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

https://doi.org/10.1016/j.preghy.2014.09.003Get rights and content

Highlights

  • 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.

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.

Introduction

Preeclampsia is a serious complication of pregnancy where the anti-angiogenic factors sFlt-1 and soluble endoglin (sEng) are released from the placenta into the maternal circulation. Together these factors inflict widespread endothelial dysfunction, ultimately necessitating delivery of the baby and placenta [1], [2]. They are perhaps the most studied molecules in the field of preeclampsia. Although they were identified almost a decade ago [3], [4], our understanding of the molecular mechanisms regulating their release from the placenta is still limited. Furthermore, identifying potential drugs that can decrease release of these factors from primary placental cells could be a strategy to discover candidate therapeutics for preeclampsia. For these reasons, a robust trophoblast cell system that can be used to undertake functional experiments examining the production and release of sFlt-1 and sEng is important to develop.

Choriocarcinoma derived cells and immortalized trophoblast cells (e.g. BeWos, JAR or JEG3 cells) are commonly used to functionally examine placental biology, including by us [5], [6], [7], [8], [9]. However, while convenient to use, there are concerns arising from the fact they are immortalized and may not reflect true trophoblast biology. Furthermore, we have screened commonly used trophoblast cells and found they secrete low or undetectable levels of sFlt-1 and sEng (see Table 1). As such, they are perhaps particularly unhelpful to functionally interrogate the mechanisms of sFlt-1 and sEng production in placenta.

Primary placental explants are another alternative. They have the advantage of being primary tissue and therefore better reflect trophoblast biology than cell lines. However, they are highly challenging to functionally manipulate. While protocols to transfect siRNAs into primary explants have been published, we have not been able to consistently obtain gene silencing efficiencies following these protocols [10]. Furthermore, we have found sflt-1 and sEng secretion between different placental explants can be extremely variable, making results difficult to interpret.

As such, studying the molecular regulation of sFlt-1 and sEng in the placenta might be best done using primary trophoblast cells. These cells should produce both factors and be amenable to siRNA gene silencing. Surprisingly, there has been very little primary trophoblast based functional interrogation of sflt-1 and sEng production. Most cell studies of sFlt-1 and sEng production have used human umbilical vein endothelial cells (HUVECs), cell lines [6], [11], [12], [13], [14] or placental explants.

Therefore, we set out to characterize a specific protocol for isolating primary term trophoblast cells, optimized for functional interrogation of the anti-angiogenic factors sFlt-1 and sEng. Specifically, we report the levels of sFlt-1 and sEng production and examine the effects of length of time in culture, forskolin and hypoxia. We also set out to confirm genes can be readily silenced using the protocol we describe and to demonstrate the protocol we have developed provides primary trophoblast cells that are readily amenable for functional studies.

Furthermore, while protocols of trophoblast isolation have been previously published [15], [16], [17], [18], [19], there are some important issues that have not been specifically examined. For instance, some investigators have dispensed with further purification from contaminating fibroblasts after percoll gradient separation, raising the question whether the negative selection step does yield significantly more pure populations, or can be dispensed with. Secondly, given the trophoblast cells spontaneously syncytialize, it is not clear whether adding forskolin significantly enhances syncytialization further, or is unnecessary. Therefore, in this technical report, we also set out to specifically resolve these questions. We examined the secretion of hCG, sFlt1 and sEng in cells before and after CD9 negative selection (the purification step), and also assessed the effects of adding forskolin.

Section snippets

Tissue collection

Women presenting to the Mercy Hospital for women gave informed written consent for placental tissue collection. Placentas were obtained from normal term pregnancies at caesarian section. Tissue was collected within 30 min of delivery for isolation of primary trophoblasts.

Primary trophoblast isolation

Approximately 150 g of placental tissue was washed thoroughly in ice-cold phosphate buffered saline (PBS) before both the maternal and fetal surfaces were dissected away and discarded. Placental cotyledons were scraped with a

sFlt-1 and sEng production by commonly utilized trophoblast cell lines

We measured levels of sFlt-1 and sEng in the media obtained from the following trophoblast cell lines: JAR, AC1M188, AC1M32, HTR8, JEG-3 and BeWo cells (Table 1). sEng was produced by JAR, JEG-3 and BeWo cells and only the immortalized HTR8 cells produced measurable levels of sFlt-1. Significantly, none produced both sFlt-1 and sEng. Thus, none of the commonly used trophoblast cell lines appear well suited to examine the molecular biology regulating both sFlt-1 and sEng production.

CD9 negative selection produces a pure trophoblast population

As discussed,

Discussion

In this study we present a specific protocol for primary trophoblast isolation and purification optimized for the functional investigation of sFlt-1 and sEng. We have shown this protocol can be used to isolate a pure population of trophoblast cells of high purity utilizing percoll gradient separation and CD9 negative selection. Interestingly, we have demonstrated differential production of sEng and hCG (but unchanged sFlt1 levels) in cells obtained after CD9 negative selection. At a functional

Ethics Statement

Human Ethics approval was obtained for this study from the Mercy Health Human Research Ethics Committee.

Sources of funding

The National Health and Medical Research Council of Australia provided salary (#1062418 to T.K, #628927 to N.H, #1050765 to S.T.) and Australian Postgraduate Award to F.B.

Disclosure statement

The authors have nothing to disclose.

Competing interest

The authors have declared that no competing interests exist.

Acknowledgements

The authors acknowledge Clinical Research midwives Gabrielle Pell, Debra Jinks, Rachel Murdoch and Genevieve Christophers and the Obstetrics midwifery staff and patients at the Mercy Hospital for Women (Heidelberg) for their provision of placental tissue.

References (21)

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