title: Isolation of syncytiotrophoblast microvesicles and exosomes and their characterisation by multicolour flow cytometry and fluorescence Nanoparticle Tracking Analysis.
abstract: The human placenta releases multiple types and sizes of syncytiotrophoblast (STB) extracellular vesicles (EV) into the maternal circulation that exhibit diverse biological activities. The placental perfusion technique enables isolation of these STBEV, but conventional flow cytometry can only be used to phenotype EV down to ∼300 nm in size. Fluorescence Nanoparticle Tracking Analysis (fl-NTA) has the potential to phenotype EV down to ∼50 nm, thereby improving current characterisation techniques. The aims of this study were to prepare microvesicle and exosome enriched fractions from human placental perfusate (n=8) and improve fl-NTA STBEV detection. Differential centrifugation and filtration effectively removed contaminating red blood cells from fresh placental perfusates and pelleted a STB microvesicle (STBMV) fraction (10,000×g pellet - 10KP; NTA modal size 395±12 nm), enriched for the STB marker placental alkaline phosphatase (PLAP) and a STB exosome (STBEX) fraction (150,000×g pellet - 150KP; NTA modal size 147±6 nm), enriched for PLAP and exosome markers Alix and CD63. The PLAP positivity of 'standard' 10KP and 150KP pools (four samples/pool), determined by immunobead depletion, was used to optimise fl-NTA camera settings. Individual 10KP and 150KP samples (n=8) were 54.5±5.7% (range 17.8-66.9%) and 30.6±5.6% (range 3.3-51.7%) PLAP positive, respectively. We have developed a reliable method for enriching STBMV and STBEX from placental perfusate. We also standardised fl-NTA settings and improved measurement of PLAP positive EV in STBMV. However, fl-NTA is not as sensitive as anti-PLAP Dynabead capture for STBEX detection, possibly due to STBEX having lower surface expression of PLAP. These important developments will facilitate more detailed studies of the role of STBMV and STBEX in normal and pathological pregnancies.
authors.affiliation: Nuffield Department of Obstetrics & Gynaecology, University of Oxford, Womens Centre Level 3, John Radcliffe Hospital, Oxford OX3 9DU, UK. Electronic address: rebecca.dragovic@obs-gyn.ox.ac.uk.
authors.forename: R A
authors.initials: RA
authors.lastname: Dragovic
authors.affiliation: Nuffield Department of Obstetrics & Gynaecology, University of Oxford, Womens Centre Level 3, John Radcliffe Hospital, Oxford OX3 9DU, UK. Electronic address: gavin.collett@obs-gyn.ox.ac.uk.
authors.forename: G P
authors.initials: GP
authors.lastname: Collett
authors.affiliation: Malvern Instruments, London Road, Minton Park, Amesbury SP4 7RT, UK. Electronic address: Patrick.Hole@malvern.com.
authors.forename: P
authors.initials: P
authors.lastname: Hole
authors.affiliation: Nuffield Department of Clinical Laboratory Science, University of Oxford, John Radcliffe Hospital, OX3 9DU Oxford, UK. Electronic address: david.ferguson@ndcls.ox.ac.uk.
authors.forename: D J P
authors.initials: DJ
authors.lastname: Ferguson
authors.affiliation: Nuffield Department of Obstetrics & Gynaecology, University of Oxford, Womens Centre Level 3, John Radcliffe Hospital, Oxford OX3 9DU, UK. Electronic address: christopher.redman@obs-gyn.ox.ac.uk.
authors.forename: C W
authors.initials: CW
authors.lastname: Redman
authors.affiliation: Nuffield Department of Obstetrics & Gynaecology, University of Oxford, Womens Centre Level 3, John Radcliffe Hospital, Oxford OX3 9DU, UK. Electronic address: ian.sargent@obs-gyn.ox.ac.uk.
authors.forename: I L
authors.initials: IL
authors.lastname: Sargent
authors.affiliation: Nuffield Department of Obstetrics & Gynaecology, University of Oxford, Womens Centre Level 3, John Radcliffe Hospital, Oxford OX3 9DU, UK. Electronic address: dionne.tannetta@obs-gyn.ox.ac.uk.
authors.forename: D S
authors.initials: DS
authors.lastname: Tannetta
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002498:Centrifugation; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism; D055354:Exosomes / Q000737:chemistry*; D005260:Female; D005374:Filtration; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005453:Fluorescence; D015870:Gene Expression; D006801:Humans; D046529:Microscopy, Electron, Transmission; D053758:Nanoparticles / Q000737:chemistry / Q000648:ultrastructure; D010477:Perfusion; D011247:Pregnancy; D060149:Tetraspanin 30 / Q000235:genetics / Q000378:metabolism; D014327:Trophoblasts / Q000737:chemistry* / Q000378:metabolism
chemical_list: D015415:Biomarkers; C554586:CD63 protein, human; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D056827:Endosomal Sorting Complexes Required for Transport; C117020:PDCD6IP protein, human; D060149:Tetraspanin 30; D000469:Alkaline Phosphatase
keywords: Exosomes; Extracellular vesicles; Flow cytometry; Fluorescence NTA; Microvesicles; Placenta perfusion
references.citation: Placenta. 2014 Feb;35 Suppl:S74-80
references.pmid: 24239157
references.citation: Biochim Biophys Acta. 1994 Jul 13;1193(1):85-92
references.pmid: 8038198
references.citation: Proc Natl Acad Sci U S A. 2013 Jul 16;110(29):12048-53
references.pmid: 23818581
references.citation: Biol Reprod. 2013 Dec 26;89(6):151
references.pmid: 24227753
references.citation: Contrib Gynecol Obstet. 1985;13:40-7
references.pmid: 3995981
references.citation: J Extracell Vesicles. 2014 Dec 22;3:26913
references.pmid: 25536934
references.citation: PLoS One. 2011;6(5):e20245
references.pmid: 21633494
references.citation: Placenta. 2012 Dec;33(12):982-90
references.pmid: 23107341
references.citation: J Reprod Immunol. 2012 Jun;94(2):131-41
references.pmid: 22542910
references.citation: J Extracell Vesicles. 2013 May 27;2:
references.pmid: 24009894
references.citation: PLoS One. 2013;8(2):e56754
references.pmid: 23437230
references.citation: Cell Mol Immunol. 2014 Nov;11(6):548-63
references.pmid: 24954226
references.citation: Placenta. 2008 Aug;29(8):680-90
references.pmid: 18653232
references.citation: J Extracell Vesicles. 2013 Feb 15;2:
references.pmid: 24009893
references.citation: J Cell Biol. 2013 Feb 18;200(4):373-83
references.pmid: 23420871
references.citation: J Reprod Immunol. 2005 Dec;68(1-2):15-26
references.pmid: 16233919
references.citation: Placenta. 2010 Feb;31(2):106-12
references.pmid: 20005571
references.citation: Nanomedicine. 2011 Dec;7(6):780-8
references.pmid: 21601655