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National Institute of Environmental Health Sciences, NIH

11 ARTICLES PUBLISHED IN JoVE

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Medicine

Combined In vivo Optical and µCT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice
Nicholas M. Bernthal 1, Brad N. Taylor 2, Jeffrey A. Meganck 2, Yu Wang 3, Jonathan H. Shahbazian 3, Jared A. Niska 1, Kevin P. Francis 2, Lloyd S. Miller 3,4
1Orthopaedic Hospital Research Center, Orthopaedic Hospital Department of Orthopaedic Surgery, David Geffen School of Medicine at University of California, Los Angeles (UCLA), 2PerkinElmer, 3Department of Dermatology, Johns Hopkins University School of Medicine, 4Department of Medicine, Division of Infectious Diseases, Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine

Combined optical and μCT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

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Immunology and Infection

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses
Con Sullivan 1,2, Denise Jurcyzszak 1, Michelle F. Goody 3, Kristin A. Gabor 4, Jacob R. Longfellow 1, Paul J. Millard 2,5, Carol H. Kim 1,2
1Department of Molecular and Biomedical Sciences, University of Maine, 2Graduate School of Biomedical Sciences and Engineering, University of Maine, 3School of Biology and Ecology, University of Maine, 4Division of Intramural Research, Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, NIH, 5Department of Chemical and Biological Engineering, University of Maine

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

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Developmental Biology

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells
Yu Wang 1, Sharmin Alhaque 1, Kate Cameron 1, Jose Meseguer-Ripolles 1, Baltasar Lucendo-Villarin 1, Hassan Rashidi 1, David C. Hay 1
1MRC Centre for Regenerative Medicine, University of Edinburgh

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

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Immunology and Infection

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells
Miranda R. Lyons-Cohen 1, Seddon Y. Thomas 1, Donald N. Cook 1, Hideki Nakano 1
1Immunity, Inflammation, and Disease Laboratory, Division of Intramural Research, National Institute of Environmental Health Sciences, NIH

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

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Biology

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis
Caleb Sutherland *1, Yu Wang *2, Robert V. Brown *1, Julie Foley 2, Beth Mahler 2, Kyathanahalli S. Janardhan 2,3, Ramesh C. Kovi 2,4, Anton M. Jetten 1
1Immunity, Inflammation, and Disease Laboratory, Division of Intramural Research, National Institute of Environmental Health Sciences, NIH, 2Cellular and Molecular Pathology Branch, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, NIH, 3Integrated Laboratory Systems Inc., 4Experimental Pathology Laboratories Inc.

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

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Developmental Biology

Semi-automated Production of Hepatocyte Like Cells from Pluripotent Stem Cells
Jose Meseguer-Ripolles 1, Baltasar Lucendo-Villarin 1, Yu Wang 1, David C. Hay 1
1MRC Centre for Regenerative Medicine, University of Edinburgh

This protocol describes a semi-automated approach to produce hepatocyte-like cells from human pluripotent stem cells in a 96 well plate format. This process is rapid and cost-effective, allowing the production of quality assured batches of hepatocyte-like cells for basic and applied human research.

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Biology

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues
Daniels Konja 1, Cuiting Luo 1, Wai Yan Sun 1, Kangmin Yang 1, Andy W.C. Man 1, Aimin Xu 1, Paul M. Vanhoutte 1, Yu Wang 1
1The State Key Laboratory of Pharmaceutical Biotechnology and the Department of Pharmacology and Pharmacy, University of Hong Kong

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

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Developmental Biology

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells
Balta Lucendo-Villarin 1, Hassan Rashidi 1,2, Sharmin Alhaque 1,3, Lena Fischer 1,4, Jose Meseguer-Ripolles 1, Yu Wang 1, Cliona O'Farrelly 4, Michael Themis 3, David C. Hay 1
1MRC Centre for Regenerative Medicine, University of Edinburgh, 2UCL Great Ormond Street Institute of Child Health, University College London, 3Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, 4School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

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Genetics

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing
Yu Wang 1, Andrew J. H. Smith 1, David C. Hay 1
1MRC Centre for Regenerative Medicine, University of Edinburgh

Here, we describe a detailed method for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and the Cas9 nickase mutant. Two point mutations were introduced into exon 8 of the hepatocyte nuclear factor 4 alpha (HNF4α) locus in human embryonic stem cells (hESCs).

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Biology

Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System
Jose Meseguer-Ripolles 1, Yu Wang 1, Agnes Sorteberg 1, Aishwariya Sharma 2, Nan-Linda Ding 2, Baltasar Lucendo-Villarin 1, Philipp Kramer 2, Charis-Patricia Segeritz 2, David C. Hay 1
1MRC Centre for Regenerative Medicine, University of Edinburgh, 2Research & Development, STEMCELL Technologies Inc

The goal of this article is to provide a standardized approach to induce human hepatic progenitor differentiation from pluripotent stem cells. The development of this procedure with ready-to-use media formulations offer the user a facile system to generate human liver cells for biomedical research and translation.

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Medicine

Acupuncture in a Rat Model of Asthma
Dong-Dong Zhou 1, Guang-Quan Zhang 1, Qing-Yi Zhao 1, Mi Cheng 1, Jin Lu 1, Yu Wang 1, Yu-Dong Xu 1, Yan-Jiao Chen 1, Yong-Qing Yang 1, Lei-Miao Yin 1,2
1Laboratory of Molecular Biology, Shanghai Research Institute of Acupuncture and Meridian, Shanghai University of Traditional Chinese Medicine, 2Shanghai Innovation Center of Traditional Chinese Medicine Health Service

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes methods for the fabrication of the high platform, establishes a rat model of asthma and measures changes in respiratory function using a noninvasive and real-time whole-body plethysmography (WBP) system.

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