Characterization of protocols for primary trophoblast purification, optimized for functional investigation of sFlt-1 and soluble endoglin

• Choriocarcinoma cell lines produce low/undetectable sFlt-1 or soluble endoglin.
• Isolated term trophoblast cells were highly pure and secrete both sFlt1 and sEng.
• Forskolin cannot enhance syncytialization, sFlt-1 or soluble endoglin secretion.
• siRNA induces high silencing efficiencies in term trophoblast.
• Term trophoblast are a good model to study the biology of sFlt-1 and sEng.

ObjectivesSoluble 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 designPrimary 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.ResultsCD9 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.ConclusionIn 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.