Friedrich-Alexander University of Erlangen-Nuremberg 18 articles published in JoVE Biology Generation of Patient-Derived Podocytes from Skin Biopsies Victoria Rose1, Janina Müller-Deile1,2 1Department of Nephrology and Hypertension, University Hospital Erlangen, Friedrich-Alexander University (FAU) Erlangen-Nürnberg, 2Research Center on Rare Kidney Diseases (RECORD), University Hospital Erlangen This manuscript describes a two-step protocol to generate patient-specific podocytes from dermal fibroblasts via episomal reprogramming into human-induced pluripotent stem cells (hiPSCs) and subsequent differentiation into podocytes. Medicine Polytetrafluoroethylene (PTFE) as a Suture Material in Tendon Surgery Elias Polykandriotis1,2, Marcus Himmler3, Shirin Mansouri2, Florian Ruppe4, Jasmin Grüner1, Lars Bräeuer5, Dirk W. Schubert3, Raymund E. Horch1 1Department of Plastic and Hand Surgery, University of Erlangen Medical Center, 2Department of Plastic, Hand, and Microsurgery, Sana Hospital Hof, 3Department of Materials Science and Engineering, Friedrich-Alexander-University, Erlangen-Nürnberg, 4Department of Plastic Surgery, Bayreuth Municipal Hospital, 5Institute of Anatomy, Chair II, Friedrich Alexander University Erlangen-Nuremberg The present protocol illustrates a method for assessing the biophysical properties of tendon repairs ex vivo. A polytetrafluoroethylene (PTFE) suture material was evaluated by this method and compared to other materials under different conditions. Immunology and Infection A Modified Surgical Technique for Kidney Transplantation in Mice Decheng Yin*1,2, Jian Fu*1,3, Rongjun Chen4, Nelli Shushakova5, Ida Allabauer1, Xin-Yi Wei1, Mario Schiffer6, Diana Dudziak7, Song Rong*5, André Hoerning*1 1Department of Pediatric and Adolescent Medicine, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nürnberg, 2Department of Pediatric Surgery, Chengdu Women’s and Children's Central Hospital, 3The Key Laboratory of Hainan Trauma and Disaster Rescue, The first affiliated Hospital of Hainan Medical University, 4Department of Department of Pediatric Kidney, Liver, and Metabolic Diseases, Hannover Medical School, 5Department of Nephrology, Hannover Medical School, 6Department of Nephrology, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nürnberg, 7Department of Dermatology, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nürnberg This protocol presents a new surgical technique of mouse kidney transplantation focusing on a modified arterial anastomosis strategy. A vascular suture technique including a simple and safer ureter-bladder anastomosis method is also presented. These modifications shorten the operation time and improve the success rate of the mouse kidney transplantation procedure. Medicine Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model Decheng Yin*1,2, Jian Fu*1,3, Ida Allabauer1, Oliver Witzke4, Song Rong5, André Hoerning1 1Department of Pediatric and Adolescent Medicine, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nürnberg, 2Department of Pediatric Surgery, Chengdu Women's and Children's Central Hospital, 3The Key Laboratory of Hainan Trauma and Disaster Rescue, The first affiliated Hospital of Hainan Medical University, 4Department of Infectious Diseases, West German Centre of Infectious Diseases, Universitätsmedizin Essen, University Duisburg-Essen, 5Department of Nephrology, Hannover Medical School A novel technique for blood circuit reconstruction in a heterotopic abdominal mouse heart transplantation model is demonstrated. Biology Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices Dominik J. Fiegle1, Tilmann Volk1,2, Thomas Seidel1,2 1Institute of Cellular and Molecular Physiology, Friedrich-Alexander University Erlangen-Nürnberg, 2Muscle Research Center Erlangen (MURCE), Friedrich-Alexander-Universität Erlangen-Nürnberg Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (<50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h. Engineering Cryogenic Liquid Jets for High Repetition Rate Discovery Science Chandra B. Curry*1,2, Christopher Schoenwaelder*1,3, Sebastian Goede4, Jongjin B. Kim1, Martin Rehwald5,6, Franziska Treffert1,7, Karl Zeil5, Siegfried H. Glenzer1, Maxence Gauthier1 1SLAC National Accelerator Laboratory, 2University of Alberta, 3Friedrich-Alexander-Universität Erlangen-Nürnberg, 4European XFEL, 5Helmholtz-Zentrum Dresden-Rossendorf, 6Technische Universität Dresden, 7Technische Universität Darmstadt This protocol presents the operation and principles of micron-scale cylindrical and planar cryogenic liquid jets. Until now, this system has been used as a high repetition rate target in laser-plasma experiments. Anticipated cross-disciplinary applications range from laboratory astrophysics to material science, and eventually next-generation particle accelerators. Medicine Investigating Intestinal Barrier Breakdown in Living Organoids Marco Bardenbacher1, Barbara Ruder2, Natalie Britzen-Laurent1, Elisabeth Naschberger1, Christoph Becker2, Ralph Palmisano3, Michael Stürzl*1, Philipp Tripal*1,3 1Division of Molecular and Experimental Surgery, Department of Surgery, Translational Research Center, Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg and Universitätsklinikum Erlangen, 2Department of Medicine 1, Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg and Universitätsklinikum Erlangen, 3Optical Imaging Centre Erlangen (OICE), Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner. Biochemistry Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays Judith A. Stolwijk1, Anne-Kathrin Mildner1, Christian Kade1, Michael Skiba1, Guenther Bernhardt2, Armin Buschauer2, Harald Huebner3, Peter Gmeiner3, Joachim Wegener1,4 1Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, 2Institute of Pharmacy, University of Regensburg, 3Department of Chemistry and Pharmacy, Friedrich-Alexander University Erlangen-Nuernberg, 4Fraunhofer Research Institution for Microsystems and Solid State Technologies EMFT This protocol demonstrates real-time recording of full dose-response relationships for agonist-induced GPCR activation from a single cell layer grown on a single microelectrode using label-free impedance measurements. The new dosing scheme significantly increases throughput without loss in time resolution. Chemistry Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy Andreas Hutzler*1, Birk Fritsch*1, Michael P. M. Jank2, Robert Branscheid3, Erdmann Spiecker3, Martin März1,2,4 1Electron Devices (LEB), Department of Electrical, Electronic and Communication Engineering, Friedrich-Alexander University Erlangen-Nürnberg, 2Fraunhofer Institute for Integrated Systems and Device Technology (IISB), 3Institute of Micro- and Nanostructure Research (IMN) and Center for Nanoanalysis and Electron Microscopy (CENEM), Department of Materials Science and Engineering, Friedrich-Alexander University Erlangen-Nürnberg, 4Power Electronics (LEE), Department of Electrical, Electronic and Communication Engineering, Friedrich-Alexander University Erlangen-Nürnberg A protocol for preparation of graphene-supported microwell liquid cells for in situ electron microscopy of gold nanocrystals from HAuCl4 precursor solution is presented. Furthermore, an analysis routine is presented to quantify observed etching and growth dynamics. Chemistry Isolating Free Carbenes, their Mixed Dimers and Organic Radicals Annette Grünwald1, Stephen J. Goodner1, Dominik Munz1 1Department of Chemistry and Pharmacy, Friedrich-Alexander-Universität Erlangen-Nürnberg We present protocols for the isolation of stable heterocyclic carbenes. The synthesis of a cyclic (alkyl)(amino) carbene (CAAC) and an N-heterocyclic carbene (NHC) is demonstrated using filter cannulas and Schlenk technique. We furthermore present the synthesis of the related oxygen-sensitive, electron-rich mixed “Wanzlick dimer” and the reduced stable organic radical. Engineering Label-Free Imaging of Single Proteins Secreted from Living Cells via iSCAT Microscopy André Gemeinhardt1, Matthew P. McDonald1,2, Katharina König1,3, Michael Aigner4, Andreas Mackensen4, Vahid Sandoghdar1,3 1Max Planck Institute for the Science of Light (MPL), 2Area of Scientific Learning, Milligan College, 3Department of Physics, Friedrich Alexander University Erlangen-Nuremberg, 4Department of Internal Medicine 5, Hematology and Oncology, Friedrich Alexander University Erlangen-Nuremberg (FAU), University Hospital Erlangen We present a protocol for the real-time optical detection of single unlabeled proteins as they are secreted from living cells. This is based on interferometric scattering (iSCAT) microscopy, which can be applied to a variety of different biological systems and configurations. Cancer Research Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol Elisabeth Naschberger1, Daniela Regensburger1, Clara Tenkerian1, Melanie Langheinrich2, Felix B. Engel3, Carol Geppert4, Arndt Hartmann4, Robert Grützmann2, Vera S. Schellerer*2, Michael Stürzl*1 1Division of Molecular and Experimental Surgery, Department of Surgery, University Medical Center Erlangen, Friedrich-Alexander University of Erlangen-Nuremberg, 2Department of Surgery, University Medical Center Erlangen, Friedrich-Alexander University of Erlangen-Nuremberg, 3Division of Nephropathology, Department of Pathology, University Medical Center Erlangen, Friedrich-Alexander University of Erlangen-Nuremberg, 4Department of Pathology, University Medical Center Erlangen, Friedrich-Alexander University of Erlangen-Nuremberg Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described. Medicine Drug-Induced Sleep Endoscopy (DISE) with Target Controlled Infusion (TCI) and Bispectral Analysis in Obstructive Sleep Apnea Maximilian Traxdorf1, Klaus Tschaikowsky2, Claudia Scherl1, Judith Bauer1, Heinrich Iro1, Florian Angerer1 1Department of Otorhinolaryngology, Head & Neck Surgery, Friedrich-Alexander University Erlangen-Nürnberg (FAU), 2Department of Anesthesiology, Friedrich-Alexander University Erlangen-Nürnberg (FAU) The aim of this study was to establish a standardized protocol for sleep endoscopy to differentiate obstruction patterns in obstructive sleep apnea (OSA). Target-controlled infusion (TCI) of the sedative was combined with real-time monitoring of the depth of sedation using bispectral analysis. Bioengineering The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering Annika Weigand1, Justus P. Beier1, Andreas Arkudas1, Majida Al-Abboodi1,2, Elias Polykandriotis3, Raymund E. Horch1, Anja M. Boos1 1Department of Plastic and Hand Surgery and Laboratory for Tissue Engineering and Regenerative Medicine, University Hospital of Erlangen, Friedrich-Alexander University of Erlangen-Nürnberg (FAU), 2Genetic Engineering and Biotechnology Institute for Postgraduate Studies, Baghdad University, 3Department of Plastic, Hand and Microsurgery, Sana Klinikum Hof GmbH We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues. Medicine The Influence of Liver Resection on Intrahepatic Tumor Growth Hannes H. Brandt1, Valérie Nißler2, Roland S. Croner3 1Friedrich-Alexander-University Erlangen-Nürnberg (FAU), 2Department of Surgery, University Hospital Regensburg, 3Department of Surgery, University Hospital Erlangen A high incidence of tumor recurrence after resection of liver metastases remains an unsolved problem. The illustrated mouse model may be useful to investigate the reasons for such recurrences. It combines a liver resection model with intrahepatic tumor cell injection for the first time. Immunology and Infection Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling Christian Berens1,2, Stephanie Bisle3, Leonie Klingenbeck3, Anja Lührmann3 1Department Biologie, Friedrich-Alexander-Universität, 2Institut für Molekulare Pathogenese, Friedrich-Loeffler-Institut, 3Mikrobiologisches Institut - Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways. Biology Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments Matteus Krappitz1, Christoph Korbmacher1, Silke Haerteis1 1Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) Proteolytic activation of the epithelial sodium channel (ENaC) heterologously expressed in Xenopus laevis oocytes can be demonstrated by combining current measurements with a biotinylation approach to investigate the appearance of ion channel cleavage products at the cell surface. Functionally important cleavage sites can be identified by using site-directed mutagenesis. Neuroscience Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons Marisa Karow1, Christian Schichor2, Ruth Beckervordersandforth3, Benedikt Berninger1,4 1Department of Physiological Genomics, Institute of Physiology, Ludwig Maximilians University Munich, 2Tumor Biology Lab, Neurosurgical Clinic, Ludwig-Maximilians University Munich, 3Institut für Biochemie, Emil-Fischer-Zentrum, Friedrich-Alexander-Universität Erlangen-Nürnberg, 4Institute of Physiological Chemistry and Focus Program Translational Neuroscience, Johannes Gutenberg University Mainz Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.