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Cedars-Sinai Medical Center

27 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)
Ching Wen Tseng 1, Marisel Sanchez-Martinez 1, Andrea Arruda 1, George Y. Liu 1
1Department of Pediatrics, Cedars-Sinai Medical Center

Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.

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Bioengineering

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors
Jae Youn Hwang 1, Daniel L. Farkas 1, Lali K. Medina-Kauwe 2,3
1Department of Biomedical Engineering, University of Southern California, 2Department of Biomedical Sciences, Cedars-Sinai Medical Center, 3Geffen School of Medicine, University of California, Los Angeles

This article details the procedures for optical imaging analysis of the tumor-targeted nanoparticle, HerDox. In particular, detailed use of the multimode imaging device for detecting tumor targeting and assessing tumor penetration is described here.

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Chemistry

Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?
Julia Y. Ljubimova 1, Hui Ding 1, Jose Portilla-Arias 1, Rameshwar Patil 1, Pallavi R. Gangalum 1, Alexandra Chesnokova 1, Satoshi Inoue 1, Arthur Rekechenetskiy 1, Tala Nassoura 1, Keith L. Black 1, Eggehard Holler 1
1Nanomedicine Research Center, Department of Neurosurgery, Cedars-Sinai Medical Center

An example of a nano drug based on polymalic acid is presented towards the rational design of personalized medicine that is applicable to cancer. It describes the synthesis of a nano drug to treat Her2-positive human breast cancer in a nude mouse.

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Neuroscience

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue
Brandon C. Shelley 1, Geneviève Gowing 1, Clive N. Svendsen 1
1Regenerative Medicine Institute, Cedars-Sinai Medical Center

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.  Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

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Neuroscience

Neonatal Pial Surface Electroporation
Rachelle Levy 1, Jessica Molina 2, Moise Danielpour *1, Joshua J. Breunig *2
1Department of Neurosurgery, Regenerative Medicine Institute, Cedars-Sinai Medical Center, 2Department of Biomedical Sciences, Regenerative Medicine Institute, Cedars-Sinai Medical Center

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

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Immunology and Infection

A Protocol for Analyzing Hepatitis C Virus Replication
Songyang Ren *1, Deisy Contreras *1, Vaithilingaraja Arumugaswami 1,2
1Liver Program at Regenerative Medicine Institute, Department of Biomedical Sciences, Department of Surgery, Cedars-Sinai Medical Center, 2Department of Surgery, David Geffen School of Medicine at UCLA

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

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Immunology and Infection

Influenza Virus Propagation in Embryonated Chicken Eggs
Rena Brauer 1, Peter Chen 1
1Department of Medicine, Division of Pulmonary and Critical Care Medicine, Women's Guild Lung Institute, Cedars-Sinai Medical Center

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

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Bioengineering

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts
Doron Cohn Yakubovich 1, Wafa Tawackoli 2,3,4, Dmitriy Sheyn 2,3, Ilan Kallai 1, Xiaoyu Da 4, Gadi Pelled 1,2,3,4, Dan Gazit 1,2,3,4, Zulma Gazit 1,2,3
1Skeletal Biotech Laboratory, The Hebrew University–Hadassah Faculty of Dental Medicine, 2Department of Surgery, Cedars-Sinai Medical Center, 3Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, 4Biomedical Imaging Research Institute, Cedars-Sinai Medical Center

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

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Medicine

An Improved Method for Rapid Intubation of the Trachea in Mice
Tyler C. Vandivort 1,2, Dowon An 3, William C. Parks 2
1Department of Environmental and Occupational Health Sciences, University of Washington, 2Department of Medicine, Cedars-Sinai Medical Center, 3Division of Pulmonary and Critical Care Medicine, University of Washington

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

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Developmental Biology

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells
Andrei A. Kramerov 1, Mehrnoosh Saghizadeh 1,2, Alexander V. Ljubimov 1,2
1Eye Program, Board of Governors Regenerative Medicine Institute, Departments of Biomedical Sciences and Neurosurgery, Cedars-Sinai Medical Center, 2David Geffen School of Medicine, University of California, Los Angeles

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

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Immunology and Infection

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons
Deisy Contreras 1, Vaithilingaraja Arumugaswami 1,2,3
1Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, 2Department of Surgery, Cedars-Sinai Medical Center, 3Department of Surgery, David Geffen School of Medicine, University of California, Los Angeles

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

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Bioengineering

Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model
Galina Shapiro 1, Maxim Bez 1, Wafa Tawackoli 2,3,4, Zulma Gazit 1,2,3,5, Dan Gazit 1,2,3,4,5, Gadi Pelled 1,2,3,4
1Skeletal Biotech Laboratory, Hebrew University-Hadassah Faculty of Dental Medicine, 2Department of Surgery, Cedars-Sinai Medical Center, 3Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, 4Biomedical Imaging Research Institute, Cedars-Sinai Medical Center, 5Department of Orthopedics, Cedars-Sinai Medical Center

The goal of this protocol is to generate a nude rat osteoporosis-related vertebral compression fracture model that can be longitudinally evaluated in vivo using a semiautomated microcomputed tomography-based quantitative structural analysis.

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JoVE Journal

Gene Regulation and Targeted Therapy in Gastric Cancer Peritoneal Metastasis: Radiological Findings from Dual Energy CT and PET/CT
Bowen Shi 1, Huimin Lin 1, Miao Zhang 2, Wei Lu 3, Ying Qu 4, Huan Zhang 1
1Department of Radiology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 2Department of Nuclear Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 3GE Healthcare China, 4Department of Surgery, Cedars-Sinai Medical Center

This protocol describes the value of dual energy CT and PET/CT imaging methods in tumor imaging and efficacy evaluation. This article demonstrates the research methods and results acquired by dual energy CT and PET/CT to evaluate the gene regulation and targeted treatment of gastric cancer peritoneal metastasis.

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Biochemistry

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species
Joseph Ochaba 1,2, Eva L. Morozko 2,3, Jacqueline G. O’Rourke 4, Leslie M. Thompson 1,2,3
1Department of Psychiatry and Human Behavior, University of California Irvine, 2UCI MIND, University of California Irvine, 3Department of Neurobiology and Behavior, University of California Irvine, 4Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center

A method is described for fractionation of insoluble and soluble mutant huntingtin species from mouse brain and cell culture. The method described is useful for characterization and quantification of huntingtin protein flux and aids in analyzing protein homeostasis in disease pathogenesis and in the presence of perturbations

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Immunology and Infection

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow
Alberto Yáñez 1,2, Helen S. Goodridge 1,2
1Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, 2Research Division of Immunology, Cedars-Sinai Medical Center

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

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Biochemistry

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion
Miroslava Stastna 1,2,3, Amandine Thomas 1, Juliana Germano 1, Somayeh Pourpirali 1, Jennifer E. Van Eyk 1,2, Roberta A. Gottlieb 1
1The Smidt Heart Institute, Cedars-Sinai Medical Center, 2Advanced Clinical Biosystems Research Institute, Cedars-Sinai Medical Center, 3Institute of Analytical Chemistry of the Czech Academy of Sciences

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

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Immunology and Infection

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique
Tanyalak Parimon 1, Norman E. Garrett III 1, Peter Chen 1,2, Travis J. Antes 3
1Department of Medicine, Division of Pulmonary and Critical Care, Women's Guild Lung Institute, Cedars-Sinai Medical Center, 2Department of Biomedical Sciences, Cedars-Sinai Medical Center, 3Department of Medicine, Smidt Heart Institute, Cedars-Sinai Medical Center

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

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Behavior

Simultaneous Eye Tracking and Single-Neuron Recordings in Human Epilepsy Patients
Shuo Wang 1, Nand Chandravadia 2, Adam N. Mamelak 2, Ueli Rutishauser 2,3,4
1Department of Chemical and Biomedical Engineering, and Rockefeller Neuroscience Institute, West Virginia University, 2Departments of Neurosurgery and Neurology, Cedars-Sinai Medical Center, 3Center for Neural Science and Medicine, Department of Biomedical Sciences, Cedars-Sinai Medical Center, 4Division of Biology and Biological Engineering, California Institute of Technology

We describe a method to conduct single-neuron recordings with simultaneous eye tracking in humans. We demonstrate the utility of this method and illustrate how we used this approach to obtain neurons in the human medial temporal lobe that encode targets of a visual search.

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Behavior

Working Memory Training for Older Participants: A Control Group Training Regimen and Initial Intellectual Functioning Assessment
Olga Matysiak 1, Wanda Zarzycka 1, Aleksandra Bramorska 1, Aneta Brzezicka 1,2
1Department of Psychology, SWPS University of Social Sciences and Humanities, 2Department of Neurosurgery, Cedars-Sinai Medical Center

A cognitive training intervention in elderly population together with the assessment of the pre training cognitive abilities is presented. We show two versions of training - experimental and active control - and demonstrate their effects on the array of cognitive tests.

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Developmental Biology

Generation of a Human iPSC-Based Blood-Brain Barrier Chip
Srikanth Jagadeesan 1,2,3, Michael J. Workman 4, Anna Herland 5,6, Clive N. Svendsen 4, Gad D. Vatine 1,2,3
1The Department of Physiology and Cell Biology, Faculty of Health Sciences, Ben-Gurion University of the Negev, 2The Regenerative Medicine and Stem Cell (RMSC) Research Center, Ben-Gurion University of the Negev, 3The Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, 4The Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, 5Division of Micro and Nanosystems, KTH Royal Institute of Technology, 6AIMES, Department of Neuroscience, Karolinska Institutet

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

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Biology

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture
Bindu Konda 1, Apoorva Mulay 1, Changfu Yao 1, Stephen Beil 1, Edo Israely 1, Barry R. Stripp 1
1Lung and Regenerative Medicine Institutes, Department of Medicine, Cedars-Sinai Medical Center

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

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Biology

Specimen Collection and Analysis of the Duodenal Microbiome
Benjamin W. Dreskin 1, Kayti Luu 1, Tien S. Dong 1,2,3, Jihane Benhammou 1,2,4, Venu Lagishetty 2,3, John Vu 1, Daniel Sanford 1, Francisco Durazo 2,4, Vatche G. Agopian 4, Jonathan P. Jacobs 1,2,3, Joseph R. Pisegna 1,5, Shehnaz K. Hussain 6
1Division of Gastroenterology, Hepatology and Parenteral Nutrition, Veterans Administration Greater Los Angeles Healthcare System, 2The Vatche and Tamar Manoukian Division of Digestive Diseases, Department of Medicine, David Geffen School of Medicine at University of California, 3UCLA Microbiome Center, David Geffen School of Medicine at University of California, 4Department of Surgery, University of California, Los Angeles, 5Department of Medicine and Human Genetics, David Geffen School of Medicine at University of California, 6Cedars-Sinai Cancer and Department of Medicine, Cedars-Sinai Medical Center

In this manuscript, we discuss a novel method to sample and analyze the duodenal microbiome. This method provides an accurate depiction of microbial diversity and composition in the duodenum and could be useful for further investigation of the duodenal microbiome.

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Biology

Removal of an Internal Translational Start Site from mRNA While Retaining Expression of the Full-Length Protein
Daisuke Shimura 1, Jennifer Hunter 1, Makoto Katsumata 2, Robin M. Shaw 1
1Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, 2Department of Biomedical Sciences, Cedars-Sinai Medical Center

The present protocol describes a single M213L mutation in Gja1 that retains full-length Connexin43 generation but prevents translation of the smaller GJA1-20k internally translated isoform.

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Cancer Research

Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures
Katie B. Grausam *1, Joshua J. Breunig *1,2,3,4,5
1Board of Governor’s Regenerative Medicine Institute, Cedars-Sinai Medical Center, 2Center for Neural Sciences in Medicine, Cedars-Sinai Medical Center, 3Department of Biomedical Sciences, Cedars-Sinai Medical Center, 4Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, 5Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles

Utilizing an immunocompetent, autochthonous tumor model driven by common patient mutations for preclinical testing is critical for immunotherapeutic testing. This protocol describes a method to generate brain tumor mouse models using electroporation-based delivery of plasmid DNA that represent common patient mutations, thus providing an accurate, reproducible, and consistent mouse model.

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Bioengineering

Generation of Induced Pluripotent Stem Cell-Derived iTenocytes via Combined Scleraxis Overexpression and 2D Uniaxial Tension
Victoria Yu 1,2,3, Angela Papalamprou 1,2,3, Dmitriy Sheyn 1,2,3,4,5
1Orthopaedic Stem Cell Research Laboratory, Cedars-Sinai Medical Center, 2Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, 3Department of Biomedical Sciences, Cedars-Sinai Medical Center, 4Department of Orthopedics, Cedars-Sinai Medical Center, 5Department of Surgery, Cedars-Sinai Medical Center

This article describes a procedure to produce iTenocytes by generating iPSC-derived mesenchymal stromal cells with combined overexpression of Scleraxis using a lentiviral vector and uniaxial stretching via a 2D bioreactor.

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Neuroscience

In Vivo Visualization of Spontaneous Activity in Neonatal Mouse Sensory Cortex at a Single-Neuron Resolution
Nao Nakagawa-Tamagawa *1, Takamitsu Egashira *2,3, Madhura S. Rao 2,3,4, Hidenobu Mizuno 2,3
1Department of Physiology, Graduate School of Medical and Dental Sciences, Kagoshima University, 2Laboratory of Multi-Dimensional Imaging, International Research Center for Medical Sciences (IRCMS), Kumamoto University, 3Graduate School of Medical Sciences, Kumamoto University, 4Cedars-Sinai Medical Center

Primary sensory areas in the neocortex exhibit unique spontaneous activities during development. This article describes how to visualize individual neuron activities and primary sensory areas to analyze area-specific synchronous activities in neonatal mice in vivo.

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Biochemistry

Split-Luciferase Reassembly Assay to Measure Endoplasmic Reticulum-Mitochondria Contacts in Live Cells
Connie Chen *1, Katherine A. Rafael *1, Ginam Cho 1, Youngshin Lim 1,2
1Department of Pathology, Cedars-Sinai Medical Center, 2Department of Biomedical Science Education, College of Medicine, Charles R. Drew University of Medicine and Science

We have established a split-luciferase reassembly assay to monitor the endoplasmic reticulum-mitochondria contacts in live cells. Using this assay, we describe a protocol to quantitatively measure the level of these inter-organelle couplings in HEK293T cells, under the condition of chemical treatment.

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