Icahn School of Medicine at Mount Sinai 47 articles published in JoVE Neuroscience Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease Camille Goldman*1,2,3,4,5, Natalie Suhy*1,2,3,4,5, Jessica E. Schwarz1,2,3,4,5, Emily Ruth Sartori1,2,3,4,5, Rikki B. Rooklin1,2,3,4,5, Braxton R. Schuldt1,2,3,4,5, Louise A. Mesentier-Louro1,2,3,4,5, Joel W. Blanchard1,2,3,4,5 1Icahn School of Medicine at Mount Sinai, 2Black Family Stem Cell Institute at Mount Sinai, 3Ronald M. Loeb Center for Alzheimer’s Disease at Mount Sinai, 4Friedman Brain Institute at Mount Sinai, 5Nash Family Department of Neuroscience at Mount Sinai The blood-brain barrier (BBB) has a crucial role in sustaining a stable and healthy brain environment. BBB dysfunction is associated with many neurological diseases. We have developed a 3D, stem-cell-derived model of the BBB to investigate cerebrovascular pathology, BBB integrity, and how the BBB is altered by genetics and disease. Bioengineering Semi-Automatic Graphical Tool for Measuring Coronary Artery Spatially Weighted Calcium Score from Gated Cardiac Computed Tomography Images Heli J. Patel1, Audrey E. Kaufman1, Marco Pereañez1, Georgios Soultanidis1, Sarayu Ramachandran1, Sonum Naidu1, Venkatesh Mani1, Zahi A. Fayad1, Philip M. Robson1 1Biomedical Engineering and Imaging Institute, Icahn School of Medicine at Mount Sinai This video demonstrates the use of a novel graphical tool for measuring the spatially weighted calcium score (SWCS), an alternative to the Agatston score, for quantifying coronary artery calcification. The graphical tool computes SWCS based on image data from gated cardiac computed tomography and user-defined paths of the coronary arteries. Bioengineering Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues Camille C. van Neste1, Karlo A. Wiley1, Serena W. Chang1, Joseph Borrello1, Irene C. Turnbull1, Kevin D. Costa1 1Cardiovascular Research Institute, Icahn School of Medicine at Mount Sinai Three-dimensional cardiac tissues bioengineered using stem-cell-derived cardiomyocytes have emerged as promising models for studying healthy and diseased human myocardium in vitro while recapitulating key aspects of the native cardiac niche. This manuscript describes a protocol for fabricating and analyzing high-content engineered cardiac tissues generated from human induced pluripotent stem-cell-derived cardiomyocytes. Biology Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria Rihab Bouchareb1, Liping Yu1, Emelie Lassen1, Ilse S. Daehn1 1Department of Medicine, Division of Nephrology, The Icahn School of Medicine at Mount Sinai The article describes the method for isolating conditionally immortalized glomerular endothelial cells from the kidneys of transgenic mice expressing the thermolabile simian virus 40 and photo-activatable mitochondria, PhAMexcised. We describe the procedure for glomeruli isolation from whole kidneys using beads, digestion steps, seeding, and culturing of GECs-CD31 positive. Neuroscience Minimally Invasive Endoscopic Intracerebral Hemorrhage Evacuation Jonathan Pan1, Alexander G. Chartrain1, Jacopo Scaggiante1, Olivia S. Allen1, Danny Hom1, Joshua B. Bederson1, J Mocco1, Christopher P. Kellner1 1Department of Neurosurgery, Icahn School of Medicine at Mount Sinai This paper details the surgical protocol for minimally invasive endoscopic intracerebral hemorrhage evacuation using the SCUBA technique. Neuroscience Generation of Human Brain Organoids for Mitochondrial Disease Modeling Stephanie Le1, Laura Petersilie2, Gizem Inak1,4, Carmen Menacho-Pando1, Karl W. Kafitz2, Agnieszka Rybak-Wolf3, Nikolaus Rajewsky3, Christine R. Rose2, Alessandro Prigione1,5 1Department of General Pediatrics, Neonatology and Pediatric Cardiology, Duesseldorf University Hospital, Medical Faculty, Heinrich Heine University, 2Institute of Neurobiology, Heinrich Heine University, 3Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrueck Center for Molecular Medicine (MDC), 4Seaver Autism Center for Research and Treatment, Icahn School of Medicine at Mount Sinai, 5Max Delbrueck Center for Molecular Medicine (MDC) We describe a detailed protocol for the generation of human induced pluripotent stem cell-derived brain organoids and their use in modeling mitochondrial diseases. Bioengineering Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging Mariah R. R. Daal1, Gustav J. Strijkers1,2, Claudia Calcagno2, Ruslan R. Garipov3, Rob C. I. Wüst1,4, David Hautemann5, Bram F. Coolen1 1Department of Biomedical Engineering & Physics, Amsterdam University Medical Centers, Amsterdam Cardiovascular Sciences, University of Amsterdam, 2BioMedical Engineering and Imaging Institute, Icahn School of Medicine at Mount Sinai, 3MR Solutions Ltd., 4Laboratory for Myology, Department of Human Movement Sciences, Faculty of Behavioral and Movement Sciences, Vrije Universiteit Amsterdam, 5Medis medical imaging systems B.V. This study describes a comprehensive cardiovascular magnetic resonance imaging (CMR) protocol to quantify the left ventricular functional parameters of the mouse heart. The protocol describes the acquisition, post-processing, and analysis of the CMR images as well as assessment of different cardiac functional parameters. Immunology and Infection Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform Tracey Freeman1, Christina Andreou2, Robert P. Sebra3,4, Kristin G. Beaumont2, Talia H. Swartz1 1Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, 2Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, Icahn Institute of Data Science and Genomic Technology, 3Black Family Stem Cell Institute, Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, Icahn Institute of Data Science and Genomic Technology, 4Sema4, a Mount Sinai venture Here, we present a protocol in which single cells are monitored for acute events and productive HIV-1 infection on a nanofluidic device. Imaging data define virus-host receptor interactions and signaling pathway dynamics. This is the first method for nanofluidic high-throughput longitudinal single-cell culture and imaging to study signaling kinetics and molecular interactions. Biology Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro Rihab Bouchareb1, Djamel Lebeche1,2,3 1Cardiovascular Research Center, The Icahn School of Medicine at Mount Sinai, 2Diabetes, Obesity and Metabolism Institute, Department of Medicine, The Icahn School of Medicine at Mount Sinai, 3Graduate School of Biomedical Sciences, The Icahn School of Medicine at Mount Sinai This article describes the isolation of mouse aortic valve cells by a two-step collagenase procedure. Isolated mouse valve cells are important for performing different assays, such as this in vitro calcification assay, and for investigating the molecular pathways leading to aortic valve mineralization. Neuroscience Objectively Assessing Sports Concussion Utilizing Visual Evoked Potentials Daryl H. Fong1, Adrian J. Cohen2, Dylan E. Mahony2, Neil G. Simon4, Joseph E. Herrera3, Rebecca B. Baron3, David Putrino3 1School of Aerospace, Mechanical and Mechatronic Engineering, Faculty of Engineering and Information Technologies, University of Sydney, 2HeadsafeIP, 3Department of Rehabilitation Medicine, Icahn School of Medicine at Mount Sinai, 4St Vincent's Clinical School, Faculty of Medicine, University of New South Wales A portable system capable of measuring steady-state visual-evoked potentials was developed and trialed on 65 amateur rugby players over 18 weeks to investigate SSVEP as a potential electrophysiological biomarker for concussion. Players' baselines were measured pre-season, with retesting for reliability, concussion, and recovery assessment being conducted within controlled time-periods, respectively. Immunology and Infection Analysis of Somatic Hypermutation in the JH4 intron of Germinal Center B cells from Mouse Peyer's Patches Emily Sible1, Simin Zheng2, Jee Eun Choi1, Bao Q. Vuong1 1Department of Biology, The City College of New York and The Graduate Center of The City University of New York, 2Department of Microbiology, Icahn School of Medicine at Mount Sinai Presented here is an assay to quantify somatic hypermutation within the immunoglobulin heavy chain gene locus using germinal center B cells from mouse Peyer’s patches. Neuroscience Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting Zarmeen Mussa1,2, Jessica Tome-Garcia1,2, Yan Jiang3, Schahram Akbarian2,4, Nadejda M. Tsankova1,2 1Department of Pathology, Icahn School of Medicine at Mount Sinai, 2Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, 3Institutes of Brian Science, Fudan University, 4Department of Psychiatry, Icahn School of Medicine at Mount Sinai We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses. Genetics Delivery of Modified mRNA in a Myocardial Infarction Mouse Model Keerat Kaur1,2,3, Nishat Sultana1,2,3, Yoav Hadas1,2,3, Ajit Magadum1,2,3, Mohammad Tofael Kabir Sharkar1,2,3, Elena Chepurko1,2,3, Lior Zangi1,2,3 1Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, 2Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, 3Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai This protocol presents a simple and coherent way to transiently upregulate a gene of interest using modRNA after myocardial infarction in mice. Medicine Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model Olympia Bikou1, Roger J. Hajjar2, Lahouaria Hadri1, Yassine Sassi1 1Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, 2Phospholamban Foundation This protocol describes the induction of pulmonary hypertension (PH) in mice based on the exposure to hypoxia and the injection of a VEGF receptor antagonist. The animals develop PH and right ventricular (RV) hypertrophy 3 weeks after the initiation of the protocol. The functional and morphometrical characterization of the model is also presented. Genetics Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin (sh)RNA Nandaraj Taye1, Sarah Stanley1, Dirk Hubmacher1 1Orthopaedic Research Laboratories, Leni & Peter W. May Department of Orthopaedics, Icahn School of Medicine at Mount Sinai We provide a protocol to stably knock down genes encoding extracellular matrix (ECM) proteins in C2C12 myoblasts using small-hairpin (sh) RNA. Targeting ADAMTSL2 as an example, we describe the methods for the validation of the knockdown efficiency on the mRNA, protein, and cellular level during C2C12 myoblast to myotube differentiation. Biochemistry A Rapidly Incremented Tethered-Swimming Maximal Protocol for Cardiorespiratory Assessment of Swimmers Dalton M. Pessôa Filho*1,2, Danilo A. Massini*2, Leandro O. C. Siqueira*2, Luiz Gustavo A. Santos*2, Camila M. T. Vasconcelos*2, Tiago A. F. Almeida*1,3, Mário A. C. Espada*4,5, Joana F. Reis*3,6, Francisco B. Alves*3, Fred J. DiMenna*7,8 1Department of Physical Education, São Paulo State University (UNESP) at Bauru, 2Institute of Bioscience, Graduate Program in Human Development and Technology, São Paulo State University (UNESP) at Rio Claro, 3Ciper, Faculdade de Motricidade Humana, Universidade de Lisboa, 4Department of Science and Technology, School of Education, Polytechnic Institute of Setúbal, 5Quality of Life Research Center, Polytechnic Institute of Santarem, 6Universidade Europeia at Lisbon, 7Division of Endocrinology, Diabetes and Bone Disease, Icahn School of Medicine at Mount Sinai, 8Department of Biobehavioral Sciences, Teachers College, Columbia University As opposed to measurement during free swimming, which presents inherent challenges and limitations, determination of important parameters of cardiorespiratory function for swimmers can be made using a more feasible and easier to administer tethered-swimming rapidly incremented protocol with gas exchange and ventilatory data collection. Medicine Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization Jae Gyun Oh1, Jaydev Dave1, Changwon Kho1, Francesca Stillitano1 1Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai Here we describe and validate a method to consistently generate robust human induced pluripotent stem cell-derived cardiomyocytes and characterize their function. These techniques may help in developing mechanistic insight into signaling pathways, provide a platform for large-scale drug screening, and reliably model cardiac diseases. Developmental Biology Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis Meng Wu1, Divya Kriti1, Harm van Bakel1,2, Ethylin Wang Jabs1, Greg Holmes1 1Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, 2Icahn Institute for Data Science and Genomic Technology, Icahn School of Medicine at Mount Sinai This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis. Developmental Biology Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors Rita Silvério-Alves1,2,3, Andreia M. Gomes3, Ilia Kurochkin4, Kateri A. Moore5,6, Carlos-Filipe Pereira1,2,3 1Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, 2Wallenberg Center for Molecular, Lund University, 3Center for Neuroscience and Cell Biology, University of Coimbra, 4Skolkovo Institute of Science and Technology, 5Department of Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, 6Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells. Immunology and Infection Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets Rebecca L.R. Powell1, Alisa Fox1 1Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai Breast milk transmits human immunodeficiency virus (HIV), though only ~15% of infants breastfed by HIV-infected mothers become infected. Breastfed infants ingest ~105−108 maternal leukocytes daily, though these cells are understudied. Here we describe the isolation of breast milk leukocytes and an analysis of their phagocytic capacity. Cancer Research Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts Dan Han1, Veronica Rodriguez-Bravo1, Sudeh Izadmehr1, Josep Domingo-Domenech1, Carlos Cordon-Cardo1 1Department of Pathology, Icahn School of Medicine at Mount Sinai We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells. Medicine Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid Luena Papa1, Mansour Djedaini1, Ronald Hoffman1 1Division of Hematology/Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications. Medicine The Left Pneumonectomy Combined with Monocrotaline or Sugen as a Model of Pulmonary Hypertension in Rats Michael G. Katz1, Anthony S. Fargnoli1, Sarah M. Gubara1, Malik Bisserier1, Yassine Sassi1, Charles R. Bridges1, Roger J. Hajjar1, Lahouaria Hadri1 1Cardiovascular Research Center, Department of Cardiology, Icahn School of Medicine at Mount Sinai The rodent left pneumonectomy is a valuable technique in pulmonary hypertension research. Here, we present a protocol to describe the rat pneumonectomy procedure and postoperative care to ensure minimal morbidity and mortality. Bioengineering A Tripeptide-Stabilized Nanoemulsion of Oleic Acid Sylwia A. Dragulska1, Marek T. Wlodarczyk1,2, Mina Poursharifi1,3, John A. Martignetti4,5,6, Aneta J. Mieszawska1,2,3 1Department of Chemistry, Brooklyn College, The City University of New York, 2Ph.D. Program in Chemistry, The Graduate Center of The City University of New York, 3Ph.D. Program in Biochemistry, The Graduate Center of The City University of New York, 4Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, 5Women's Health Research Institute, Icahn School of Medicine at Mount Sinai, 6Laboratory for Translational Research, Western Connecticut Health Network This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate. Bioengineering Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers Anna M. Chiarella1, Tiffany A. Wang2, Kyle V. Butler3, Jian Jin3, Nathaniel A. Hathaway4 1Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, Curriculum in Genetics and Molecular Biology, University of North Carolina, 2College of Arts and Sciences, Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, University of North Carolina, 3Chemical Biology and Drug Discovery, Pharmacological Sciences and Oncological Sciences, Icahn School of Medicine at Mount Sinai, 4Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, Curriculum in Genetics and Molecular Biology, Lineberger Comprehensive Cancer Center, University of North Carolina Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner. Immunology and Infection A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion Anthony M. Esposito1,2, Alexandra Y. Soare1, Foramben Patel1, Namita Satija1, Benjamin K. Chen1, Talia H. Swartz1 1Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, Immunology Institute, 2Department of Biology, New Jersey City University We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems. Immunology and Infection A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies Mark J. Bailey1,2, Felix Broecker1, Paul E. Leon1,2, Gene S. Tan3 1Department of Microbiology, Icahn School of Medicine at Mount Sinai, 2Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, 3Department of Infectious Disease, J. Craig Venter Institute We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity. Developmental Biology Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos Evan Bardot1,2,3, Nikos Tzavaras4, Deanna L. Benson5,6, Nicole C. Dubois1,2,3 1Cell, Developmental, and Regenerative Biology Department, Icahn School of Medicine at Mount Sinai, 2Mindich Child Health and Development Institute, Icahn School of Medicine at Mount Sinai, 3Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, 4Microscopy Core, Icahn School of Medicine at Mount Sinai, 5Department of Neuroscience, Icahn School of Medicine at Mount Sinai, 6Friedman Brain Institute, Icahn School of Medicine at Mount Sinai Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems. Cancer Research Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel Lisa Mohr1, Marc Carceles-Cordon1, Jungreem Woo1, Carlos Cordon-Cardo1, Josep Domingo-Domenech1, Veronica Rodriguez-Bravo1,2 1Department of Pathology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, 2Department of Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai Resistance to cancer therapies contributes to disease progression and death. Determining the mechanistic underpinnings of resistance is crucial for improving therapeutic response. This manuscript details the protocol to generate taxane-resistant cell models of prostate cancer (PC) to help dissecting the pathways involved in progression to Docetaxel resistance in PC patients. Behavior Novel Object Recognition Test for the Investigation of Learning and Memory in Mice Lindsay M. Lueptow1 1Pharmacological Sciences, Icahn School of Medicine at Mount Sinai The object recognition test (ORT) is a simple and efficient assay for evaluating learning and memory in mice. The methodology is described below. Immunology and Infection Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies Paul E. Leon1,2, Teddy John Wohlbold1,2, Wenqian He1,2, Mark J. Bailey1,2, Carole J. Henry3, Patrick C. Wilson3, Florian Krammer1, Gene S. Tan1 1Department of Microbiology, Icahn School of Medicine at Mount Sinai, 2Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, 3The Department of Medicine, Section of Rheumatology, The Knapp Center for Lupus and Immunology Research, The University of Chicago We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies. Immunology and Infection Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity Jordi Ochando1,2, Patricia Conde1,2 1Department of Medicine, Icahn School of Medicine at Mount Sinai, 2Immunología de Trasplantes, Centro Nacional de Microbiología, Instituto de Salud Carlos III Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions. Cancer Research A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines Jun Tang1, Carlos Pérez-Medina1,2, Yiming Zhao2, Ahmad Sadique1, Willem J. M. Mulder2, Thomas Reiner1 1Department of Radiology, Memorial Sloan Kettering Cancer Center, 2Translational and Molecular Imaging Institute, Icahn School of Medicine at Mount Sinai The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation. Developmental Biology Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program Michael G. Daniel1,2,3, Carlos-Filipe Pereira4, Jeffrey M. Bernitz1,2,3, Ihor R. Lemischka1,3,5, Kateri Moore1,3 1Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, 2The Graduate School of Biomedical Science, Icahn School of Medicine at Mount Sinai, 3Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, 4Center for Neuroscience and Cell Biology, University of Coimbra, 5Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant. Developmental Biology Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae Theresa A. Reimels1,2, Cathie M. Pfleger1,2 1The Graduate School of Biomedical Sciences, The Icahn School of Medicine at Mount Sinai, 2Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells. Neuroscience HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice Terrell Holloway1, Jose L. Moreno1, Javier González-Maeso1,2,3,4 1Department of Psychiatry, Icahn School of Medicine at Mount Sinai, 2Department of Neurology, Icahn School of Medicine at Mount Sinai, 3Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, 4Department of Physiology and Biophysics, Virginia Commonwealth University Medical School This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation. Neuroscience Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for Optical Detection of Neurotransmitters In Vivo Emre Lacin1, Arnaud Muller2, Marian Fernando1, David Kleinfeld2,3, Paul A. Slesinger1 1Department of Neuroscience, Icahn School of Medicine at Mount Sinai, 2Department of Physics, University of California, San Diego, 3Section of Neurobiology, University of California, San Diego We present a protocol to create cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) for the optical detection of volumetric neurotransmitter release. Bioengineering Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy Timothy J. Cashman1, Rebecca Josowitz2, Bruce D. Gelb2, Ronald A. Li1,3, Nicole C. Dubois2, Kevin D. Costa1 1Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, 2The Mindich Child Health and Development Institute, Icahn School of Medicine at Mount Sinai, 3Stem Cell & Regenerative Medicine Consortium, LKS Faculty of Medicine, University of Hong Kong This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart. Biology Identification of Kinesin-1 Cargos Using Fluorescence Microscopy Clement M. Lee1 1Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai Here, a protocol is presented to identify Kinesin-1 cargos. A motorless mutant of the Kinesin-1 heavy chain (KIF5B) aggregates in the cytoplasm and induces aggregation of its cargos. Both aggregates are detected under fluorescence microscopy. A similar strategy can be employed to identify cargos of other motor proteins. Medicine Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells Estrelania S. Williams1, Veronica Rodriguez-Bravo3, Uma Chippada-Venkata2, Janis De Ia Iglesia-Vicente1, Yixuan Gong2, Matthew Galsky2, William Oh2, Carlos Cordon-Cardo1, Josep Domingo-Domenech1 1Department of Pathology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, 2Department of Hematology/Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, 3Molecular Biology Program, Memorial Sloan-Kettering Cancer Center This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men. Medicine The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma Michael Li1,2, Ravi R. Pathak5, Esther Lopez-Rivera3, Scott L. Friedman1, Julio A. Aguirre-Ghiso4, Andrew G. Sikora5 1Department of Medicine, Division of Liver Diseases, Icahn School of Medicine at Mount Sinai, 2Department of Otolaryngology, Icahn School of Medicine at Mount Sinai, 3Division of Nephrology, Columbia University College of Physicians and Surgeons, 4Departments of Medicine, Hematology and Medical Oncology, Icahn School of Medicine at Mount Sinai, 5Bobby R. Alford Department of Otolaryngology - Head and Neck Surgery, Baylor College of Medicine The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay. Medicine A Multicenter MRI Protocol for the Evaluation and Quantification of Deep Vein Thrombosis Venkatesh Mani1, Nadia Alie1, Sarayu Ramachandran1, Philip M. Robson1, Cecilia Besa1, Gregory Piazza2, Michele Mercuri3, Michael Grosso3, Bachir Taouli1, Samuel Z. Goldhaber2, Zahi A. Fayad1 1Department of Radiology, Translational and Molecular Imaging Institute, Icahn School of Medicine at Mount Sinai, 2 The goal of this study is to use magnetic resonance venography with long-circulating gadolinium-based contrast agent and direct thrombus imaging for quantitative evaluation of DVT volume in a multicenter, clinical trial setting. Inter- and intra-observer variability assessments were conducted, and reproducibility of the protocol was determined. Medicine A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases Aaron Topol1, Ngoc N. Tran1, Kristen J. Brennand1,2 1Department of Psychiatry, Icahn School of Medicine at Mount Sinai, 2Department of Neuroscience, Icahn School of Medicine at Mount Sinai This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders. Bioengineering Diagnosis of Neoplasia in Barrett’s Esophagus using Vital-dye Enhanced Fluorescence Imaging Daniel P. Perl1, Neil Parikh1, Shannon Chang1, Paul Peng1, Nadhi Thekkek3, Michelle H. Lee1, Alexandros D. Polydorides2, Josephine Mitcham1, Rebecca Richards-Kortum3, Sharmila Anandasabapathy1 1Department of Gastroenterology, Icahn School of Medicine at Mount Sinai, 2Department of Pathology, Icahn School of Medicine at Mount Sinai, 3Department of Bioengineering, Rice University Vital-dye enhanced fluorescence imaging (VFI) is a novel in vivo technique that combines high-resolution epithelial imaging with exogenous topical fluorescent contrast to highlight glandular morphology and delineate neoplasia (high grade dysplasia and cancer) in the distal esophagus. Medicine Isolation of Cancer Stem Cells From Human Prostate Cancer Samples Samuel J. Vidal1, S. Aidan Quinn1, Janis de la Iglesia-Vicente1, Dennis M. Bonal1, Veronica Rodriguez-Bravo2, Adolfo Firpo-Betancourt1, Carlos Cordon-Cardo1, Josep Domingo-Domenech1 1Department of Pathology, Icahn School of Medicine at Mount Sinai, 2Molecular Biology Program, Memorial Sloan-Kettering Cancer Center The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps. Immunology and Infection Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System Irina Margine1,2, Peter Palese1,3, Florian Krammer1 1Department of Microbiology, Icahn School of Medicine at Mount Sinai, 2Graduate School of Biological Sciences, Icahn School of Medicine at Mount Sinai, 3Department of Medicine, Icahn School of Medicine at Mount Sinai Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins. Immunology and Infection Rescue of Recombinant Newcastle Disease Virus from cDNA Juan Ayllon1,2, Adolfo García-Sastre1,2,3, Luis Martínez-Sobrido4 1Department of Microbiology, Icahn School of Medicine at Mount Sinai, 2Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, 3Department of Medicine, Icahn School of Medicine at Mount Sinai, 4Department of Microbiology and Immunology, School of Medicine and Dentistry, University of Rochester Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.
