Nationwide Children's Hospital View Institution's Website 17 articles published in JoVE Medicine Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography Qiang Zeng1, Doug A. Gouchoe2,3, Mahboubeh Nabavinia4, Yong Gyu Lee2, Xi Wang2, Terri A. Shaffer5, Mitchel R. Stacy4,5,6, Blake R. Peterson7,8, Bryan A. Whitson2,9, Christopher Breuer1,4, Sylvester M. Black1,10 1Center for Regenerative Medicine, The Research Institute at Nationwide Children’s Hospital, 5Division of Vascular Diseases and Surgery, The Ohio State University College of Medicine, 6Interdisciplinary Biophysics Graduate Program, The Ohio State University, 7Division of Medicinal Chemistry and Pharmacognosy, The Ohio State University College of Pharmacy, 8The Ohio State University Comprehensive Cancer Center, 9Division of Cardiac Surgery, Department of Surgery, The Ohio State Wexner Medical Center, 10Division of Transplantation, Department of Surgery, The Ohio State Wexner Medical Center In this protocol, we discuss the implementation of a model of successful orthotopic liver transplantation (OLT) in mice. Additionally, adjuvants to further analyze allograft patency after successful OLT in a mouse are discussed as well, specifically utilizing microcomputed tomography (microCT) scans. Engineering Surgery and Sample Processing for Correlative Imaging of the Murine Pulmonary Valve Yifei Liu1, Yong-Ung Lee2, Tai Yi2, Ken Wu3, Cedric Bouchet-Marquis3, Han Chan3, Christopher K. Breuer2, David W. McComb1 1Center for Electron Microscopy and Analysis, The Department of Materials Science and Engineering, Ohio State University, 2 Here, we describe a correlative workflow for the excision, pressurization, fixation, and imaging of the murine pulmonary valve to determine the gross conformation and local extracellular matrix structures. Genetics Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders Camila F. Almeida1, Emma C. Frair1, Nianyuan Huang1, Reid Neinast2, Kim L. McBride2,3,4,6, Robert B. Weiss5, Kevin M. Flanigan1,6, Nicolas Wein1,6 1Center for Cardiovascular Research, The Research Institute at Nationwide Children’s Hospital, 3Division of Genetic and Genomic Medicine, Nationwide Children’s Hospital, 5Department of Human Genetics, The University of Utah School of Medicine, 6Department of Pediatrics, The Ohio State University This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies. Developmental Biology Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells Shiqiao Ye1,2, Xiaoping Wan3, Juan Su1,2, Akshar Patel1,2,4, Blake Justis1,2,5, Isabelle Deschênes3, Ming-Tao Zhao1,2,5,6 1The Heart Center, Nationwide Children’s Hospital, 3Department of Physiology and Cell Biology, The Ohio State University College of Medicine, 4Department of Anatomy, The Ohio State University College of Medicine, 5MCDB Graduate Program, The Ohio State University, 6Department of Pediatrics, The Ohio State University College of Medicine Here, we present a protocol to robustly generate and expand human cardiomyocytes from patient peripheral blood mononuclear cells. Cancer Research Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis Iftekhar A. Showpnil1,2, Kyle R. Miller1, Cenny Taslim1, Kathleen I. Pishas1, Stephen L. Lessnick1,3, Emily R. Theisen1,4 1 Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma. Developmental Biology Analysis of Hematopoietic Stem Progenitor Cell Metabolism Giorgia Scapin1,2,3, Marie C. Goulard1,2,3, Priyanka R. Dharampuriya1,2,3, Jennifer L. Cillis1,2,3, Dhvanit I. Shah1,2,3 1 Hematopoietic stem progenitor cells (HSPCs) transition from a quiescent state to a differentiation state due to their metabolic plasticity during blood formation. Here, we present an optimized method for measuring mitochondrial respiration and glycolysis of HSPCs. Bioengineering Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model Matthew G. Wiet*1,2, Sayali Dharmadhikari*1,3, Audrey White1,2, Susan D. Reynolds4, Jed Johnson5, Christopher K. Breuer3,6, Tendy Chiang1,3 1 Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation. Immunology and Infection Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins Meisam Naeimi Kararoudi1, Hamid Dolatshad2, Prashant Trikha1, Syed-Rehan A. Hussain3, Ezgi Elmas1, Jennifer A. Foltz1, Jena E. Moseman1, Aarohi Thakkar1, Robin J. Nakkula1, Margaret Lamb1, Nitin Chakravarti1, K. John McLaughlin3, Dean A. Lee1 1 Here, we present a protocol to genetically modify primary or expanded human natural killer (NK) cells using Cas9 Ribonucleoproteins (Cas9/RNPs). By using this protocol, we generated human NK cells deficient for transforming growth factor–b receptor 2 (TGFBR2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1). Neuroscience Adaptation of Microelectrode Array Technology for the Study of Anesthesia-induced Neurotoxicity in the Intact Piglet Brain Emily D. Geyer*1, Prithvi A. Shetty*1, Christopher J. Suozzi*1, David Z. Allen*1,2, Pamela P. Benavidez*1,2, Joseph Liu*1,3, Charles N. Hollis1, Greg A. Gerhardt4, Jorge E. Quintero4, Jason J. Burmeister4, Emmett E. Whitaker1,3 1Department of Anesthesiology, Ohio State University College of Medicine, 2Medical Student Research Program, Ohio State University College of Medicine, 3 This study explores the novel use of enzyme-based microelectrode array (MEA) technology to monitor in vivo neurotransmitter activity in piglets. The hypothesis was that glutamate dysregulation contributes to the mechanism of anesthetic neurotoxicity. Here, we present a protocol to adapt MEA technology to study the mechanism of anesthesia-induced neurotoxicity. Medicine Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity (AIDN): A Translational Neuroscience Approach Emmett E. Whitaker1,2, Christopher Z. Zheng1, Bruno Bissonnette1,2,3, Andrew D. Miller4, Tanner L. Koppert1,2, Joseph D. Tobias1,2, Christopher R. Pierson5,6, Fedias L. Christofi1 1Department of Anesthesiology, Ohio State University College of Medicine, 2 Anesthesia-induced developmental neurotoxicity (AIDN) research has focused on rodents, which are not broadly applicable to humans. Non-human primate models are more relevant, but are cost-prohibitive and difficult to use for experimentation. The piglet, in contrast, is a clinically relevant, practical animal model ideal for the study of anesthetic neurotoxicity. Biology Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins Yohei Niikura1, Katsumi Kitagawa1 1 Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation). Chemistry Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues R. Wolfgang Rumpf1, William C. Ray1 1 Synthetic protein sequences based on consensus motifs typically ignore co-evolving residues, that imply interpositional dependencies (IPDs). IPDs can be essential to activity, and designs that disregard them may result in suboptimal results. This protocol uses StickWRLD to identify IPDs and help inform rational protein design, resulting in more efficient results. Medicine Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program Kevin Nelson1,2, Christopher Bobba1,2, Emre Eren3, Tyler Spata4, Malak Tadres2, Don Hayes, Jr.5,6, Sylvester M. Black3,7, Samir Ghadiali*1,2,3, Bryan A. Whitson*2,3,4 1Department of Biomedical Engineering, Ohio State University Wexner Medical Center, 2Davis Heart & Lung Research Institute, Ohio State University Wexner Medical Center, 3The Collaboration for Organ Perfusion, Protection, Engineering and Regeneration (COPPER) Laboratory, Ohio State University Wexner Medical Center, 4Division of Cardiac Surgery, Department of Surgery, Ohio State University Wexner Medical Center, 5Departments of Pediatrics and Internal Medicine, Ohio State University, 6 Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion. Biology Isolation of Murine Valve Endothelial Cells Lindsey J. Miller1,2, Joy Lincoln2,3 1Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University, 2 The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes. Medicine Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation Yong-Ung Lee1, Tai Yi1, Iyore James1, Shuhei Tara1, Alexander J. Stuber1, Kejal V. Shah1, Avione Y. Lee1, Tadahisa Sugiura1, Narutoshi Hibino2, Toshiharu Shinoka1, Christopher K. Breuer1,3 1 In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique. Medicine Implantation of Inferior Vena Cava Interposition Graft in Mouse Model Yong-Ung Lee1, Tai Yi1, Shuhei Tara1, Avione Y. Lee1, Narutoshi Hibino1, Toshiharu Shinoka2, Christopher K. Breuer1,3 1Department of Cardiothoracic Surgery, Nationwide Children's Hospital, 3 To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course. Immunology and Infection In vitro Biofilm Formation in an 8-well Chamber Slide Joseph A. Jurcisek1, Amanda C. Dickson1, Molly E. Bruggeman1, Lauren O. Bakaletz1 1Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide