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Ottawa Hospital Research Institute

12 ARTICLES PUBLISHED IN JoVE

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Biology

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells
Carmen G. Palii 1, Roya Pasha 1, Marjorie Brand 1,2
1The Sprott Center for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

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Medicine

Ex Vivo Infection of Live Tissue with Oncolytic Viruses
Jean-Simon Diallo 1, Dominic Roy 1, Hesham Abdelbary 1, Naomi De Silva 1, John C. Bell 1
1Center for Innovative Cancer Research, Ottawa Hospital Research Institute (OHRI)

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

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Neuroscience

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues
Ryan W. O'Meara 1,2, Scott D. Ryan 1, Holly Colognato 3, Rashmi Kothary 1,2,4
1Regenerative Medicine Program, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa , 3Department of Pharmacological Sciences, Stony Brook University, 4Department of Medicine, University of Ottawa

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

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Biology

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle
Alessandra Pasut 1,2, Andrew E. Jones 1,2, Michael A. Rudnicki 1,2
1Sprott Center for Stem Cell Research, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

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Neuroscience

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis
Lyndsay Murray 1, Thomas H Gillingwater 2, Rashmi Kothary 1
1Regenerative Medicine Program, Ottawa Hospital Research Institute, 2Centre for Integrative Physiology, University of Edinburgh

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

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Medicine

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies
Lee-Hwa Tai 1, Christiano Tanese de Souza 1, Shalini Sahi 1, Jiqing Zhang 1,2,4, Almohanad A Alkayyal 1,3,5, Abhirami Anu Ananth 1,3, Rebecca A.C. Auer 1,6
1Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa, 3Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 4Department of Neurosurgery, The Second Hospital of Shandong University, 5Department of Medical Laboratory Technology, University of Tabuk, 6Department of Surgery, Ottawa General Hospital

A mouse tumor model of surgical stress is used to explore how postoperative immune suppression promotes metastatic disease and to evaluate immunostimulatory perioperative therapies.

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Biology

High-throughput Titration of Luciferase-expressing Recombinant Viruses
Vanessa Garcia 1,2, Ramya Krishnan 1,2, Colin Davis 1,2, Cory Batenchuk 1,2, Fabrice Le Boeuf 1,3, Hesham Abdelbary 1,3, Jean-Simon Diallo 1,2
1Center for Innovative Cancer Research, Ottawa Hospital Research Institute, 2Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 3Faculty of Medicine, University of Ottawa

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

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Biology

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs
Jennifer J.P. Collins 1,2, Marius A. Möbius 1,3,4, Bernard Thébaud 1,2,5
1Sinclair Centre for Regenerative Medicine, Ottawa Hospital Research Institute, 2University of Ottawa, 3Department of Neonatology and Pediatric Critical Care Medicine, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, 4DFG Research Center and Cluster of Excellence for Regenerative Therapies (CRTD), Technische Universität, Dresden, 5Children's Hospital of Eastern Ontario Research Institute

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

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Cancer Research

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor
Taha Azad *1, Kazem Nouri *1, Helena J. Janse van Rensburg 1, Yawei Hao 1, Xiaolong Yang 1
1Department of Pathology and Molecular Medicine, Queen's University

Here we present a luciferase-based biosensor to quantify the kinase activity of large tumor suppressor (LATS)-a central kinase in the Hippo signaling pathway. This biosensor has diverse applications in basic and translational research aimed at investigating Hippo pathway regulators in vitro and in vivo.

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Immunology and Infection

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection
Taylor R. Jamieson 1,2, Joanna Poutou 1,2, Ricardo Marius 1,2, Xiaohong He 1, Reza Rezaei 1,2, Taha Azad 1,2, Carolina S. Ilkow 1,2
1Ottawa Hospital Research Institute, 2Department of Biochemistry, Microbiology and Immunology, University of Ottawa

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

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Immunology and Infection

Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter
Reza Rezaei 1,2, Abera Surendran 1,2, Ragunath Singaravelu 1,2, Taylor R. Jamieson 1,2, Parisa Taklifi 3, Joanna Poutou 1,2, Taha Azad 1,2, Carolina S. Ilkow 1,2
1Ottawa Hospital Research Institute, 2Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 3Department of Biotechnology, University of Tehran

The outlined protocol describes the procedure for producing the HiBiT-receptor-binding domain protein complex and its application for fast and sensitive detection of SARS-CoV-2 antibodies.

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Cancer Research

Scalable Biomanufacturing Workflow to Produce and Isolate Natural Killer Cell-Derived Extracellular Vesicle-Based Cancer Biotherapeutics
Frederic St-Denis-Bissonnette *1,2, Melanie Kirkby *1,2, Lisheng Wang 2,3,4, Jessie R. Lavoie 1,2
1Biologic and Radiopharmaceutical Drugs Directorate, 2Department of Biochemistry, Microbiology and Immunology Institute, University of Ottawa, 3Centre for Infection, Immunity and Inflammation Institute, University of Ottawa, 4Regenerative Medicine Program, Ottawa Hospital Research Institute

Natural killer cell-derived extracellular vesicles (NK-EVs) hold promising potential as cancer biotherapeutics. This methodology-based study presents a scalable closed-loop biomanufacturing workflow designed to continuously produce and isolate large quantities of high-purity NK-EVs. In-process control testing is performed throughout the biomanufacturing workflow, ensuring the EVs meet quality standards for product release.

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