Annual Conference of the German Society for Cytometry Date : October 13 - 15 , 2010 Location : Leipzig Kubus Helmholtz - Centre for Environmental Research Leipzig

Abstract

S (ORAL PRESENTATIONS) Wednesday, 13/Oct/2010 12:30pm 1:30pm OPENER: CYTOMETRIC RESEARCH IN GERMANY – TWENTY YEARS OF EXPERIENCE Chair: Susann Müller Chair: Laura Teodori Speaker: Günther Valet 20 years DGfZ: Past and future concepts Speaker: Andreas Radbruch Immunologie und Zytometrie: 35 Jahre Ko-Evolution Wednesday, 13/Oct/2010 1:30pm 3:00pm AGING IN HEALTH AND DISEASE Chair: Leoni Kunz-Schughart IS CELL SENESCENCE A CAUSE OF MAMMALIAN AGEING ? Thomas von Zglinicki Institute for Ageing and Health, Newcastle University, United Kingdom Cell senescence is characterized by the loss of proliferative capacity after a (generally well reproducible) number of cell divisions. However, recently it has become clear that senescence is much more than just a permanent growth arrest observed in cells cultured in vitro. Senescent cells develop a completely different phenotype, including mitochondrial dysfunction, production of reactive oxygen species and secretion of pro-inflammatory cytokines and other bioactive substances. The signalling pathways governing this phenotypic change are triggered as late responses to lasting DNA damage. Moreover, senescent cells are found in tissues of ageing animals and humans at significant frequencies. In many tissues, these frequencies increase with ageing and decrease under caloric restriction, which postpones ageing. This suggests that cell senescence might be among the causes of organismic ageing. We are beginning to unravel the complex signalling pathway networks that govern the changes to the senescent phenotype in the hope to find novel targets for preventive intervention. A FLOW CYTOMETRY APPROACH FOR THE DETECTION OF AMYLOID β-OLIGOMERS IN CEREBROSPINAL FLUID Alexander Navarrete Santos Department of Cardiothoracic Surgery, Martin Luther University Halle-Wittenberg, Halle, Germany Alzheimer’s disease (AD) is the most common form of neurodegenerative dementia with an average survival of 7 years after prognosis. Ongoing clinical studies point out promising possibilities for the treatment of this disease. For ideal therapy and timely conservation of essential cognitive functions, however, a diagnostic tool for the early detection of AD is a pre-requisite. In this regard, there are evidences pointing to Aβ oligomers as the neurotoxic species in AD. For example the concentration of these structures in human brain was found to be up to 70-fold higher in AD patients than in non-demented controls. Furthermore, it was shown that the severity of the disease correlates with the oligomer concentration rather than with the number of plaques (a hallmark of AD in the brain). Actually, a reliable method for the measurement of oligomers in cerebrospinal fluid (CSF) does not exist. In our work we developed a reliable method for the detection of Aß oligomers in CSF. The method is based on the measurement of fluorescence energy transfer signals (FRET) from two different A binding antibodies labelled with Alexa fluor 488 and Alexa fluor 594  (donor/acceptor pair). The detection of the oligomers is achieved by flow cytometry. The accuracy of the method for the discrimination between AD and healthy controls is ongoing. FIBROBLASTS: THE DRIVING FORCE FOR SKIN AGING? Sabrina Gundermann, Hans-Jürgen-Stark, and Petra Boukamp Deutsches Krebsforschungszentrum (DKFZ), Germany One presently favoured “aging hypothesis” is the telomere hypothesis of cellular aging. For human skin, telomere loss was postulated as a major driving force for the aging process. However, we recently demonstrated that neither the continuously proliferating epidermis nor the largely resting dermal fibroblasts show significant agedependent decline (Krunic et al., 2009). Instead, we now show that UV-dependent telomere loss is more crucial and more likely contributes to skin cancer development (Krunic et al., in prep.). It is further proposed that the epidermal stem cells decline in number or loose functional competence with age thereby cause the aging phenotype. We however, propose that not the epidermis but the dermis with its matrix and cellular components, the dermal fibroblasts are responsible for skin aging in humans. Their age-dependent modulation has in turn consequences on epidermal function and leads to the known phenotype of the aging skin, i.e. loss of rete ridges, thinning of the epidermis, reduced barrier function, and impaired wound healing. To test our hypothesis, we studied fibroblasts from skin of different aged donors (in vivo aging) and in the first step compared the in vivo aged fibroblasts with fibroblasts aged by continuous culturing close to cellular senescence (in vitro aged fibroblasts). These studies demonstrated that the in vivo aged fibroblasts and in vitro aged fibroblasts differ in their telomere length, in their gene expression profile, and also in their ability to support epidermal regeneration in organotypic cocultures (OTCs). From these findings we postulate that in vivo aging and in vitro aging are two independent processes. Most importantly, when studying fibroblasts from different aged donors, we did not yet identify senescent cells. Instead, we found a massive increase in α-smooth muscle actin in in vivo aged fibroblasts correlating with an increased number of differentiated fibroblasts, so called myofibroblasts. Additionally, gene expression array analyses showed that fibroblasts from aged donors overexpressed a number of common extracellular matrix proteins and also expressed matrix proteins not found in fibroblasts from a young donor. Verification of the expression of these proteins in the OTCs as well as in skin sections from different aged donors confirmed our expression analysis, thus strongly arguing for the functional relevance of these proteins for the skin aging process. Altogether, we conclude that it may not be the accumulation of senescent fibroblasts that may be causal for skin aging but rather that the fibroblasts differentiate into myofibroblasts. Thereby they express a different set of proteins, which modifies the extracellular matrix and in turn modifies the paracrine interaction with the keratinocytes that cause the phenotypic characteristics associated with skin aging. ASSAY OF CELL LINES DEVOID OF MTDNA COMPARED TO THEIR PARENTAL WILD TYPE Sandra Heller, Susanna Schubert, Peter Seibel Universität Leipzig, Germany The mitochondrion, one important organelle of eukaryotic cells harbours important biochemical processes and acts as key-player in the ageing process and the programmed cell death. The human mitochondrial genome displays a size of 16569bp and contains 37 genes that are important for normal mitochondrial function. Apart from genes for rRNA and tRNA the mitochondrial DNA (mtDNA) encodes 13 polypeptides that are essential enzymatic subunits of the respiratory chain. Genes of other mitochondrial peptides are located in the nuclear genome so that these peptides have to be transported into mitochondria. The respiratory chain consists of the four enzyme complexes NADH:ubiquinone oxidoreductase (complex I), succinate:ubiquinone oxidoreductase (complex II), ubiquinol:cytochrome c oxidoreductase (complex III) and cytochrome c oxidase (complex IV) that function as electron transport complexes and the ATP synthesising complex ATP synthase respectively. Only complex II is completely encoded by the nuclear genome whereas genes of the other respiratory chain complexes are located on the nuclear and mitochondrial genome. Cells without mitochondrial DNA are termed ρ0-cells according to the genetics of yeast. Therefore, cells with ρ0-genotype lack a functional respiratory chain and require metabolic supplementation for cell viability. Cells devoid of mitochondrial DNA are generated by cultivating them on growth medium that includes chemicals like ethidiumbromide or ditercalinium that interfere with DNA replication. A new method takes advantage of a restrictionendonuclease directed to the mitochondrial matrix. This allowed us to generate ρ0-cells without the toxicological side effects caused by the chemical method. Wednesday, 13/Oct/2009 3:30pm 5:00pm REGENERATION Chair: Ulrich Sack Chair: Nicole I. zur Nieden CELL SURFACE MARKER EXPRESSION IN EMBRYONIC STEM CELLS OVEREXPRESSING A STABLE FORM OF BETA-CATENIN Nicole I. zur Nieden1,2 1Department of Cell Biology & Neuroscience and Stem Cell Center, University of California Riverside, Riverside, CA 92521, USA; 2Fraunhofer Institute for Cell Therapy and Immunology, Department of Cell Therapy, Applied Stem Cell Technologies Unit, Perlickstrasse 1, 04103 Leipzig, Germany Although immune-compatible autologous adult stem cells have been proven to repair bone defects, the practical use of these cells is limited by the scarcity of available progenitors or the quality of their genetic composition. New approaches to skeletal repair involving pluripotent stem cells have thus emerged. Our lab has previously explored the in vitro osteogenic differentiation of murine, non-human primate and human embryonic stem cells (ESCs) as a model of skeletal development from pluripotent stem cells. One of the major bottlenecks on the path to successful clinical implementation of ESC technology in the area of degenerative bone diseases, however, is the poorly defined strategies that are currently available to induce directed differentiation of ESCs into osteoblasts. In the past, we have identified the Wnt/beta-catenin (CatnB) signaling pathway as a major regulator of osteogenesis, which may be manipulated to enhance mineralization and maturation as well as early mesendodermal specification. We have found that Wnt signaling crass-talks to a variety of other signaling pathways, such as the ones activated by nitric oxide, bone morphogenetic protein-2 and retinoic acid, in the regulation of these processes. PRODUCTION OF SKELETAL MUSCLE CONSTRUCTS FOR IN VIVO TRANSPLANTATION: PRELIMINARY RESULTS

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@inproceedings{Mller2010AnnualCO, title={Annual Conference of the German Society for Cytometry Date : October 13 - 15 , 2010 Location : Leipzig Kubus Helmholtz - Centre for Environmental Research Leipzig}, author={Susann M{\"{u}ller and Annette G Beck-Sickinger and Wolfgang Beisker and Dirk Bumann and Thomas Egli and Thomas Kroneis and Andreas Radbruch and A. Robitzki and Alexander Scheffold and Frank A Schildberg and G{\"{u}nter K. Valet and Torsten Viergutz and Christine S{\"{u}ring and Laura Teodori and G{\"{u}nther K Valet}, year={2010} }