Cedars-Sinai Medical Center View Institution's Website 27 articles published in JoVE Bioengineering Generation of Induced Pluripotent Stem Cell-Derived iTenocytes via Combined Scleraxis Overexpression and 2D Uniaxial Tension Victoria Yu1,2,3, Angela Papalamprou1,2,3, Dmitriy Sheyn1,2,3,4,5 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. 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. 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. Shaw1 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. Behavior Working Memory Training for Older Participants: A Control Group Training Regimen and Initial Intellectual Functioning Assessment Olga Matysiak1, Wanda Zarzycka1, Aleksandra Bramorska1, Aneta Brzezicka1,2 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. 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. Stripp1 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. 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 Eyk1 1Advanced 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 Jagadeesan1,2,3, Michael J. Workman4, Anna Herland5,6, Clive N. Svendsen4, Gad D. Vatine1,2,3 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. Behavior Simultaneous Eye Tracking and Single-Neuron Recordings in Human Epilepsy Patients Shuo Wang1, Nand Chandravadia2, Adam N. Mamelak2, Ueli Rutishauser2,3,4 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. Immunology and Infection Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique Tanyalak Parimon1, Norman E. Garrett III1, Peter Chen1,2, Travis J. Antes3 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. Biochemistry Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion Miroslava Stastna1,2,3, Amandine Thomas1, Juliana Germano1, Somayeh Pourpirali1, Jennifer E. Van Eyk1,2, Roberta A. Gottlieb1 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. Immunology and Infection Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow Alberto Yáñez1,2, Helen S. Goodridge1,2 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. Biochemistry Fractionation for Resolution of Soluble and Insoluble Huntingtin Species Joseph Ochaba1,2, Eva L. Morozko2,3, Jacqueline G. O’Rourke4, Leslie M. Thompson1,2,3 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 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 Zhang1 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. Bioengineering Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model Galina Shapiro1, Maxim Bez1, Wafa Tawackoli2,3,4, Zulma Gazit1,2,3,5, Dan Gazit1,2,3,4,5, Gadi Pelled1,2,3,4 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. Immunology and Infection Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons Deisy Contreras1, Vaithilingaraja Arumugaswami1,2,3 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. 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 Saghizadeh1,2, Alexander V. Ljubimov1,2 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. Medicine An Improved Method for Rapid Intubation of the Trachea in Mice Tyler C. Vandivort1,2, Dowon An3, William C. Parks2 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. Bioengineering Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts Doron Cohn Yakubovich1, Wafa Tawackoli2,3,4, Dmitriy Sheyn2,3, Ilan Kallai1, Xiaoyu Da4, Gadi Pelled1,2,3,4, Dan Gazit1,2,3,4, Zulma Gazit1,2,3 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. Immunology and Infection Influenza Virus Propagation in Embryonated Chicken Eggs Rena Brauer1, Peter Chen1 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. Immunology and Infection A Protocol for Analyzing Hepatitis C Virus Replication Songyang Ren*1, Deisy Contreras*1, Vaithilingaraja Arumugaswami1,2 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. 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. Svendsen1 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. 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 Holler1 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. Neuroscience Neonatal Pial Surface Electroporation Rachelle Levy1, Jessica Molina2, 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. Bioengineering Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors Jae Youn Hwang1, Daniel L. Farkas1, Lali K. Medina-Kauwe2,3 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. 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 Crystal4 1Department 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. Liu1 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. Neuroscience Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method Han-peng Xu1,2, Lin Gou3,4, Hong-Wei Dong3 1Department 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.