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Icahn School of Medicine at Mount Sinai

60 ARTICLES PUBLISHED IN JoVE

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Biology

A Chromatin Assay for Human Brain Tissue
Anouch Matevossian 1, Schahram Akbarian 1
1Psychiatry, Brudnick Neuropsychiatric Research Institute, University of Massachusetts Medical School

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

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Biology

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue
Anouch Matevossian 1, Schahram Akbarian 1
1Psychiatry, Brudnick Neuropsychiatric Research Institute, University of Massachusetts Medical School

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

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Biology

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation
Oshri Avraham *1, Sophie Zisman *1, Yoav Hadas 1, Lilach Vald 1, Avihu Klar 1
1Department of Medical Neurobiology, Institute for Medical Research Israel Canada, Hebrew University-Hadassah Medical School

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

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Immunology and Infection

Generation of Recombinant Influenza Virus from Plasmid DNA
Luis Martínez-Sobrido 1, Adolfo García-Sastre 2
1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 2Departments of Microbiology and Medicine, and Global Health and Emerging Pathogens Institute, Mount Sinai School of Medicine

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

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Biology

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells
Bartosz Balana 1, Natalie Taylor 1, Paul A. Slesinger 1
1Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

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Neuroscience

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection
Tatiana Hurtado de Mendoza 1, Bartosz Balana 2, Paul A. Slesinger 2, Inder M. Verma 1
1Laboratory of Genetics, The Salk Institute for Biological Studies, 2Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

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Medicine

Gene Transfer for Ischemic Heart Failure in a Preclinical Model
Kiyotake Ishikawa 1, Dennis Ladage 1, Lisa Tilemann 1, Kenneth Fish 1, Yoshiaki Kawase 1, Roger J. Hajjar 1
1Cardiovascular Research Center, Mount Sinai School of Medicine

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

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Medicine

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice
Saeed Ansari *1, Hassan Azari *1,2, Douglas J. McConnell 1, Aqeela Afzal 1, J Mocco 1
1Department of Neurosurgery, University of Florida, 2Department of Anatomical Sciences, Shiraz University of Medical Sciences

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

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Medicine

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice
Michael T. Kuhlmann 1, Simon Cuhlmann 2,3, Irmgard Hoppe 1, Rob Krams 3, Paul C. Evans 2, Gustav J. Strijkers 4, Klaas Nicolay 4, Sven Hermann 1, Michael Schäfers 1
1European Institute for Molecular Imaging, Westfälische Wilhelms-University Münster, 2British Heart Foundation Cardiovascular Sciences Unit, Imperial College London , 3Department of Bioengineering, Imperial College London , 4Biomedical Engineering, Eindhoven University of Technology

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

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Clinical Skills

COVID-19 / Coronavirus Outbreak: A Rapidly Deployable Medical Facility Optimized for Epidemics (Smart Pod)
Elena V. Petrova 1,2, Huan (Teena) Xu 3, Brodus A. Franklin 1, Laila E. Woc-Colburn 3, Sharmila Anandasabapathy 1,2
1Baylor Global Health, Baylor College of Medicine, 2Department of Medicine-Gastroenterology, Baylor College of Medicine, 3Department of Medicine-Infection Disease, Baylor College of Medicine

COVID-19 / Coronavirus Outbreak: A Rapidly Deployable Medical Facility Optimized for Epidemics (Smart Pod)

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Clinical Skills

COVID-19 / Coronavirus Outbreak: Guidance for Hand Hygiene for Healthcare Providers to Ensure a Safe and Healthy Environment
Elena V. Petrova 1,2, Huan (Teena) Xu 3, Brodus A. Franklin 1, Laila E. Woc-Colburn 3, Sharmila Anandasabapathy 1,2
1Baylor Global Health, Baylor College of Medicine, 2Department of Medicine-Gastroenterology, Baylor College of Medicine, 3Department of Medicine-Infection Disease, Baylor College of Medicine

COVID-19 / Coronavirus Outbreak: Guidance for Hand Hygiene for Healthcare Providers to Ensure a Safe and Healthy Environment

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Clinical Skills

COVID-19 / Coronavirus Outbreak: Donning and Doffing Personal Protective Equipment (PPE) for Healthcare Providers
Elena V. Petrova 1,2, Huan (Teena) Xu 3, Brodus A. Franklin 1, Laila E. Woc-Colburn 3, Sharmila Anandasabapathy 1,2
1Baylor Global Health, Baylor College of Medicine, 2Department of Medicine-Gastroenterology, Baylor College of Medicine, 3Department of Medicine-Infection Disease, Baylor College of Medicine

COVID-19 / Coronavirus Outbreak: Donning and Doffing Personal Protective Equipment (PPE) for Healthcare Providers

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Clinical Skills

COVID-19 / Coronavirus Outbreak: Performing a Nasal Swab Test on Patients inside a Rapidly Deployable Facility Optimized for Epidemics
Elena V. Petrova 1,2, Huan (Teena) Xu 3, Brodus A. Franklin 1, Laila E. Woc-Colburn 3, Sharmila Anandasabapathy 1,2
1Baylor Global Health, Baylor College of Medicine, 2Department of Medicine-Gastroenterology, Baylor College of Medicine, 3Department of Medicine-Infection Disease, Baylor College of Medicine

COVID-19 / Coronavirus Outbreak: Performing a Nasal Swab Test on Patients inside a Rapidly Deployable Facility Optimized for Epidemics

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Neuroscience

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo
Ayelet Kohl 1, Yoav Hadas 2, Avihu Klar 2, Dalit Sela-Donenfeld 1
1Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, 2Department of Medical Neurobiology, The Hebrew University of Jerusalem

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox–plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

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Immunology and Infection

Rescue of Recombinant Newcastle Disease Virus from cDNA
Juan Ayllon 1,2, Adolfo García-Sastre 1,2,3, Luis Martínez-Sobrido 4
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.

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Bioengineering

Diagnosis of Neoplasia in Barrett’s Esophagus using Vital-dye Enhanced Fluorescence Imaging
Daniel P. Perl 1, Neil Parikh 1, Shannon Chang 1, Paul Peng 1, Nadhi Thekkek 3, Michelle H. Lee 1, Alexandros D. Polydorides 2, Josephine Mitcham 1, Rebecca Richards-Kortum 3, Sharmila Anandasabapathy 1
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.

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Immunology and Infection

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System
Irina Margine 1,2, Peter Palese 1,3, Florian Krammer 1
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.

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Medicine

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples
Samuel J. Vidal 1, S. Aidan Quinn 1, Janis de la Iglesia-Vicente 1, Dennis M. Bonal 1, Veronica Rodriguez-Bravo 2, Adolfo Firpo-Betancourt 1, Carlos Cordon-Cardo 1, Josep Domingo-Domenech 1
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.

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Medicine

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma
Michael Li 1,2, Ravi R. Pathak 5, Esther Lopez-Rivera 3, Scott L. Friedman 1, Julio A. Aguirre-Ghiso 4, Andrew G. Sikora 5
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.

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Medicine

A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases
Aaron Topol 1, Ngoc N. Tran 1, Kristen J. Brennand 1,2
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.

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Medicine

A Multicenter MRI Protocol for the Evaluation and Quantification of Deep Vein Thrombosis
Venkatesh Mani 1, Nadia Alie 1, Sarayu Ramachandran 1, Philip M. Robson 1, Cecilia Besa 1, Gregory Piazza 2, Michele Mercuri 3, Michael Grosso 3, Bachir Taouli 1, Samuel Z. Goldhaber 2, Zahi A. Fayad 1
1Department of Radiology, Translational and Molecular Imaging Institute, Icahn School of Medicine at Mount Sinai, 2Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, 3Daiichi Sankyo Pharma Development

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.

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Medicine

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells
Estrelania S. Williams 1, Veronica Rodriguez-Bravo 3, Uma Chippada-Venkata 2, Janis De Ia Iglesia-Vicente 1, Yixuan Gong 2, Matthew Galsky 2, William Oh 2, Carlos Cordon-Cardo 1, Josep Domingo-Domenech 1
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.

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Neuroscience

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for Optical Detection of Neurotransmitters In Vivo
Emre Lacin 1, Arnaud Muller 2, Marian Fernando 1, David Kleinfeld 2,3, Paul A. Slesinger 1
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.

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Bioengineering

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy
Timothy J. Cashman 1, Rebecca Josowitz 2, Bruce D. Gelb 2, Ronald A. Li 1,3, Nicole C. Dubois 2, Kevin D. Costa 1
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.

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Biology

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy
Clement M. Lee 1
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.

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Neuroscience

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice
Terrell Holloway 1, Jose L. Moreno 1, Javier González-Maeso 1,2,3,4
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.

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Immunology and Infection

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity
Jordi Ochando 1,2, Patricia Conde 1,2
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.

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Developmental Biology

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program
Michael G. Daniel 1,2,3, Carlos-Filipe Pereira 4, Jeffrey M. Bernitz 1,2,3, Ihor R. Lemischka 1,3,5, Kateri Moore 1,3
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.

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Cancer Research

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines
Jun Tang 1, Carlos Pérez-Medina 1,2, Yiming Zhao 2, Ahmad Sadique 1, Willem J. M. Mulder 2, Thomas Reiner 1
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.

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Behavior

Novel Object Recognition Test for the Investigation of Learning and Memory in Mice
Lindsay M. Lueptow 1
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.

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Immunology and Infection

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies
Paul E. Leon 1,2, Teddy John Wohlbold 1,2, Wenqian He 1,2, Mark J. Bailey 1,2, Carole J. Henry 3, Patrick C. Wilson 3, Florian Krammer 1, Gene S. Tan 1
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.

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Immunology and Infection

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies
Mark J. Bailey 1,2, Felix Broecker 1, Paul E. Leon 1,2, Gene S. Tan 3
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.

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Cancer Research

Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel
Lisa Mohr 1, Marc Carceles-Cordon 1, Jungreem Woo 1, Carlos Cordon-Cardo 1, Josep Domingo-Domenech 1, Veronica Rodriguez-Bravo 1,2
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.

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Developmental Biology

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos
Evan Bardot 1,2,3, Nikos Tzavaras 4, Deanna L. Benson 5,6, Nicole C. Dubois 1,2,3
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.

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Cancer Research

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts
Dan Han 1, Veronica Rodriguez-Bravo 1, Sudeh Izadmehr 1, Josep Domingo-Domenech 1, Carlos Cordon-Cardo 1
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.

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Immunology and Infection

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion
Anthony M. Esposito 1,2, Alexandra Y. Soare 1, Foramben Patel 1, Namita Satija 1, Benjamin K. Chen 1, Talia H. Swartz 1
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.

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Neuroscience

Minimally Invasive Endoscopic Intracerebral Hemorrhage Evacuation
Jonathan Pan 1, Alexander G. Chartrain 1, Jacopo Scaggiante 1, Olivia S. Allen 1, Danny Hom 1, Joshua B. Bederson 1, J Mocco 1, Christopher P. Kellner 1
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.

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JoVE Journal

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers
Anna M. Chiarella 1, Tiffany A. Wang 2, Kyle V. Butler 3, Jian Jin 3, Nathaniel A. Hathaway 4
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.

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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

Remote Laboratory Management: Respiratory Virus Diagnostics
Elena V. Petrova 1,2, Vasanthi Avadhanula 3, Sarah Michel 1, Karen E. Gincoo 3, Pedro A. Piedra 3, Sharmila Anandasabapathy 1,2
1Baylor Global Health, Baylor College of Medicine, 2Department of Medicine, Baylor College of Medicine, 3Department of Molecular Virology and Microbiology, Baylor College of Medicine

A rapidly-deployable, off-grid laboratory has been designed and built for remote, resource-constrained global settings. The features and critical aspects of the logistically-enhanced, expandable, multifunctional laboratory modules are explored. A checklist for a basic laboratory workflow and a protocol for a respiratory viral diagnostic test are developed and presented.

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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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Cancer Research

Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids
Po Hien Ear 1, Guiying Li 1, Meng Wu 2, Ellen Abusada 3, Andrew M. Bellizzi 3, James R. Howe 1
1Department of Surgery, University of Iowa Carver College of Medicine, 2High Throughput Screening Facility, University of Iowa Carver College of Medicine, 3Department of Pathology, University of Iowa Carver College of Medicine

Neuroendocrine tumors (NETs) originate from neuroendocrine cells of the neural crest. They are slow growing and challenging to culture. We present an alternative strategy to grow NETs from the small bowel by culturing them as spheroids. These spheroids have small bowel NET markers and can be used for drug testing.

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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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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. 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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