Trophoblast lineage specification, differentiation and their regulation by oxygen tension

Abstract

Development of the early embryo takes place under low oxygen tension. Under such conditions, the embryo implants and the trophectoderm, the outer layer of blastocyst, proliferate, forming the cytotrophoblastic shell, the early placenta. The cytotrophoblasts (CTBs) are the so-called epithelial 'stem cells' of the placenta, which, depending on the signals they receive, can differentiate into either extravillous trophoblast (EVT) or syncytiotrophoblast (STB). EVTs anchor the placenta to the uterine wall and remodel maternal spiral arterioles in order to provide ample blood supply to the growing fetus. STBs arise through CTB fusion, secrete hormones necessary for pregnancy maintenance and form a barrier across which nutrient and gas exchange can take place. The bulk of EVT differentiation occurs during the first trimester, before the onset of maternal arterial blood flow into the intervillous space of the placenta, and thus under low oxygen tension. These conditions affect numerous signaling pathways, including those acting through hypoxia-inducible factor, the nutrient sensor mTOR and the endoplasmic reticulum stress-induced unfolded protein response pathway. These pathways are known to be involved in placental development and disease, and specific components have even been identified as directly involved in lineage-specific trophoblast differentiation. Nevertheless, much controversy surrounds the role of hypoxia in trophoblast differentiation, particularly with EVT. This review summarizes previous studies on this topic, with the intent of integrating these results and synthesizing conclusions that resolve some of the controversy, but then also pointing to remaining areas, which require further investigation.

Keywords: cytotrophoblast; extravillous trophoblast; hypoxia; hypoxia-inducible factor; placenta; syncytiotrophoblast.

Figure 1

Early human embryo (~gestational age of 30 days), highlighting the proliferative cytotrophoblastic shell. E=embryo, C=cytotrophoblastic shell. Top panels are H&E stained, while bottom panels show Ki67 immunostaining. Magnification of 20× (left panels) or 50× (right panels).

Figure 2

Schematic representation of the change of oxygen tension in human placenta from early to late first trimester pregnancy. During this period, two types of extravillous trophoblasts with migratory and invasive capacity are found, including endovascular and interstitial trophoblasts. Endovascular trophoblasts invade into spiral arteries to form EVT plugs that prevent maternal blood flow into the intervillous space, thereby creating a low oxygen environment. Toward the end of the first trimester, these plugs gradually dissipate and endovascular trophoblasts begin migrating proximally along the vessel to cause spiral artery dilation, an important process for establishing the maternal-placental blood circulation, and therefore rapidly increasing the oxygen levels in support of fetal growth.

Figure 3

Three main pathways mediating effects downstream of low oxygen sensing in placenta and trophoblast.

Figure 4

Trophoblast lineage specification is not affected in low oxygen tension. Human embryonic stem cell line (WA09/H9), adapted to StemPro (day −2) was switched to minimal media for two days (day 0), then treated with 10 ng/ml BMP4 over a 4 day period (days 1–4, respectively) in either normoxia (20% oxygen) or hypoxia (5% oxygen) (for more experimental details, see Horii et al. 2016). Cells were collected and RNA isolated for quantitative PCR for the pluripotency marker, OCT4, as well as cytotrophoblast stem cell markers p63 and CDX2. Values are normalized to 18S rRNA, and shown as fold change relative to undifferentiated StemPro-adapted cells (day −2).

Figure 5

Schematic representation of the relationship between oxygen level and human trophoblast cell fate decisions during early placental development. NOTCH1 expression identifies an EVT progenitor cell population in early first trimester placenta. Under low oxygen tension, the hypoxia-inducible factor (HIF) pathway is triggered and initiates EVT lineage commitment and survival of EVT progenitor cells; as oxygen tensions rise later in the first trimester, EVT progenitors mature and give rise to invasive EVT which further invade and remodel maternal spiral arterioles. By contrast, low oxygen tension prevents STB differentiation by downregulating the expression of GCM1, a transcription factor required for cytotrophoblastic cell fusion.