Matteo Moretto-Zita

BMP4-directed trophoblast differentiation of human embryonic stem cells is mediated through a ΔNp63+ cytotrophoblast stem cell state

The placenta is a transient organ that is necessary for proper fetal development. Its main functional component is the trophoblast, which is derived from extra-embryonic ectoderm. Little is known about early trophoblast differentiation in the human embryo, owing to lack of a proper in vitro model system. Human embryonic stem cells (hESCs) differentiate into functional trophoblast following BMP4 treatment in the presence of feeder-conditioned media; however, this model has not been widely accepted, in part owing to a lack of proof for a trophoblast progenitor population. We have previously shown that p63, a member of the p53 family of nuclear proteins, is expressed in proliferative cytotrophoblast (CTB), precursors to terminally differentiated syncytiotrophoblast (STB) in chorionic villi and extravillous trophoblast (EVT) at the implantation site. Here, we show that BMP4-treated hESCs differentiate into bona fide CTB by direct comparison with primary human placental tissues and isolated CTB through gene expression profiling. We show that, in primary CTB, p63 levels are reduced as cells differentiate into STB, and that forced expression of p63 maintains cyclin B1 and inhibits STB differentiation. We also establish that, similar to in vivo events, hESC differentiation into trophoblast is characterized by a p63+/KRT7+ CTB stem cell state, followed by formation of functional KLF4+ STB and HLA-G+ EVT. Finally, we illustrate that downregulation of p63 by shRNA inhibits differentiation of hESCs into functional trophoblast. Taken together, our results establish that BMP4-treated hESCs are an excellent model of human trophoblast differentiation, closely mimicking the in vivo progression from p63+ CTB stem cells to terminally differentiated trophoblast subtypes.

Hypoxia and trophoblast differentiation: A key role for PPARγ

Tissue oxygen tension regulates differentiation of multiple types of stem cells. In the placenta, hypoxia has been associated with abnormal trophoblast differentiation and placental insufficiency syndromes of preeclampsia (PE) and intrauterine growth restriction (IUGR). Peroxisome proliferator-activated receptor-γ (PPARγ) is a ligand-activated transcription factor involved in many cellular processes, including differentiation. We have previously shown that PPARγ-null trophoblast stem (TS) cells show a defect in differentiation to labyrinthine trophoblast, instead differentiating preferentially to trophoblast giant cells (TGC). Since PPARγ is known to be regulated by hypoxia in adipose tissue, we hypothesized that there may be a link between oxygen tension, PPARγ expression, and trophoblast differentiation. We found that hypoxia reduced PPARγ expression by a mechanism independent of both hypoxia-inducible factor (HIF) and histone deacetylases (HDACs). In addition, PPARγ partially rescued hypoxia-induced inhibition of labyrinthine differentiation in wild-type TS cells but was not required for hypoxia-induced inhibition of TGC differentiation. Finally, we show that induction of labyrinthine trophoblast differentiation by HDAC inhibitor treatment is independent of both PPARγ and Gcm1. We propose a model with two pathways for labyrinthine trophoblast differentiation of TS cells, one of which is dependent on PPARγ and inhibited by hypoxia. Since hypoxia is associated with PE and IUGR, we propose that PPARγ may at least partially mediate hypoxia-induced placental insufficiency and as such may be a promising therapeutic target for these disorders.

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.

p63 Inhibits Extravillous Trophoblast Migration and Maintains Cells in a Cytotrophoblast Stem Cell-Like State

Proper differentiation of placental epithelial cells, called trophoblast, is required for implantation. Early during placentation, trophoblast cell columns help anchor the developing embryo in the uterine wall. Although proximally continuous with villous cytotrophoblast (CTB) distally, these cells differentiate into invasive extravillous trophoblast. We previously reported that p63, a p53 family member, is highly expressed in proliferative villous CTB and required for induction of the trophoblast lineage in human pluripotent stem cells. We now further explore its function in human trophoblast by using both primary CTB from the early placenta and established trophoblast cell lines. We show that p63 is expressed in epidermal growth factor receptor-positive CTB and that its expression decreases with differentiation into HLA-G(+) extravillous trophoblast. In trophoblast cell lines, p63 is expressed in JEG3 cells but absent from HTR8 cells. Overexpression of p63 in both cell lines enhances cell proliferation and significantly reduces cell migration; conversely, down-regulation of p63 in JEG3 cells reduces cell proliferation and restores cell migration. Analysis of epithelial-to-mesenchymal transition, cell adhesion, and matrix degradation pathways shows that p63 blocks epithelial-to-mesenchymal transition, promotes a CTB-specific cell adhesion profile, and inhibits expression of matrix metalloproteinases. Taken together, these data show that p63 maintains the proliferative CTB state, at least partially through regulation of epithelial-to-mesenchymal transition, cell adhesion, and matrix degradation pathways.

Sirtuin1 is required for proper trophoblast differentiation and placental development in mice

INTRODUCTION Placental insufficiency, arising from abnormal trophoblast differentiation and function, is a major cause of fetal growth restriction. Sirtuin-1 (Sirt1) is a ubiquitously-expressed NAD-dependent protein deacetylase which plays a key role in numerous cellular processes, including cellular differentiation and metabolism. Though Sirt1 has been widely studied, its role in placentation and trophoblast differentiation is unclear. METHOD Sirt1-heterozygous mice were mated and evaluated at various points during embryogenesis. In situ hybridization and immunohistochemistry were used to further characterize the placental phenotype of Sirt1-null mice. Wild-type (WT) and Sirt1-null mouse trophoblast stem cell (TSC) lines were derived from e3.5 littermate blastocysts. These cells were then evaluated at various points following differentiation. Differentiation was evaluated by expression of lineage specific markers using qPCR and flow cytometry, as well as Matrigel invasion assays. Global gene expression changes were evaluated using microarray-based RNA profiling; changes in specific pathways were validated using qPCR and western blot. RESULTS In the absence of Sirt1, both embryos and placentas were small, with placentas showing abnormalities in both the labyrinthine layer and junctional zone. Sirt1-null TSCs exhibited an altered phenotype in both undifferentiated and differentiated states, phenotypes which corresponded to changes in pathways relevant to both TSC maintenance and differentiation. Specifically, Sirt1-null TSC showed blunted differentiation, and appeared to be suspended in an Epcamhigh trophoblast progenitor state. DISCUSSION Our results suggest that Sirt1 is required for proper TSC differentiation and placental development.

Comparative analysis of mouse and human placentae across gestation reveals species-specific regulators of placental development

ABSTRACT An increasing body of evidence points to significant spatio-temporal differences in early placental development between mouse and human, but a detailed comparison of placentae in these two species is missing. We set out to compare placentae from both species across gestation, with a focus on trophoblast progenitor markers. We found that CDX2 and ELF5, but not EOMES, are expressed in early post-implantation trophoblast subpopulations in both species. Genome-wide expression profiling of mouse and human placentae revealed clusters of genes with distinct co-expression patterns across gestation. Overall, there was a closer fit between patterns observed in the placentae when the inter-species comparison was restricted to human placentae through gestational week 16 (thus, excluding full-term samples), suggesting that the developmental timeline in mouse runs parallel to the first half of human placental development. In addition, we identified VGLL1 as a human-specific marker of proliferative cytotrophoblast, where it is co-expressed with the transcription factor TEAD4. As TEAD4 is involved in trophectoderm specification in the mouse, we posit a regulatory role for VGLL1 in early events during human placental development. Highlighted Article: A large-scale comparison of mouse and human placentae from early post-implantation to full term identifies species-specific regulatory networks of genes involved in early placental development.

IFPA Meeting 2013 Workshop Report I: diabetes in pregnancy, maternal dyslipidemia in pregnancy, oxygen in placental development, stem cells and pregnancy pathology

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2013 there were twelve themed workshops, four of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of pregnancy pathologies and placental metabolism: 1) diabetes in pregnancy; 2) lipids, fatty acids and the placenta; 3) oxygen in placental development and pathologies; 4) stem cells and pathologies.