Boston University School of Medicine 15 articles published in JoVE Developmental Biology Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells Andrew Berical1,2, Mary Lou Beermann2, Shingo Suzuki3, Jake LeSuer2, Taylor Matte2, Brian Davis3, Darrell Kotton1,2, Finn Hawkins1,2 1Pulmonary Center, Boston University School of Medicine, 2Center for Regenerative Medicine, Boston University and Boston Medical Center, 3Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center Recent advances in human induced pluripotent stem cell differentiation protocols allow for the stepwise derivation of organ-specific cell types. Here, we provide detailed steps for the maintenance and expansion of iPSC-derived airway basal cells and their differentiation into a mucociliary epithelium in air-liquid interface cultures. Developmental Biology Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells Rhiannon B. Werder*1,2,3, Jessie Huang*1,2, Kristine M. Abo1,2, Olivia T. Hix1,2, Kasey Minakin1,2, Konstantinos-Dionysios Alysandratos1,2, Carly Merritt1,2, Kayleigh Berthiaume1,2, Andrea B. Alber1,2, Claire L. Burgess1,2, Darrell N. Kotton*1,2, Andrew A. Wilson*1,2 1Center for Regenerative Medicine, Boston University and Boston Medical Center, 2The Pulmonary Center and Department of Medicine, Boston University School of Medicine, 3QIMR Berghofer Medical Research Institute The present protocol describes human induced pluripotent stem cell-derived type 2 alveolar epithelial-like cells (iAT2s). These cells can be cultured as self-renewing spheres in 3D culture or adapted to air-liquid interface (ALI) culture. Biology Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion Jing Zhang*1, Jing Huang*2, Ishani Majumdar1, Ryan C. James3, Julia Gichimu1, Manikandan Paramasivam4, Durga Pokharel5, Himabindu Gali6, Marina A. Bellani1, Michael M Seidman1 1Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, 2Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, 3Department of Molecular Biology and Genetics, Cornell University, 4Department of Cellular and Molecular Medicine, University of Copenhagen, 5Horizon Discovery, 6Boston University School of Medicine While replication fork collisions with DNA adducts can induce double strand breaks, less is known about the interaction between replisomes and blocking lesions. We have employed the proximity ligation assay to visualize these encounters and to characterize the consequences for replisome composition. Cancer Research In Vivo Targeting of Xenografted Human Cancer Cells with Functionalized Fluorescent Silica Nanoparticles in Zebrafish Xiaodan Qin*1, Fabrice F. J. Laroche*1, Saquib Ahmed M. A. Peerzade2, Andrew Lam1, Igor Sokolov2,3,4, Hui Feng1 1Departments of Pharmacology and Medicine, Section of Hematology and Medical Oncology, The Cancer Research Center, Boston University School of Medicine, 2Department of Biomedical Engineering, Tufts University, 3Department of Mechanical Engineering, Tufts University, 4Department of Physics, Tufts University Described here is a method for utilizing zebrafish embryos to study the ability of functionalized nanoparticles to target human cancer cells in vivo. This method allows for the evaluation and selection of optimal nanoparticles for future testing in large animals and in clinical trials. Neuroscience Real-time Video Projection in an MRI for Characterization of Neural Correlates Associated with Mirror Therapy for Phantom Limb Pain Faddi G. Saleh Velez1,2, Camila B. Pinto1,3, Emma S. Bailin4, Marionna Münger1, Andrew Ellison5, Beatriz T. Costa1, David Crandell6, Nadia Bolognini7,8, Lotfi B. Merabet4, Felipe Fregni1 1Laboratory of Neuromodulation & Center for Clinical Research Learning, Department of Physical Medicine and Rehabilitation, Harvard Medical School, Spaulding Rehabilitation Hospital, 2University of Chicago Medical Center, Department of Neurology, University of Chicago, 3Department of Neuroscience and Behavior, Psychology Institute, University of Sao Paulo, 4The Laboratory for Visual Neuroplasticity, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, 5Center for Biomedical Imaging, Department of Anatomy and Neurobiology, Boston University School of Medicine, 6Spaulding Rehabilitation Hospital, Harvard Medical School, 7Department of Psychology & Milan Center for Neuroscience, University of Milano-Bicocca, 8Neuropsychological Laboratory, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Istituto Auxologico Italiano We present a novel combined behavioral and neuroimaging protocol employing real-time video projection for the purpose of characterizing the neural correlates associated with mirror therapy within the magnetic resonance imaging scanner environment in leg amputee subjects with phantom limb pain. Immunology and Infection Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells Sultan Asad1,2, Fabiana Feitosa-Suntheimer1,2, Alexander Gold1,2, Berlin Londono-Renteria2, Tonya M. Colpitts1,2 1Department of Microbiology, Boston University School of Medicine, 2National Emerging Infectious Diseases Laborator, Boston University School of Medicine, 3Department of Entomology, Kansas State University We describe a method to evaluate the effect of pre-existing immunity against dengue virus on the Zika virus infection by using human serum, primary human cells, and infection quantification by quantitative real-time polymerase chain reaction. Genetics Chromatin Immunoprecipitation of Murine Brown Adipose Tissue Maria Dafne Cardamone1, Joseph Orofino1, Adam Labadorf2, Valentina Perissi1 1Biochemistry Department, Boston University School of Medicine, 2Bioinformatic Hub, Boston University Here we describe a protocol for efficient chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of brown adipose tissue (BAT) isolated from a mouse. This protocol is suitable for both mapping histone modifications and investigating genome-wide localization of non-histone proteins of interest in vivo. Bioengineering Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT) Weiye Song*1, Libo Zhou*1, Ji Yi1,2 1Department of Medicine, Boston University School of Medicine, 2Department of Biomedical Engineering, Boston University Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided. Cancer Research Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel Amanda F. Bolgioni1, Marc A. Vittoria1, Neil J. Ganem1,2 1Department of Pharmacology & Experimental Therapeutics, Boston University School of Medicine, 2Department of Medicine, Section of Hematology and Oncology, Boston University School of Medicine Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described. Biochemistry Detection of Detergent-sensitive Interactions Between Membrane Proteins Nava Zaarur1, Xiang Pan1, Konstantin V. Kandror1 1Department of Biochemistry, Boston University School of Medicine We describe a protocol for detection of detergent-sensitive interactions between membrane proteins using binding of the sorting receptor, sortilin, to the first luminal loop of the glucose transporter protein, GLUT4, as an example. Biochemistry Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling Carine Beaupere1, Rosalyn B. Chen1, William Pelosi1, Vyacheslav M. Labunskyy1 1Department of Dermatology, Boston University School of Medicine Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae. Medicine Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy Melissa G. Farb*1, Song-Young Park*1, Shakun Karki1, Noyan Gokce1 1Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans. Immunology and Infection A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites George Papadopoulos*1, Carolyn D. Kramer*1, Connie S. Slocum1, Ellen O. Weinberg1, Ning Hua2, Cynthia V. Gudino1, James A. Hamilton1, Caroline A. Genco1 1Department of Medicine, Section of Infectious Disease, Boston University School of Medicine, 2Department of Biophysics, Boston University School of Medicine Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites. Immunology and Infection Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression Daniel K. Rozelle1, Claire Marie Filone1, Ken Dower1, John H. Connor1 1Department of Microbiology, Boston University School of Medicine We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition. Medicine Utilizing Repetitive Transcranial Magnetic Stimulation to Improve Language Function in Stroke Patients with Chronic Non-fluent Aphasia Gabriella Garcia1,2, Catherine Norise1,2, Olufunsho Faseyitan1,2, Margaret A. Naeser3,4,5, Roy H. Hamilton1,2 1Department of Neurology, Perelman School of Medicine, University of Pennsylvania, 2Center for Cognitive Neuroscience, University of Pennsylvania, 3Veterans Affairs Boston Healthcare System, 4Harold Goodglass Aphasia Research Center, Boston University School of Medicine, 5Department of Neurology, Boston University School of Medicine We explore the use of repetitive transcranial magnetic stimulation (rTMS) to improve language abilities in patients with chronic stroke and non-fluent aphasia. After identifying a site in the right frontal gyrus for each patient that responds optimally to stimulation, we target this site during ten days of rTMS treatment.
