Sanford-Burnham Medical Research Institute View Institution's Website 10 articles published in JoVE Developmental Biology Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids Viviana Meraviglia*1, Alessandra Zanon*1, Alexandros A. Lavdas1, Christine Schwienbacher1, Rosamaria Silipigni2, Marina Di Segni2, Huei-Sheng Vincent Chen3, Peter P. Pramstaller1, Andrew A. Hicks1, Alessandra Rossini1 1Center for Biomedicine, European Academy Bozen/Bolzano (EURAC), 2 Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids. Biology Generation of Myospheres From hESCs by Epigenetic Reprogramming Sonia Albini1, Pier Lorenzo Puri1,2 1Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, 2IRCCS Fondazione Santa Lucia Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles. Medicine Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry Wenhong Zhu1, Richard L. Gallo2,3, Chun-Ming Huang2,3,4 1Sanford-Burnham Medical Research Institute, 2Division of Dermatology, University of California, San Diego, 3VA San Diego Healthcare Center, 4Moores Cancer Center, University of California, San Diego Considering saliva sampling for future clinical application, a lollipop-like ultrafiltration (LLUF) probe was fabricated to fit in the human oral cavity. Direct analysis of undigested saliva by NanoLC-LTQ mass spectrometry demonstrated the ability of LLUF probes to remove large proteins and high abundance proteins, and make low-abundant peptides more detectable. Medicine Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice Julio E. Ayala1, Deanna P. Bracy2,3, Carlo Malabanan3, Freyja D. James2,3, Tasneem Ansari3, Patrick T. Fueger4, Owen P. McGuinness2,3, David H. Wasserman2,3 1Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute at Lake Nona, 2Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 3Vanderbilt Mouse Metabolic Phenotyping Center, Vanderbilt University School of Medicine, 4Department of Pediatrics and Cellular and Integrative Physiology, Indiana University School of Medicine The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress. Biology Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction Xuejun H. Parsons1,2, Yang D. Teng3,4, James F. Parsons1,2, Evan Y. Snyder1,2,5, David B. Smotrich1,2,6, Dennis A. Moore1,2 1San Diego Regenerative Medicine Institute, 2Xcelthera, 3Department of Neurosurgery, Harvard Medical School, 4Division of SCI Research, VA Boston Healthcare System, 5Program in Stem Cell & Regenerative Biology, Sanford-Burnham Medical Research Institute, 6La Jolla IVF We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair. Neuroscience Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction Xuejun H. Parsons1,2, Yang D. Teng3,4, James F. Parsons1,2, Evan Y. Snyder1,2,5, David B. Smotrich1,2,6, Dennis A. Moore1,2 1San Diego Regenerative Medicine Institute, 2Xcelthera, 3Department of Neurosurgery, Harvard Medical School, 4Division of SCI Research, VA Boston Healthcare System, 5Program in Stem Cell & Regenerative Biology, Sanford-Burnham Medical Research Institute, 6La Jolla IVF We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair. Biology Fluorescent Labeling of Drosophila Heart Structures Nakissa N. Alayari1,2, Georg Vogler2, Ouarda Taghli-Lamallem2, Karen Ocorr2, Rolf Bodmer2, Anthony Cammarato1,2 1Biology Department, San Diego State University, 2Development and Aging Program, NASCR Center, The Sanford Burnham Institute for Medical Research Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism. Biology Visualizing the Beating Heart in Drosophila Georg Vogler1, Karen Ocorr1 1Development and Aging Program, The Sanford Burnham Institute for Medical Research Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology. Biology Semi-automated Optical Heartbeat Analysis of Small Hearts Karen Ocorr1, Martin Fink2, Anthony Cammarato1,3, Sanford I. Bernstein3, Rolf Bodmer1 1Development and Aging Program, The Sanford Burnham Institute for Medical Research, 2Cardiac Electrophysiology Group, Dept. of Physiology, Anatomy and Genetics, The Sanford Burnham Institute for Medical Research, 3Biology Department and Heart Institute, San Diego State University We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts. Biology Detection of Protein Ubiquitination Yeun Su Choo1, Zhuohua Zhang1 1Signal Transduction Program, The Sanford Burnham Institute for Medical Research Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.