Tu'uhevaha J Kaitu'u-Lino1, Stephen Tong2, Sally Beard2, Roxanne Hastie2, Laura Tuohey2, Fiona Brownfoot2, Kenji Onda2, Natalie J Hannan2. 1. 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. 2. Translational Obstetrics Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne, Heidelberg, Victoria, Australia.
Abstract
OBJECTIVES: 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. STUDY DESIGN: 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. CONCLUSION: 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.
OBJECTIVES: 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. STUDY DESIGN: 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. CONCLUSION: 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: Rebecca L Wilson; Maxime François; Tanja Jankovic-Karasoulos; Dale McAninch; Dylan McCullough; Wayne R Leifert; Claire T Roberts; Tina Bianco-Miotto Journal: Epigenetics Date: 2019-04-30 Impact factor: 4.528
Authors: Feng Li; Masao Kakoki; Marcela Smid; Kim Boggess; Jennifer Wilder; Sylvia Hiller; Carol Bounajim; Scott E Parnell; Kathleen K Sulik; Oliver Smithies; Nobuyo Maeda-Smithies Journal: Hypertension Date: 2018-04-02 Impact factor: 10.190