title: Characterization of protocols for primary trophoblast purification, optimized for functional investigation of sFlt-1 and soluble endoglin.
abstract: OBJECTIVE
  Soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng) are the most studied molecules in preeclampsia. However, most trophoblast cell lines do not secrete both these factors. Thus, we set out to characterize protocols to functionally investigate sFlt-1 and sEng from primary trophoblast.
  
  
  METHODS
  Primary trophoblasts were isolated from term placenta by percoll gradient, then negative selection using a CD9 antibody. Purity was assessed by cytokeratin 7 immunostaining. We first examined the effects of CD9 negative selection on sFlt-1, sEng and hCG secretion and the ability of forskolin to enhance syncytialization. We then examined the effects of hypoxia on sFlt-1 production and assessed gene knockdown using siRNA.
  
  
  RESULTS
  CD9 negative selection produced a pure population of primary trophoblasts. Secretion of sEng was 5-fold lower when CD9-positive cells were removed, sFlt1 was unchanged, and hCG was significantly increased. hCG analysis of the purified population indicated spontaneous syncytialization, which was not enhanced by forskolin. Forskolin similarly did not alter sFlt-1 secretion. Hypoxia significantly increased sFlt-1 secretion as expected. Importantly, high gene silencing efficiencies were readily achieved.
  
  
  CONCLUSIONS
  In conclusion, we present a protocol that yields primary trophoblasts of high purity that produce abundant sFlt-1 and low but detectable levels of sEng. Furthermore, these cells are readily amenable to gene silencing by siRNAs and hence suitable for functional studies.
authors.affiliation: Translational Obstetrics Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria, Australia. Electronic address: t.klino@unimelb.edu.au.
authors.forename: Tu'uhevaha J
authors.initials: TJ
authors.lastname: Kaitu'u-Lino
authors.affiliation: Translational Obstetrics Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria, Australia.
authors.forename: Stephen
authors.initials: S
authors.lastname: Tong
authors.affiliation: Translational Obstetrics Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria, Australia.
authors.forename: Sally
authors.initials: S
authors.lastname: Beard
authors.affiliation: Translational Obstetrics Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria, Australia.
authors.forename: Roxanne
authors.initials: R
authors.lastname: Hastie
authors.affiliation: Translational Obstetrics Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria, Australia.
authors.forename: Laura
authors.initials: L
authors.lastname: Tuohey
authors.affiliation: Translational Obstetrics Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria, Australia.
authors.forename: Fiona
authors.initials: F
authors.lastname: Brownfoot
authors.affiliation: Translational Obstetrics Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria, Australia.
authors.forename: Kenji
authors.initials: K
authors.lastname: Onda
authors.affiliation: Translational Obstetrics Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria, Australia.
authors.forename: Natalie J
authors.initials: NJ
authors.lastname: Hannan
keywords: Placenta; Soluble endoglin; Trophoblast; sFlt-1