Nationwide Children's Hospital

17 articles published in JoVE

• Medicine

  
  Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography Qiang Zeng¹, Doug A. Gouchoe^2,3, Mahboubeh Nabavinia⁴, Yong Gyu Lee², Xi Wang², Terri A. Shaffer⁵, Mitchel R. Stacy^4,5,6, Blake R. Peterson^7,8, Bryan A. Whitson^2,9, Christopher Breuer^1,4, Sylvester M. Black^{1,10 1}Center 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. McComb^{1 1}Center 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. McBride^2,3,4,6, Robert B. Weiss⁵, Kevin M. Flanigan^1,6, Nicolas Wein^{1,6 1}Center 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 Ye^1,2, Xiaoping Wan³, Juan Su^1,2, Akshar Patel^1,2,4, Blake Justis^1,2,5, Isabelle Deschênes³, Ming-Tao Zhao^{1,2,5,6 1}The 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. Showpnil^1,2, Kyle R. Miller¹, Cenny Taslim¹, Kathleen I. Pishas¹, Stephen L. Lessnick^1,3, Emily R. Theisen^{1,4 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 Scapin^1,2,3, Marie C. Goulard^1,2,3, Priyanka R. Dharampuriya^1,2,3, Jennifer L. Cillis^1,2,3, Dhvanit I. Shah^{1,2,3 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 White^1,2, Susan D. Reynolds⁴, Jed Johnson⁵, Christopher K. Breuer^3,6, Tendy Chiang^{1,3 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. Lee^{1 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. Whitaker^{1,3 1}Department 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. Whitaker^1,2, Christopher Z. Zheng¹, Bruno Bissonnette^1,2,3, Andrew D. Miller⁴, Tanner L. Koppert^1,2, Joseph D. Tobias^1,2, Christopher R. Pierson^5,6, Fedias L. Christofi^{1 1}Department 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 Kitagawa^{1 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. Ray^{1 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 Nelson^1,2, Christopher Bobba^1,2, Emre Eren³, Tyler Spata⁴, Malak Tadres², Don Hayes, Jr.^5,6, Sylvester M. Black^3,7, Samir Ghadiali*^1,2,3, Bryan A. Whitson*^{2,3,4 1}Department 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. Miller^1,2, Joy Lincoln^{2,3 1}Molecular, 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. Breuer^{1,3 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. Breuer^{1,3 1}Department 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. Bakaletz^{1 1}Center 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