Stanford University School of Medicine 47 articles published in JoVE Medicine Corneal and Limbal Alkali Injury Induction Using a Punch-Trephine Technique in a Mouse Model Athar Shadmani1, Hala Shakib Dhowre1, Ozlem Ercal1, Xiang Qi Meng2, Albert Y. Wu1 1Department of Ophthalmology, Stanford University School of Medicine, 2McGill University Faculty of Medicine and Health Sciences This protocol describes a method to induce an accurate and reproducible corneal and limbal alkali injury in a mouse model. The protocol is advantageous as it allows for an evenly distributed injury to the highly curved mouse cornea and limbus. Medicine Chronic Ovine Model of Right Ventricular Failure and Functional Tricuspid Regurgitation Boguslaw Gaweda1,2, Artur Iwasieczko1,2, Manikantam Gaddam3,4, Jared D. Bush3, Brian MacDougal3, Tomasz A. Timek1 1Division of Cardiothoracic Surgery, Spectrum Health, 2Clinical Department of Cardiac Surgery, Clinical District Hospital no 2, Faculty of Medicine, University of Rzeszow, 3Research Department, Meijer Heart and Vascular Institute at Spectrum Health, 4Department of Urology, Stanford University School of Medicine Right ventricular failure and functional tricuspid regurgitation are associated with left-sided heart disease and pulmonary hypertension, which contribute significantly to morbidity and mortality in patients. Establishing a chronic ovine model to study right ventricular failure and functional tricuspid regurgitation will help in understanding their mechanisms, progression, and possible treatments. Cancer Research Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells Tommaso Sconocchia1, Johannes Foßelteder1, Thomas Köhnke2, Ravindra Majeti2, Andreas Reinisch1,3 1Division of Hematology, Department of Internal Medicine, Medical University of Graz, 2Division of Hematology, Stanford Cancer Institute, Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 3Department of Blood Group Serology and Transfusion Medicine, Medical University of Graz Novel strategies to faithfully model somatic mutations in hematopoietic stem and progenitor cells (HSPCs) are necessary to better study hematopoietic stem cell biology and hematological malignancies. Here, a protocol to model heterozygous gain-of-function mutations in HSPCs by combining the use of CRISPR/Cas9 and dual rAAV donor transduction is described. Medicine Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Shane Rui Zhao1,2, Gema Mondéjar-Parreño1,2, Dong Li1,2, Mengcheng Shen1,2, Joseph C. Wu1,2,3 1Stanford Cardiovascular Institute, Stanford University School of Medicine, 2Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, 3Department of Radiology, Stanford University School of Medicine Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising in vitro model for drug-induced cardiotoxicity screening and disease modeling. Here, we detail a protocol for measuring the contractility and electrophysiology of hiPSC-CMs. Neuroscience Investigating Drivers of Antireward in Addiction Behavior with Anatomically Specific Single-Cell Gene Expression Methods Sean J. O'Sullivan*1,2,3, Ankita Srivastava*1, Rajanikanth Vadigepalli1, James S. Schwaber1 1Daniel Baugh Institute for Functional Genomics and Computational Biology, Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, 2Sidney Kimmel Medical College, Thomas Jefferson University, 3Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine The combination of laser capture microdissection and microfluidic RT-qPCR provides anatomic and biotechnical specificity in measuring the transcriptome in single neurons and glia. Applying creative methods with a system's biology approach to psychiatric disease may lead to breakthroughs in understanding and treatment such as the neuroinflammation antireward hypothesis in addiction. Biochemistry Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology Yana Lerner*1, Surya Sukumaran*1, Mei-Sze Chua2, Samuel K. So2, Nir Qvit1 1The Azrieli Faculty of Medicine in the Galilee, Bar-Ilan University, 2Asian Liver Center, Department of Surgery, Stanford University School of Medicine Field-effect biosensing (FEB) is a label-free technique for detecting biomolecular interactions. It measures the electric current through the graphene biosensor to which the binding targets are immobilized. The FEB technology was used to evaluate biomolecular interactions between Hsp90 and Cdc37 and a strong interaction between the two proteins was detected. Neuroscience Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice Caitlyn M. Gaffney1, Gabriella Muwanga2, Huaishuang Shen2, Vivianne L. Tawfik2, Andrew J. Shepherd2 1Laboratories of Neuroimmunology, Department of Symptom Research, and the MD Anderson Pain Research Consortium, University of Texas MD Anderson Cancer Center, 2Department of Anesthesiology, Perioperative and Pain Medicine, Stanford University School of Medicine The mechanical conflict-avoidance assay is used as a non-reflexive readout of pain sensitivity in mice which can be used to better understand affective-motivational responses in a variety of mouse pain models. Biology Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization Xianrui Cheng1,2, James E. Ferrell, Jr.1,3 1Department of Chemical and Systems Biology, Stanford University School of Medicine, 2Current Address: Department of Biological Sciences, University of Southern California, 3Department of Biochemistry, Stanford University School of Medicine We describe a method for the preparation and live imaging of undiluted cytoplasmic extracts from Xenopus laevis eggs. Biology Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission György Vámosi1, Sarah Miller2, Molika Sinha2, Maria Kristha Fernandez2, Gabor Mocsár1, Malte Renz2 1Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, 2Gynecologic Oncology Division, Stanford University School of Medicine Förster Resonance Energy Transfer (FRET) between two fluorophore molecules can be used for studying protein interactions in the living cell. Here, a protocol is provided as to how to measure FRET in live cells by detecting sensitized emission of the acceptor and quenching of the donor molecule using confocal laser scanning microscopy. Bioengineering Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells Dilip Thomas1, Hyeonyu Kim1, Nicole Lopez1, Joseph C. Wu1,2,3 1Stanford Cardiovascular Institute, Stanford University School of Medicine, 2Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, 3Department of Radiology, Stanford University School of Medicine Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development. Neuroscience Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions Benedetta Assetta*1, Changyong Tang*1,2, Jing Bian*3, Ryan O'Rourke1, Kevin Connolly1, Thomas Brickler3, Sundari Chetty3,4, Yu-Wen Alvin Huang1,5,6 1Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, 2Department of Neurology, The Third Affiliated Hospital of Sun Yat-Sen University, 3Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, 4Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 5Department of Neurology, Warren Alpert Medical School of Brown University, 6Center for Translational Neuroscience, Robert J. and Nancy D. Carney Institute for Brain Science and Brown Institute for Translational Science, Brown University The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer’s disease. Medicine Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model Michaela Olthoff1, Federico Franchi1, Karen M. Peterson1, Ramasamy Paulmurugan2, Martin Rodriguez-Porcel1 1Department of Cardiovascular Medicine, Mayo Clinic, 2Department of Radiology and Molecular Imaging Program at Stanford (MIPS), Stanford University School of Medicine Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury. Developmental Biology Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart Alyssa Klein1,2,3, Bethel Bayrau1,2,3, Yifei Miao1,2,3,4,5, Mingxia Gu1,2,3,4,5 1Department of Pediatrics, Division of Cardiology, Stanford School of Medicine, 2Vera Moulton Wall Center for Pulmonary Vascular Disease, Stanford School of Medicine, 3Stanford Cardiovascular Institute, Stanford School of Medicine, 4 We present a protocol for the isolation of endocardial and coronary endothelial cells from rat hearts through sequential tissue digestion in a digestion buffer, cell collection from recurrent centrifuge cycles, and cell purification using anti-rat CD31 microbeads. Developmental Biology Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils Soah Lee*1,2,3, Huaxiao Yang*1,2,3, Caressa Chen*1,2,3, Sneha Venkatraman1,2,3, Adrija Darsha1,2,3, Sean M. Wu1,2,3, Joseph C. Wu1,2,3, Timon Seeger1,2,3,4,5 1Stanford Cardiovascular Institute, Stanford University School of Medicine, 2Department of Medicine, Division of Cardiovascular Medicine, Stanford University, 3Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, 4Department of Medicine III, University Hospital Heidelberg, 5German Centre for Cardiovascular Research (DZHK), Partner Site Heidelberg/Mannheim This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes. Neuroscience Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing Lu Zhou1, Qingyun Li1 1Department of Neurobiology, Stanford University School of Medicine We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease. Medicine A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice Jie Zhang1,2, Fang Fang1,3, Liang Li1, Haoliang Huang1, Hannah C. Webber1, Yang Sun1,4, Vinit B. Mahajan1,4, Yang Hu1 1Department of Ophthalmology, Stanford University School of Medicine, 2Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 3Department of Ophthalmology, Second Xiangya Hospital of Central South University, 4Department of Ophthalmology, Veterans Affairs Palo Alto Health Care Here, we present a protocol to induce ocular hypertension and glaucomatous neurodegeneration in mouse eyes by intracameral injection of silicone oil and the procedure for silicone oil removal from the anterior chamber to return elevated intraocular pressure to normal. Developmental Biology Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation Danielle Sambo1, Jingling Li1, Thomas Brickler1, Sundari Chetty1,2 1Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine Generating differentiated cell types from human pluripotent stem cells (hPSCs) holds great therapeutic promise but remains challenging. PSCs often exhibit an inherent inability to differentiate even when stimulated with a proper set of signals. Described here is a simple tool to enhance multilineage differentiation across a variety of PSC lines. Developmental Biology Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells Kyle M. Loh1,2, Amrita Palaria1, Lay Teng Ang1 1Institute for Stem Cell Biology & Regenerative Medicine, Stanford-UC Berkeley Siebel Stem Cell Institute, Stanford University School of Medicine, 2Department of Developmental Biology, Stanford University School of Medicine This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells. Medicine Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model Karim Sallam1,2,3, June-Wha Rhee1,2, Tony Chour1, Jessica D'addabbo1,2,3, Andrew S. Lee1,2,4,5,6, Edward Graves4,5,7, Patricia K. Nguyen1,2,3 1Stanford Cardiovascular Institute, Stanford University School of Medicine, 2Department of Medicine, Division of Cardiology, Stanford University School of Medicine, 3Medical Service, Cardiology Section, Veteran Affairs Palo Alto Health Care System, 4Department of Pathology, Stanford University School of Medicine, 5Department of Radiology, Molecular Imaging Program, Stanford University School of Medicine, 6Peking University Shenzhen Health Science Institute, 7Department of Radiation Oncology, Stanford University School of Medicine Research on treatment strategies for pluripotent stem cell-derived teratomas is important for the clinical translation of stem cell therapy. Here, we describe a protocol to, first, generate stem cell-derived teratomas in mice and, then, to selectively target and treat these tumors in vivo using a small-animal irradiator. Biochemistry How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells Vanessa Chen1, Malte Renz1 1Gynecologic Oncology Division, Stanford University School of Medicine Here, we present a protocol that involves genetically coupled spectrally distinct photoactivatable and fluorescent proteins. These fluorescent protein chimeras permit quantification of the PA-FP fraction that is photoactivated to be fluorescent, i.e., the photoactivation efficiency. The protocol reveals that different modes of photoactivation yield different photoactivation efficiencies. Genetics CRISPR-Mediated Reorganization of Chromatin Loop Structure Stefanie L. Morgan*1,2, Erin Y. Chang*1, Natasha C. Mariano1, Abel Bermudez3, Nicole L. Arruda4, Fanting Wu5, Yunhai Luo1, Gautam Shankar1, Star K. Huynh1, Chiao-Chain Huang5, Sharon J. Pitteri3, Kevin C. Wang1,2,6 1Department of Dermatology, Program in Epithelial Biology, Stanford University School of Medicine, 2Program in Cancer Biology, Stanford University School of Medicine, 3Canary Center for Cancer Early Detection, Department of Radiology, Stanford University School of Medicine, 4Department of Biology, Bridgewater State University, 5System Biosciences, 6Veterans Affairs Healthcare System Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci. Immunology and Infection A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells Effie E Bastounis1, Fabian E Ortega1, Ricardo Serrano2, Julie A Theriot3 1Department of Biochemistry, Stanford University School of Medicine, 2Department of Mechanical and Aerospace Engineering, University of California San Diego, 3Departments of Biochemistry, Microbiology and Immunology and Howard Hughes Medical Institute, Stanford University School of Medicine We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. Developmental Biology Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration Jonathan M. Tsai1,2, Irving L Weissman1,2, Yuval Rinkevich3,4 1Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 2Department of Developmental Biology, Stanford University School of Medicine, 3Comprehensive Pneumology Center, Institute of Lung Biology and Disease, Helmholtz Zentrum München, 4German Center for Lung Research (DZL) Here, we present a new method for partial resection of the left hepatic lobe in neonatal (day 0) mice. This new protocol is suitable for studying acute liver injury and injury response in the neonatal setting. Bioengineering Electrically Conductive Scaffold to Modulate and Deliver Stem Cells Byeongtaek Oh1, Alexa Levinson1, Vivek Lam1, Shang Song1, Paul George1,2 1Department of Neurology and Neurological Sciences, Stanford University School of Medicine, 2Stanford Stroke Center and Stanford University School of Medicine This protocol describes fabrication of a cell culture system to allow seeding of stem cells on a conductive polymer scaffold for in vitro electrical stimulation and subsequent in vivo implantation of the stem cell-seeded scaffold using a minimally invasive technique. Biology High-throughput Screening for Protein-based Inheritance in S. cerevisiae James S. Byers1, Daniel F. Jarosz1,2 1Department of Developmental Biology, Stanford University School of Medicine, 2Department of Chemical and Systems Biology, Stanford University School of Medicine This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae. Medicine A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs Jonathon J. Parker1, Marcela Lizarraga2, Allen Waziri2, Kara M. Foshay2 1Department of Neurosurgery, Stanford University Hospital, Stanford University School of Medicine, 2Inova Neuroscience Institute Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided. Cancer Research A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG) Grant L. Lin1, Michelle Monje2 1Graduate Program in Neuroscience, Department of Neurology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 2Departments of Neurology, Neurosurgery, Pathology and Pediatrics, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine This protocol describes a method for the rapid processing of post-mortem diffuse intrinsic pontine glioma samples for the establishment of patient-derived cell culture models or direct characterization of tumor and microenvironmental cells. Developmental Biology Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model Clement D. Marshall1, Elizabeth R. Zielins1, Elizabeth A. Brett1, Charles P. Blackshear1, Michael S. Hu1, Tripp Leavitt1, Leandra A. Barnes1, H. Peter Lorenz1, Michael T. Longaker1, Derrick C. Wan1 1Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay. Medicine Creation of Abdominal Adhesions in Mice Clement D. Marshall1, Michael S. Hu1, Tripp Leavitt1, Leandra A. Barnes1, Alexander T.M. Cheung1, Samir Malhotra1, H. Peter Lorenz1, Michael T. Longaker1 1Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine Abdominal adhesions that form after surgery are a major cause of pain, infertility, and hospitalization and reoperation for small bowel obstruction. Our surgical procedure for creating abdominal adhesions in mice is a reliable tool to study the mechanisms underlying the formation of adhesions. Medicine A High-content In Vitro Pancreatic Islet β-cell Replication Discovery Platform Zhengshan Zhao1, Yassan Abdolazimi1, Neali A. Armstrong1, Justin P. Annes1 1Department of Medicine, Division of Endocrinology, Stanford University School of Medicine Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet β-cell replication and to develop methods for stimulating β-cell regeneration. Herein a high-content screening method to identify and assess the β-cell replication-promoting activity of small molecules is presented. Chemistry Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation Nir Qvit1, Opher S. Kornfeld1 1Department of Chemical and Systems Biology, Stanford University School of Medicine A simple and general method for the synthesis of cyclic peptides using microwave irradiation is outlined. This procedure enables the synthesis of backbone cyclic peptides with a collection of different conformations while retaining the side chains and the pharmacophoric moieties., and therefore, allows to screen for the bioactive conformation. Developmental Biology Murine Dermal Fibroblast Isolation by FACS Graham G. Walmsley*1,2, Zeshaan N. Maan*1, Michael S. Hu*1,2,3, David A. Atashroo1, Alexander J. Whittam1, Dominik Duscher1, Ruth Tevlin1, Owen Marecic1, H. Peter Lorenz1, Geoffrey C. Gurtner1, Michael T. Longaker1,2 1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 3Department of Surgery, John A. Burns School of Medicine, University of Hawai'i Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture. Immunology and Infection Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice Nicolas Gaudenzio1, Riccardo Sibilano1, Philipp Starkl1, Mindy Tsai1, Stephen J. Galli1,2, Laurent L. Reber1 1Department of Pathology, Stanford University School of Medicine, 2Department of Microbiology & Immunology, Stanford University School of Medicine We describe a method for the generation of in vitro derived mast cells, their engraftment into mast cell-deficient mice, and the analysis of the phenotype, numbers and distribution of engrafted mast cells at different anatomical sites. This protocol can be used to assess the functions of mast cells in vivo. Medicine Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model Russell K. Pachynski1, Alexander Scholz3, Justin Monnier2,3, Eugene C. Butcher3, Brian A. Zabel2 1Division of Oncology, Department of Medicine, Washington University School of Medicine, 2Palo Alto Institute for Research and Education, Veterans Affairs Palo Alto Health Care System, 3Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses. Biology Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle Jared M. Schrader1, Lucy Shapiro1 1Department of Developmental Biology, Stanford University School of Medicine Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells. Neuroscience Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice Devsmita Das1,2, Cristy Phillips3, Bill Lin1, Fatemeh Mojabi1, Mehmet Akif Baktir2, Van Dang1,2, Ravikumar Ponnusamy1, Ahmad Salehi1,2 1VA Palo Alto Health Care System, 2Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, 3Department of Physical Therapy, Arkansas State University We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree. Developmental Biology Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation Arun Sharma*1, Guang Li*1, Kuppusamy Rajarajan*1, Ryoko Hamaguchi1, Paul W. Burridge1, Sean M. Wu1,2 1Stanford Cardiovascular Institute, Stanford University School of Medicine, 2Institute of Stem Cell Biology and Regenerative Medicine, Cardiovascular Medicine Division, Department of Medicine, Child Health Research Institute, Stanford University School of Medicine Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening. Medicine Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche Zachary S. Templeton1, Michael H. Bachmann1, Rajiv V. Alluri1, William J. Maloney2, Christopher H. Contag1, Bonnie L. King1 1Department of Pediatrics, Stanford University School of Medicine, 2Department of Orthopaedic Surgery, Stanford University School of Medicine Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system. Medicine A Mouse Fetal Skin Model of Scarless Wound Repair Graham G. Walmsley*1,2, Michael S. Hu*1,2,3, Wan Xing Hong1,4, Zeshaan N. Maan1, H. Peter Lorenz1, Michael T. Longaker1,2 1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 3Department of Surgery, John A. Burns School of Medicine, University of Hawai'i, 4University of Central Florida College of Medicine During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos. Developmental Biology Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis Elizabeth R. Zielins*1, Ruth Tevlin*1, Michael S. Hu1, Michael T. Chung1, Adrian McArdle1, Kevin J. Paik1, David Atashroo1, Christopher R. Duldulao1, Anna Luan1, Kshemendra Senarath-Yapa1, Graham G. Walmsley1, Taylor Wearda1, Michael T. Longaker1,2, Derrick C. Wan1 1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction. Medicine Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography David A. Atashroo*1, Kevin J. Paik*1, Michael T. Chung1, Adrian McArdle1, Kshemendra Senarath-Yapa1, Elizabeth R. Zielins1, Ruth Tevlin1, Christopher R. Duldulao1, Graham G. Walmsley1, Taylor Wearda1, Owen Marecic1, Michael T. Longaker1,2, Derrick C. Wan1,2 1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford University School of Medicine, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery. Biology Osteoclast Derivation from Mouse Bone Marrow Ruth Tevlin*1, Adrian McArdle*1,2, Charles K.F. Chan2, John Pluvinage2, Graham G. Walmsley1,2, Taylor Wearda1,2, Owen Marecic1,2, Michael S. Hu1, Kevin J. Paik1, Kshemendra Senarath-Yapa1, David A. Atashroo1, Elizabeth R. Zielins1, Derrick C. Wan1, Irving L. Weissman1,2, Michael T. Longaker1,2 1Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro. Biology High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry Subarna Bhattacharya*1, Paul W. Burridge*2, Erin M. Kropp1, Sandra L. Chuppa1, Wai-Meng Kwok3, Joseph C. Wu2, Kenneth R. Boheler4,5, Rebekah L. Gundry1,6 1Department of Biochemistry, Medical College of Wisconsin, 2Stanford Cardiovascular Institute, Stanford University School of Medicine, 3Department of Anesthesiology, Medical College of Wisconsin, 4Stem Cell and Regenerative Medicine Consortium, LKS Faculty of Medicine, Hong Kong University, 5Division of Cardiology, Johns Hopkins University School of Medicine, 6Cardiovascular Research Center, Biotechnology and Bioengineering Center, Medical College of Wisconsin The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population. Neuroscience Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells Pengbo Zhang*1, Ninuo Xia*1, Renee A. Reijo Pera1,2 1Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 2Department of Obstetrics and Gynecology, Stanford University School of Medicine We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson’s disease. Bioengineering A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL) Joseph Dragavon1, Carolyn Sinow2, Alexandra D. Holland1, Abdessalem Rekiki1, Ioanna Theodorou3, Chelsea Samson4, Samantha Blazquez1, Kelly L. Rogers5, Régis Tournebize1,6,7, Spencer L. Shorte1 1Plate-Forme d'Imagerie Dynamique, Imagopole, Institut Pasteur, 2Department of Radiation Oncology, Stanford School of Medicine, 3 Expanding the foundation and applicability of Fluorescence by Unbound Excitation from Luminescence (FUEL) by surveying the relevant principles and demonstrating its compatibility with a multitude of fluorophores and antibody-targeted conditions. Chemistry Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography Samantha M. Desmarais1, Felipe Cava2, Miguel A. de Pedro3, Kerwyn Casey Huang1,4 1Department of Bioengineering, Stanford University, 2Department of Molecular Biology and Laboratory for Molecular Infection Medicine Sweden, Umeå Centre for Microbial Research, Umeå University, 3Campus de Cantoblanco, Universidad Autonoma de Madrid, 4Department of Microbiology and Immunology, Stanford University School of Medicine The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC. Medicine Parabiosis in Mice: A Detailed Protocol Paniz Kamran1,2, Konstantina-Ioanna Sereti1,2, Peng Zhao1,2, Shah R. Ali3, Irving L. Weissman3, Reza Ardehali1,2 1Department of Medicine-Division of Cardiology, University of California, Los Angeles, 2Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, 3Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine Parabiotic joining of two organisms leads to the development of a shared circulatory system. In this protocol, we describe the surgical steps to form a parabiotic connection between a wild-type mouse and a constitutive GFP-expressing mouse.
