Trophoblast lineage specification, differentiation and their regulation by oxygen tension.

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.

Hypoxia Directs Human Extravillous Trophoblast Differentiation in a Hypoxia-Inducible Factor-Dependent Manner.

Villous cytotrophoblasts are epithelial stem cells of the early human placenta, able to differentiate either into syncytiotrophoblasts in floating chorionic villi or extravillous trophoblasts (EVTs) at the anchoring villi. The signaling pathways regulating differentiation into these two lineages are incompletely understood. The bulk of placental growth and development in the first trimester occurs under low oxygen tension. One major mechanism by which oxygen regulates cellular function is through the hypoxia-inducible factor (HIF), a transcription factor complex stabilized under low oxygen tension to mediate cellular responses, including cell fate decisions. HIF is known to play a role in trophoblast differentiation in rodents; however, its role in human trophoblast differentiation is poorly understood. Using RNA profiling of sorted populations of primary first-trimester trophoblasts, we evaluated the first stage of EVT differentiation, the transition from epidermal growth factor receptor+ villous cytotrophoblasts into human leukocyte antigen-G+ proximal column EVT (pcEVT) and identified hypoxia as a major pcEVT-associated pathway. Using primary cytotrophoblasts, we determined that culture in low oxygen directs differentiation preferentially toward human leukocyte antigen-G+ pcEVT, and that an intact HIF complex is required for this process. Finally, using global RNA profiling, we identified integrin-linked kinase and associated cytoskeletal remodeling and adhesion to be among HIF-dependent pcEVT-associated signaling pathways. Taken together, we propose that oxygen regulates EVT differentiation through HIF-dependent modulation of various cell adhesion and morphology-related pathways.

Human pluripotent stem cells as a model of trophoblast differentiation in both normal development and disease.

Trophoblast is the primary epithelial cell type in the placenta, a transient organ required for proper fetal growth and development. Different trophoblast subtypes are responsible for gas/nutrient exchange (syncytiotrophoblasts, STBs) and invasion and maternal vascular remodeling (extravillous trophoblasts, EVTs). Studies of early human placental development are severely hampered by the lack of a representative trophoblast stem cell (TSC) model with the capacity for self-renewal and the ability to differentiate into both STBs and EVTs. Primary cytotrophoblasts (CTBs) isolated from early-gestation (6-8 wk) human placentas are bipotential, a phenotype that is lost with increasing gestational age. We have identified a CDX2(+)/p63(+) CTB subpopulation in the early postimplantation human placenta that is significantly reduced later in gestation. We describe a reproducible protocol, using defined medium containing bone morphogenetic protein 4 by which human pluripotent stem cells (hPSCs) can be differentiated into CDX2(+)/p63(+) CTB stem-like cells. These cells can be replated and further differentiated into STB- and EVT-like cells, based on marker expression, hormone secretion, and invasive ability. As in primary CTBs, differentiation of hPSC-derived CTBs in low oxygen leads to reduced human chorionic gonadotropin secretion and STB-associated gene expression, instead promoting differentiation into HLA-G(+) EVTs in an hypoxia-inducible, factor-dependent manner. To validate further the utility of hPSC-derived CTBs, we demonstrated that differentiation of trisomy 21 (T21) hPSCs recapitulates the delayed CTB maturation and blunted STB differentiation seen in T21 placentae. Collectively, our data suggest that hPSCs are a valuable model of human placental development, enabling us to recapitulate processes that result in both normal and diseased pregnancies.