title: Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery.
abstract: Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called "early" EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While "early" EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas "early" EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation. Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.
authors.affiliation: Department of Intensive Care Medicine, University Hospital Aachen; Institute of Biochemistry and Molecular Biology, University Hospital Aachen.
authors.forename: Christoph
authors.initials: C
authors.lastname: Emontzpohl
authors.affiliation: Department of Radiology, German Cancer Research Center.
authors.forename: David
authors.initials: D
authors.lastname: Simons
authors.affiliation: Department of Thoracic and Cardiovascular Surgery, University Hospital Aachen.
authors.forename: Sandra
authors.initials: S
authors.lastname: Kraemer
authors.affiliation: Department of Thoracic and Cardiovascular Surgery, University Hospital Aachen.
authors.forename: Andreas
authors.initials: A
authors.lastname: Goetzenich
authors.affiliation: Department of Intensive Care Medicine, University Hospital Aachen.
authors.forename: Gernot
authors.initials: G
authors.lastname: Marx
authors.affiliation: Department of Vascular Biology, Institute for Stroke and Dementia Research (ISD), Klinikum der Universität München; Deutsches Zentrum für Herz-/Kreislaufkrankheiten (DZHK), Munich Heart Alliance.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Bernhagen
authors.affiliation: Department of Intensive Care Medicine, University Hospital Aachen; christian.stoppe@gmail.com.
authors.forename: Christian
authors.initials: C
authors.lastname: Stoppe
mesh_terms: D000328:Adult; D006348:Cardiac Surgical Procedures; D018929:Cell Culture Techniques / Q000379:methods*; D002465:Cell Movement / Q000502:physiology*; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D018925:Chemokines / Q000378:metabolism; D002633:Chemotaxis / Q000187:drug effects; D016207:Cytokines / Q000378:metabolism; D066026:Endothelial Progenitor Cells / Q000502:physiology; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000494:pharmacology; D008263:Macrophage Migration-Inhibitory Factors / Q000494:pharmacology; D018919:Neovascularization, Physiologic / Q000502:physiology*; D013234:Stem Cells / Q000166:cytology*
chemical_list: D018925:Chemokines; D016207:Cytokines; D008263:Macrophage Migration-Inhibitory Factors; D019746:Intramolecular Oxidoreductases; C515598:MIF protein, human