Icahn School of Medicine at Mount Sinai

21 ARTICLES PUBLISHED IN JoVE

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Bioengineering

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid
Sylwia A. Dragulska 1, Marek T. Wlodarczyk 1,2, Mina Poursharifi 1,3, John A. Martignetti 4,5,6, Aneta J. Mieszawska 1,2,3
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.

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Medicine

The Left Pneumonectomy Combined with Monocrotaline or Sugen as a Model of Pulmonary Hypertension in Rats
Michael G. Katz 1, Anthony S. Fargnoli 1, Sarah M. Gubara 1, Malik Bisserier 1, Yassine Sassi 1, Charles R. Bridges 1, Roger J. Hajjar 1, Lahouaria Hadri 1
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.

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Medicine

Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model
Olympia Bikou 1, Roger J. Hajjar 2, Lahouaria Hadri 1, Yassine Sassi 1
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.

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Medicine

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid
Luena Papa 1, Mansour Djedaini 1, Ronald Hoffman 1
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.

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Developmental Biology

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors
Rita Silvério-Alves 1,2,3, Andreia M. Gomes 3, Ilia Kurochkin 4, Kateri A. Moore 5,6, Carlos-Filipe Pereira 1,2,3
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.

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Medicine

Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization
Jae Gyun Oh 1, Jaydev Dave 1, Changwon Kho 1, Francesca Stillitano 1
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.

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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. Powell 1, Alisa Fox 1
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.

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Developmental Biology

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis
Meng Wu 1, Divya Kriti 1, Harm van Bakel 1,2, Ethylin Wang Jabs 1, Greg Holmes 1
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.

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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.

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Genetics

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin (sh)RNA
Nandaraj Taye 1, Sarah Stanley 1, Dirk Hubmacher 1
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.

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Genetics

Delivery of Modified mRNA in a Myocardial Infarction Mouse Model
Keerat Kaur 1,2,3, Nishat Sultana 1,2,3, Yoav Hadas 1,2,3, Ajit Magadum 1,2,3, Mohammad Tofael Kabir Sharkar 1,2,3, Elena Chepurko 1,2,3, Lior Zangi 1,2,3
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.

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Immunology and Infection

Analysis of Somatic Hypermutation in the JH4 intron of Germinal Center B cells from Mouse Peyer's Patches
Emily Sible 1, Simin Zheng 2, Jee Eun Choi 1, Bao Q. Vuong 1
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.

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Neuroscience

Objectively Assessing Sports Concussion Utilizing Visual Evoked Potentials
Daryl H. Fong 1, Adrian J. Cohen 2, Dylan E. Mahony 2, Neil G. Simon 4, Joseph E. Herrera 3, Rebecca B. Baron 3, David Putrino 3
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.

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Neuroscience

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting
Zarmeen Mussa 1,2, Jessica Tome-Garcia 1,2, Yan Jiang 3, Schahram Akbarian 2,4, Nadejda M. Tsankova 1,2
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.

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Bioengineering

Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging
Mariah R. R. Daal 1, Gustav J. Strijkers 1,2, Claudia Calcagno 2, Ruslan R. Garipov 3, Rob C. I. Wüst 1,4, David Hautemann 5, Bram F. Coolen 1
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.

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Immunology and Infection

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform
Tracey Freeman 1, Christina Andreou 2, Robert P. Sebra 3,4, Kristin G. Beaumont 2, Talia H. Swartz 1
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.

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Neuroscience

Generation of Human Brain Organoids for Mitochondrial Disease Modeling
Stephanie Le 1, Laura Petersilie 2, Gizem Inak 1,4, Carmen Menacho-Pando 1, Karl W. Kafitz 2, Agnieszka Rybak-Wolf 3, Nikolaus Rajewsky 3, Christine R. Rose 2, Alessandro Prigione 1,5
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.

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Bioengineering

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues
Camille C. van Neste 1, Karlo A. Wiley 1, Serena W. Chang 1, Joseph Borrello 1, Irene C. Turnbull 1, Kevin D. Costa 1
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.

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Bioengineering

Semi-Automatic Graphical Tool for Measuring Coronary Artery Spatially Weighted Calcium Score from Gated Cardiac Computed Tomography Images
Heli J. Patel 1, Audrey E. Kaufman 1, Marco Pereañez 1, Georgios Soultanidis 1, Sarayu Ramachandran 1, Sonum Naidu 1, Venkatesh Mani 1, Zahi A. Fayad 1, Philip M. Robson 1
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.

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JoVE Core

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. Schwarz 1,2,3,4,5, Emily Ruth Sartori 1,2,3,4,5, Rikki B. Rooklin 1,2,3,4,5, Braxton R. Schuldt 1,2,3,4,5, Louise A. Mesentier-Louro 1,2,3,4,5, Joel W. Blanchard 1,2,3,4,5
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.

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Biology

Non-Invasive Visualization of Nailbed Microvascular Morphology in Mice Using Capillaroscopy
Olivia L. Bossardet *1, Clara C. Cousins *2, Joseph M. Holden 1, Vincent Yao 3, Kristin L. Clark 1, Louis R. Pasquale 4, Emmanuel S. Buys 3, Lauren K. Wareham 1,2
1Department of Ophthalmology and Visual Sciences, Vanderbilt Eye Institute, Vanderbilt University Medical Center, 2Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, 3Department of Anesthesia and Critical Pain Medicine, Massachusetts General Hospital, Harvard Medical School, 4Department of Ophthalmology, Icahn School of Medicine at Mount Sinai

Capillaroscopy is an accessible tool for direct, inexpensive, and non-invasive visualization of microvasculature. The goal of this protocol is to enable researchers to use capillaroscopy for the visualization of peripheral microvascular morphology in the nailbeds of mice.

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