Broad institute of MIT and Harvard 10 articles published in JoVE Neuroscience Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells Sovannarath Pong1,2,3, Paulo Lizano1,2,3,4, Rakesh Karmacharya1,3,4,5 1Center for Genomic Medicine, Massachusetts General Hospital, 2Department of Psychiatry, Beth Israel Deaconess Medical Center, 3Chemical Biology and Therapeutic Science Program, Broad Institute of MIT and Harvard, 4Department of Psychiatry, Harvard Medical School, 5Schizophrenia and Bipolar Disorder Program, McLean Hospital This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model. Neuroscience Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice Ryosuke Fujiki1,2,3,4,9, Joun Y. Lee1,2,10, Julie A. Jurgens1,2,3,7, Mary C. Whitman2,5,6, Elizabeth C. Engle1,2,3,4,5,6,7,8 1 This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons. Biochemistry Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation Emily A. Brown1,2, Steven C. Neier3,4, Claudia Neuhauser5, Adam G. Schrum6,7,8, Stephen E.P. Smith1,2,9 1 Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network. Genetics Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease Allison A. Dilliott1,2, Sali M.K. Farhan3, Mahdi Ghani4, Christine Sato4, Eric Liang5, Ming Zhang4, Adam D. McIntyre1, Henian Cao1, Lemuel Racacho6,7, John F. Robinson1, Michael J. Strong1,8, Mario Masellis9,10, Dennis E. Bulman6,7, Ekaterina Rogaeva4, Anthony Lang10,11, Carmela Tartaglia4,10, Elizabeth Finger12,13, Lorne Zinman9, John Turnbull14, Morris Freedman10,15, Rick Swartz9, Sandra E. Black9,16, Robert A. Hegele1,2 1Robarts Research Institute, Schulich School of Medicine and Dentistry, Western University, 2Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, 3Analytic and Translational Genetics Unit, Center for Genomic Medicine, Harvard Medical School, Massachusetts General Hospital, Stanley Centre for Psychiatric Research, Broad Institute of MIT and Harvard, 4Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, 5 Targeted next-generation sequencing is a time- and cost-efficient approach that is becoming increasingly popular in both disease research and clinical diagnostics. The protocol described here presents the complex workflow required for sequencing and the bioinformatics process used to identify genetic variants that contribute to disease. Neuroscience Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids Max R Salick*1, Michael F Wells*2,3, Kevin Eggan2,3, Ajamete Kaykas1 1Department of Neuroscience, Novartis Institutes for BioMedical Research, 2Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, 3Department of Stem Cell and Regenerative Biology and Harvard Stem Cell Institute, Harvard University This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos. Biology Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules Anaïs Aulas*1,2, Marta M. Fay*1,2, Witold Szaflarski1,2,3, Nancy Kedersha1,2, Paul Anderson1,2, Pavel Ivanov1,2,4 1 Stress Granules (SGs) are nonmembranous cytoplasmic structures that form in cells exposed to a variety of stresses. SGs contain mRNAs, RNA-binding proteins, small ribosomal subunits, translation-related factors, and various cell signaling proteins. This protocol describes a workflow that uses several experimental approaches to detect, characterize, and quantify bona fide SGs. Medicine Unbiased Deep Sequencing of RNA Viruses from Clinical Samples Christian B. Matranga1, Adrianne Gladden-Young1, James Qu1, Sarah Winnicki1, Dolo Nosamiefan1, Joshua Z. Levin1, Pardis C. Sabeti1,2 1Broad Institute of MIT and Harvard, 2Harvard University This protocol describes a rapid and broadly applicable method for unbiased RNA-sequencing of viral samples from human clinical isolates. Immunology and Infection Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria Rachel Daniels1,2, Elizabeth J. Hamilton2, Katelyn Durfee2, Daouda Ndiaye3, Dyann F. Wirth2,5, Daniel L. Hartl1, Sarah K. Volkman2,4 1Department of Organismic and Evolutionary Biology, Harvard University, 2Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, 3Faculty of Medicine and Pharmacy, Cheikh Anta Diop University, 4School of Nursing and Health Sciences, Simmons College, 5Institute of Infectious Diseases, Broad Institute While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis. Immunology and Infection Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins Savannah E. Sanchez1, Daniel A. Cuevas2, Jason E. Rostron1, Tiffany Y. Liang3, Cullen G. Pivaroff1, Matthew R. Haynes1, Jim Nulton4, Ben Felts4, Barbara A. Bailey4, Peter Salamon4, Robert A. Edwards1,5,6, Alex B. Burgin7, Anca M. Segall1, Forest Rohwer1 1Department of Biology, San Diego State University, 2Computational Science Research Center, San Diego State University, 3Bioinformatics and Medical Informatics Research Center, San Diego State University, 4Department of Mathematics and Statistics, San Diego State University, 5Department of Computer Science, San Diego State University, 6Mathematics and Computer Science Division, Argonne National Laboratory, 7SPARC Committee, Broad Institute Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism. Immunology and Infection Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria Tal Danino*1, Arthur Prindle*2, Jeff Hasty2,3,4, Sangeeta Bhatia1,5,6,7,8 1Health Sciences and Technology, Massachusetts Institute of Technology, 2Department of Bioengineering, University of California, San Diego, 3Biocircuits Institute, University of California, San Diego, 4Molecular Biology Section, Division of Biological Science, University of California, San Diego, 5Broad Institute of Harvard and MIT, 6Department of Medicine, Brigham and Women's Hospital, 7Electrical Engineering and Computer Science and David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 8Howard Hughes Medical Institute The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. This video covers tumor cell preparation and implantation, bacteria preparation and injection, whole-animal luminescence imaging, tumor excision, and bacterial colony counting.
