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Nationwide Children's Hospital

17 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells
Srinivas S. Somanchi 1, Vladimir V. Senyukov 1, Cecele J. Denman 1, Dean A. Lee 1
1Division of Pediatrics, MD Anderson Cancer Center - University of Texas

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

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Medicine

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model
Yong-Ung Lee 1, Tai Yi 1, Shuhei Tara 1, Avione Y. Lee 1, Narutoshi Hibino 1, Toshiharu Shinoka 2, Christopher K. Breuer 1,3
1Tissue Engineering Program and Surgical Research, Nationwide Children's Hospital, 2Department of Cardiothoracic Surgery, Nationwide Children's Hospital, 3Pediatric Surgery, Nationwide Children's Hospital

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.

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Medicine

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation
Yong-Ung Lee 1, Tai Yi 1, Iyore James 1, Shuhei Tara 1, Alexander J. Stuber 1, Kejal V. Shah 1, Avione Y. Lee 1, Tadahisa Sugiura 1, Narutoshi Hibino 2, Toshiharu Shinoka 1, Christopher K. Breuer 1,3
1Tissue Engineering Program and Surgical Research, Nationwide Children's Hospital, 2Cardiothoracic Surgery, Nationwide Children's Hospital, 3Pediatric Surgery, Nationwide Children's Hospital

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.

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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 Eren 3, Tyler Spata 4, Malak Tadres 2, Don Hayes, Jr. 5,6, Sylvester M. Black 3,7, 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, 6Advanced Lung Disease Program, Lung and Heart-Lung Transplant Programs, Nationwide Children's Hospital, 7Division of Transplantation, Department of Surgery, Ohio State University Wexner Medical Center

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.

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Biology

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins
Yohei Niikura 1, Katsumi Kitagawa 1
1Center for Childhood Cancer and Blood Diseases, Nationwide Children's Hospital

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).

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Genetics

Methyl-binding DNA capture Sequencing for Patient Tissues
Rohit R. Jadhav 1, Yao V. Wang 1, Ya-Ting Hsu 1, Joseph Liu 1, Dawn Garcia 2, Zhao Lai 2, Tim H. M. Huang 1, Victor X. Jin 1
1Department of Molecular Medicine, University of Texas Health Science Center at San Antonio, 2Greehey Children's Cancer Research Institute, University of Texas Health Science Center at San Antonio

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

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Developmental Biology

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae
Elliott J. Hagedorn 1,2, Jennifer L. Cillis 3, Caitlyn R. Curley 3, Taylor C. Patch 3, Brian Li 1,2, Bradley W. Blaser 1,2,7, Raquel Riquelme 1,2, Leonard I. Zon 1,2,4,5,6, Dhvanit I. Shah 1,2,3,4,5
1Division of Hematology/Oncology, Boston Children’s Hospital, 2Harvard Medical School, 3Division of Hematology, Department of Medicine, Brigham and Women’s Hospital, 4Harvard Stem Cell Institute, 5Broad Institute of Massachusetts Institute of Technology, 6Howard Hughes Medical Institute, 7Division of Hematologic Malignancies, Dana-Farber Cancer Institute

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

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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. Zheng 1, Bruno Bissonnette 1,2,3, Andrew D. Miller 4, Tanner L. Koppert 1,2, Joseph D. Tobias 1,2, Christopher R. Pierson 5,6, Fedias L. Christofi 1
1Department of Anesthesiology, Ohio State University College of Medicine, 2Department of Anesthesiology and Pain Medicine, Nationwide Children's Hospital, 3Department of Anaesthesia and Critical Care Medicine, University of Toronto, 4Department of Biomedical Sciences, Section of Anatomic Pathology, Cornell University College of Veterinary Medicine, 5Department of Pathology and Anatomy, Ohio State University College of Medicine, 6Department of Pathology and Laboratory Medicine, Nationwide Children's Hospital

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.

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Bioengineering

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting
Chin Siang Ong 1,2, Takuma Fukunishi 1, Andrew Nashed 1, Adriana Blazeski 3, Huaitao Zhang 1, Samantha Hardy 1, Deborah DiSilvestre 2, Luca Vricella 1, John Conte 1, Leslie Tung 3, Gordon Tomaselli 2, Narutoshi Hibino 1
1Division of Cardiac Surgery, Johns Hopkins Hospital, 2Division of Cardiology, Johns Hopkins Hospital, 3Department of Biomedical Engineering, Johns Hopkins University

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

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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. Hollis 1, Greg A. Gerhardt 4, Jorge E. Quintero 4, Jason J. Burmeister 4, Emmett E. Whitaker 1,3
1Department of Anesthesiology, Ohio State University College of Medicine, 2Medical Student Research Program, Ohio State University College of Medicine, 3Department of Anesthesiology and Pain Medicine, Nationwide Children's Hospital, 4Department of Neuroscience, University of Kentucky Medical Center

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.

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Immunology and Infection

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins
Meisam Naeimi Kararoudi 1, Hamid Dolatshad 2, Prashant Trikha 1, Syed-Rehan A. Hussain 3, Ezgi Elmas 1, Jennifer A. Foltz 1, Jena E. Moseman 1, Aarohi Thakkar 1, Robin J. Nakkula 1, Margaret Lamb 1, Nitin Chakravarti 1, K. John McLaughlin 3, Dean A. Lee 1
1Center for Childhood Cancer and Blood Disease, Nationwide Children's Hospital, 2Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, 3Center for Clinical and Translational Research, Nationwide Children's Hospital Research Institute

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).

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Bioengineering

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation
Yang Bai 1,2, Enoch Yeung 2, Cecillia Lui 2, Chin Siang Ong 2, Isaree Pitaktong 2, Chenyu Huang 2, Takahiro Inoue 2, Hiroshi Matsushita 2, Chunye Ma 2, Narutoshi Hibino 2
1Department of Cardiac Surgery, The First Hospital of Jilin University, 2Division of Cardiac Surgery, Johns Hopkins Hospital

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

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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. Reynolds 4, Jed Johnson 5, Christopher K. Breuer 3,6, Tendy Chiang 1,3
1Department of Otolaryngology Head & Neck Surgery, Nationwide Children's Hospital, 2The Ohio State University College of Medicine, 3Center for Regenerative Medicine, Research Institute at Nationwide Children's Hospital, 4Center for Perinatal Research, Nationwide Children's Hospital, 5Nanofiber Solutions, Inc., 6Department of Pediatric Surgery, Nationwide Children's Hospital

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.

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JoVE Journal

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
1Nationwide Children's Hospital, 2The Ohio State University College of Medicine, 3The Ohio State University Comprehensive Cancer Center

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.

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JoVE Core

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)
Yohei Niikura *1, Lei Fang *2, Risa Kitagawa *3, Peizhao Li 1, Yao Xi 1, Ju You 1, Yan Guo 2, Katsumi Kitagawa 3
1MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing University, 2Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, 3Greehey Children's Cancer Institute, Department of Molecular Medicine, UT Health Science Center San Antonio

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

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Engineering

Surgery and Sample Processing for Correlative Imaging of the Murine Pulmonary Valve
Yifei Liu 1, Yong-Ung Lee 2, Tai Yi 2, Ken Wu 3, Cedric Bouchet-Marquis 3, Han Chan 3, Christopher K. Breuer 2, David W. McComb 1
1Center for Electron Microscopy and Analysis, The Department of Materials Science and Engineering, Ohio State University, 2Center for Regenerative Medicine, Nationwide Children’s Hospital, 3Thermo Fisher Scientific

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.

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Bioengineering

Quantifying Inferior Vena Cava Compliance and Distensibility in an In Vivo Ovine Model Using 3D Angiography
Delaney J. Villarreal *1,2, Tatsuya Watanabe *1, Kirsten Nelson 1, Adrienne Morrison 1, Eric D. Heuer 1, Anudari Ulziibayar 1, John M. Kelly 1,3,4, Christopher K. Breuer 1,5
1Center for Regenerative Medicine, Nationwide Children's Hospital, 2Biomedical Sciences Graduate Program, The Ohio State University College of Medicine, 3The Heart Center, Nationwide Children's Hospital, 4Department of Pediatrics, The Ohio State University College of Medicine, 5Department of Surgery, The Ohio State University College of Medicine

This protocol allows for the in vivo quantification of venous compliance and distensibility using catheterization and 3D angiography as a survival procedure allowing for a variety of potential applications.

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