maps we use a controlled biotin tagging approach that enables to identify protein domain contribution to chromosomal localization. We have utilized this to map all MBD domain proteins and a set of isoforms and disease causing mutants in order to better understand the readout of DNA methylation by this family of proteins. This has allowed to define methylation dependent and independent binding sites and challenged the model that these proteins recruit histone modifying enzymes to repress transcription (Baubec et al., Cell 2013). In unpublished work we have applied high-throughput genome editing to create several thousands of sequence variants of CpG islands in order to systematically ask how DNA sequence defines their DNA methylation states. These findings tightly links DNA methylation with underlying DNA sequence features and opens new possibilities in diagnostics. It further suggests that a substantial fraction of methylation changes occur downstream of gene regulation.

8 Clinical genetics of liver diseases F. Lammert Department of Medicine II, Saarland University Medical Center, Homburg, Germany By implementation of novel genotyping technologies, progress in delineating the genetic architecture of acquired liver diseases has been achieved in recent years. The rapid dissemination of genome-wide linkage and association studies has paved the way for the identification of genetic variants that cause or modify non-viral liver diseases as well as the natural and treatmentrelated outcomes in chronic viral hepatitis. Invaluable genomic data has recently been derived from additional genome-wide association studies of the archetypical cholestatic liver diseases primary sclerosing cholangitis and primary biliary cirrhosis as well as hepatocellular cancer. Beyond providing novel pathobiological insights in need of more sophisticated functional annotation, gene variation might in the future be instrumental in precise risk stratification and the development of genotypebased treatment algorithms. In this regard, the definition of subtypes of acquired liver disease and re-categorization of clinically defined disease phenotypes into a more genometype-based disease classification represents a priority.

7 Genomic patterns and readout of DNA methylation D. Schu¨beler Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland DNA methylation is a reversible epigenetic modification of DNA that is essential for development and frequently perturbed in diseases such as cancer.

© 2014 The Authors. European Journal of Clinical Investigation © 2014 Stichting European Society for Clinical Investigation Journal Foundation European Journal of Clinical Investigation, 44 (Suppl. 1), 1–3

Abstracts of the 48th Annual Scientific Meeting of the European Society for Clinical Investigation, 30 April – 3 May 2014, Utrecht, the Netherlands.

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