Cedars-Sinai Medical Center

25 articles published in JoVE

• Biology

  
  Removal of an Internal Translational Start Site from mRNA While Retaining Expression of the Full-Length Protein Daisuke Shimura¹, Jennifer Hunter¹, Makoto Katsumata², Robin M. Shaw^{1 1}Nora 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.

• Behavior

  
  Working Memory Training for Older Participants: A Control Group Training Regimen and Initial Intellectual Functioning Assessment Olga Matysiak¹, Wanda Zarzycka¹, Aleksandra Bramorska¹, Aneta Brzezicka^{1,2 1}Department 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.

• Biology

  
  Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture Bindu Konda¹, Apoorva Mulay¹, Changfu Yao¹, Stephen Beil¹, Edo Israely¹, Barry R. Stripp^{1 1}Lung 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.

• Biochemistry

  
  A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation Qin Fu¹, Casey W. Johnson¹, Bhagya K. Wijayawardena², Michael P. Kowalski², Miranda Kheradmand², Jennifer E. Van Eyk^{1 1}Advanced Clinical Biosystems Institute, Smidt Heart institute, Cedars Sinai Medical Center, ²Beckman Coulter Life Sciences Here, we present an automated plasma protein digestion method for mass spectrometry-based quantitative proteomic analysis. In this protocol, the liquid transferring and incubation steps for protein denaturation, reduction, alkylation, and trypsin digestion reactions are streamlined and automated. It takes approximately five hours to prepare a 96-well plate with desired precision.

• Developmental Biology

  
  Generation of a Human iPSC-Based Blood-Brain Barrier Chip Srikanth Jagadeesan^1,2,3, Michael J. Workman⁴, Anna Herland^5,6, Clive N. Svendsen⁴, Gad D. Vatine^{1,2,3 1}The 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.

• Behavior

  
  Simultaneous Eye Tracking and Single-Neuron Recordings in Human Epilepsy Patients Shuo Wang¹, Nand Chandravadia², Adam N. Mamelak², Ueli Rutishauser^{2,3,4 1}Department 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.

• Immunology and Infection

  
  Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique Tanyalak Parimon¹, Norman E. Garrett III¹, Peter Chen^1,2, Travis J. Antes^{3 1}Department 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.

• Biochemistry

  
  Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion Miroslava Stastna^1,2,3, Amandine Thomas¹, Juliana Germano¹, Somayeh Pourpirali¹, Jennifer E. Van Eyk^1,2, Roberta A. Gottlieb^{1 1}The 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.

• 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 1}Board 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.

• Biochemistry

  
  Fractionation for Resolution of Soluble and Insoluble Huntingtin Species Joseph Ochaba^1,2, Eva L. Morozko^2,3, Jacqueline G. O’Rourke⁴, Leslie M. Thompson^{1,2,3 1}Department 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

• Medicine

  
  Gene Regulation and Targeted Therapy in Gastric Cancer Peritoneal Metastasis: Radiological Findings from Dual Energy CT and PET/CT Bowen Shi¹, Huimin Lin¹, Miao Zhang², Wei Lu³, Ying Qu⁴, Huan Zhang^{1 1}Department 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.

• Bioengineering

  
  Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model Galina Shapiro¹, Maxim Bez¹, Wafa Tawackoli^2,3,4, Zulma Gazit^1,2,3,5, Dan Gazit^1,2,3,4,5, Gadi Pelled^{1,2,3,4 1}Skeletal 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.

• Immunology and Infection

  
  Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons Deisy Contreras¹, Vaithilingaraja Arumugaswami^{1,2,3 1}Board 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.

• 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. Kramerov¹, Mehrnoosh Saghizadeh^1,2, Alexander V. Ljubimov^{1,2 1}Eye 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.

• Medicine

  
  An Improved Method for Rapid Intubation of the Trachea in Mice Tyler C. Vandivort^1,2, Dowon An³, William C. Parks^{2 1}Department 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.

• Bioengineering

  
  Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts Doron Cohn Yakubovich¹, Wafa Tawackoli^2,3,4, Dmitriy Sheyn^2,3, Ilan Kallai¹, Xiaoyu Da⁴, Gadi Pelled^1,2,3,4, Dan Gazit^1,2,3,4, Zulma Gazit^{1,2,3 1}Skeletal 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.

• Immunology and Infection

  
  Influenza Virus Propagation in Embryonated Chicken Eggs Rena Brauer¹, Peter Chen^{1 1}Department 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.

• Immunology and Infection

  
  A Protocol for Analyzing Hepatitis C Virus Replication Songyang Ren*¹, Deisy Contreras*¹, Vaithilingaraja Arumugaswami^{1,2 1}Liver 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.

• 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. Shelley¹, Geneviève Gowing¹, Clive N. Svendsen^{1 1}Regenerative 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.

• Chemistry

  
  Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine? Julia Y. Ljubimova¹, Hui Ding¹, Jose Portilla-Arias¹, Rameshwar Patil¹, Pallavi R. Gangalum¹, Alexandra Chesnokova¹, Satoshi Inoue¹, Arthur Rekechenetskiy¹, Tala Nassoura¹, Keith L. Black¹, Eggehard Holler^{1 1}Nanomedicine 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.

• Neuroscience

  
  Neonatal Pial Surface Electroporation Rachelle Levy¹, Jessica Molina², Moise Danielpour*¹, Joshua J. Breunig*^{2 1}Department 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.

• Bioengineering

  
  Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors Jae Youn Hwang¹, Daniel L. Farkas¹, Lali K. Medina-Kauwe^{2,3 1}Department 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.

• Immunology and Infection

  
  A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures Margie A. Morgan¹, Elizabeth Marlowe², Susan Novak-Weekly², J.M. Miller², T.M. Painter³, Hossein Salimnia³, Benjamin Crystal^{4 1}Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Cente, ²Pasadena, CA, Southern California Permanente Medical Group, ³Detroit, Detroit Medical Center, ⁴Woburn, MA, AdvanDx A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

• Immunology and Infection

  
  Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA) Ching Wen Tseng¹, Marisel Sanchez-Martinez¹, Andrea Arruda¹, George Y. Liu^{1 1}Department 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.

• Neuroscience

  
  Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method Han-peng Xu^1,2, Lin Gou^3,4, Hong-Wei Dong^{3 1}Department of Neurosurgery, Cedars Sinai Medical Center, UCLA, ²Basic Medicine School, Fourth Military Medical University, ³Department of Neurology, David Geffen School of Medicine, UCLA, ⁴Aerospace Medicine School, Fourth Military Medical Univeristy In this protocol, we described a new method to study the influence of glial cell heterogeneity on axon growth with an in vitro co-culture system. Rat cortical glial cells were cultured to confluence and cocultured with highly purified rat dorsal root ganglia neurons. Different glial cell influence on neurons adhesion and axon growth was compared directly in the same culture. This method provides a new way to directly study the glial cell heterogeneity influence on neuron adhesion and axon growth.