title: Genome-Edited Human Pluripotent Stem Cell-Derived Macrophages as a Model of Reverse Cholesterol Transport--Brief Report.
abstract: OBJECTIVE
  To create isogenic human pluripotent stem cell-derived macrophages with and without ABCA1 expression as a model for reverse cholesterol transport.
  
  
  RESULTS
  The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) genome-editing system was used to introduce frameshift mutations into the coding sequence of ATP-binding cassette, subfamily A, member 1. Individual human pluripotent stem cell clones with deleterious mutations were identified, expanded, and differentiated into mature macrophages with a cytokine-based, feeder-free differentiation protocol. Wild-type cells demonstrated effective cholesterol efflux to apoAI acceptor, whereas ABCA1(-/-) cells displayed significantly reduced efflux ability and increased expression of proinflammatory cytokines.
  
  
  CONCLUSIONS
  Human pluripotent stem cell-derived macrophages capable of reverse cholesterol transport can be rapidly generated and genetically edited with CRISPR/Cas9. Introduction of homozygous frameshift mutations results in loss of ABCA1 expression in differentiated macrophages and subsequent reduction of cholesterol efflux capability. This facile genome-editing approach and differentiation protocol pave the way for future studies of the molecular determinants of reverse cholesterol transport and other macrophage properties.
authors.affiliation: From the Department of Stem Cell and Regenerative Biology, Harvard University, and Harvard Stem Cell Institute, Cambridge, MA (R.M.G., T.B.M., C.A.C., K.M.); and Division of Cardiovascular Medicine, Brigham and Women's Hospital, Boston, MA (R.M.G., K.M.).
authors.forename: Rajat M
authors.initials: RM
authors.lastname: Gupta
authors.affiliation: From the Department of Stem Cell and Regenerative Biology, Harvard University, and Harvard Stem Cell Institute, Cambridge, MA (R.M.G., T.B.M., C.A.C., K.M.); and Division of Cardiovascular Medicine, Brigham and Women's Hospital, Boston, MA (R.M.G., K.M.).
authors.forename: Torsten B
authors.initials: TB
authors.lastname: Meissner
authors.forename: Chad A
authors.initials: CA
authors.lastname: Cowan
authors.forename: Kiran
authors.initials: K
authors.lastname: Musunuru
mesh_terms: D064286:ATP Binding Cassette Transporter 1 / Q000235:genetics / Q000378:metabolism*; D016632:Apolipoprotein A-I / Q000378:metabolism; D001692:Biological Transport; D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000378:metabolism; D064113:CRISPR-Cas Systems; D002454:Cell Differentiation; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism; D016368:Frameshift Mutation; D005786:Gene Expression Regulation; D015894:Genome, Human; D005838:Genotype; D006801:Humans; D054643:INDEL Mutation; D018836:Inflammation Mediators / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism*; D010641:Phenotype; D039904:Pluripotent Stem Cells / Q000378:metabolism*
chemical_list: C577836:ABCA1 protein, human; C505693:APOA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D016632:Apolipoprotein A-I; D064130:CRISPR-Associated Proteins; D016207:Cytokines; D018836:Inflammation Mediators; D002784:Cholesterol
keywords: cell differentiation; cholesterol; genetics; macrophages; stem cells