Life Sciences Research for Lifelong Health

Publications myriam-hemberger

Title / Authors / Details Open Access Download

Mechanisms of early placental development in mouse and humans.
Hemberger M, Hanna CW, Dean W

The importance of the placenta in supporting mammalian development has long been recognized, but our knowledge of the molecular, genetic and epigenetic requirements that underpin normal placentation has remained remarkably under-appreciated. Both the in vivo mouse model and in vitro-derived murine trophoblast stem cells have been invaluable research tools for gaining insights into these aspects of placental development and function, with recent studies starting to reshape our view of how a unique epigenetic environment contributes to trophoblast differentiation and placenta formation. These advances, together with recent successes in deriving human trophoblast stem cells, open up new and exciting prospects in basic and clinical settings that will help deepen our understanding of placental development and associated disorders of pregnancy.

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Nature reviews. Genetics, , 1471-0064, , 2019

PMID: 31534202

ZFP57 regulation of transposable elements and gene expression within and beyond imprinted domains.
Shi H, Strogantsev R, Takahashi N, Kazachenka A, Lorincz MC, Hemberger M, Ferguson-Smith AC

KRAB zinc finger proteins (KZFPs) represent one of the largest families of DNA-binding proteins in vertebrate genomes and appear to have evolved to silence transposable elements (TEs) including endogenous retroviruses through sequence-specific targeting of repressive chromatin states. ZFP57 is required to maintain the post-fertilization DNA methylation memory of parental origin at genomic imprints. Here we conduct RNA-seq and ChIP-seq analyses in normal and ZFP57 mutant mouse ES cells to understand the relative importance of ZFP57 at imprints, unique and repetitive regions of the genome.

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Epigenetics & chromatin, 12, 1756-8935, 49, 2019

PMID: 31399135

Open Access

Common and distinct transcriptional signatures of mammalian embryonic lethality.
Collins JE, White RJ, Staudt N, Sealy IM, Packham I, Wali N, Tudor C, Mazzeo C, Green A, Siragher E, Ryder E, White JK, Papatheodoru I, Tang A, Füllgrabe A, Billis K, Geyer SH, Weninger WJ, Galli A, Hemberger M, Stemple DL, Robertson E, Smith JC, Mohun T, Adams DJ, Busch-Nentwich EM

The Deciphering the Mechanisms of Developmental Disorders programme has analysed the morphological and molecular phenotypes of embryonic and perinatal lethal mouse mutant lines in order to investigate the causes of embryonic lethality. Here we show that individual whole-embryo RNA-seq of 73 mouse mutant lines (>1000 transcriptomes) identifies transcriptional events underlying embryonic lethality and associates previously uncharacterised genes with specific pathways and tissues. For example, our data suggest that Hmgxb3 is involved in DNA-damage repair and cell-cycle regulation. Further, we separate embryonic delay signatures from mutant line-specific transcriptional changes by developing a baseline mRNA expression catalogue of wild-type mice during early embryogenesis (4-36 somites). Analysis of transcription outside coding sequence identifies deregulation of repetitive elements in Morc2a mutants and a gene involved in gene-specific splicing. Collectively, this work provides a large scale resource to further our understanding of early embryonic developmental disorders.

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Nature communications, 10, 2041-1723, 2792, 2019

PMID: 31243271

Open Access

Fetal and trophoblast PI3K p110α have distinct roles in regulating resource supply to the growing fetus in mice.
López-Tello J, Pérez-García V, Khaira J, Kusinski LC, Cooper WN, Andreani A, Grant I, Fernández de Liger E, Lam BY, Hemberger M, Sandovici I, Constancia M, Sferruzzi-Perri AN

Studies suggest that placental nutrient supply adapts according to fetal demands. However, signaling events underlying placental adaptations remain unknown. Here we demonstrate that phosphoinositide 3-kinase p110α in the fetus and the trophoblast interplay to regulate placental nutrient supply and fetal growth. Complete loss of fetal p110α caused embryonic death, whilst heterozygous loss resulted in fetal growth restriction and impaired placental formation and nutrient transport. Loss of trophoblast p110α resulted in viable fetuses, abnormal placental development and a failure of the placenta to transport sufficient nutrients to match fetal demands for growth. Using RNA-seq we identified genes downstream of p110α in the trophoblast that are important in adapting placental phenotype. Using CRISPR/Cas9 we showed loss of p110α differentially affects gene expression in trophoblast and embryonic stem cells. Our findings reveal important, but distinct roles for p110α in the different compartments of the conceptus, which control fetal resource acquisition and growth.

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eLife, 8, 2050-084X, , 2019

PMID: 31241463

Open Access

Inhibition of Phosphoinositide-3-Kinase Signaling Promotes the Stem Cell State of Trophoblast.
Lee CQE, Bailey A, Lopez-Tello J, Sferruzzi-Perri AN, Okkenhaug K, Moffett A, Rossant J, Hemberger M

Trophoblast stem cells (TSCs) are a heterogeneous cell population despite the presence of fibroblast growth factor (FGF) and transforming growth factor β (TGFB) as key growth factors in standard culture conditions. To understand what other signaling cascades control the stem cell state of mouse TSCs, we performed a kinase inhibitor screen and identified several novel pathways that cause TSC differentiation. Surprisingly, inhibition of phosphoinositide-3-kinase (PI3K) signaling increased the mRNA and protein expression of stem cell markers instead, and resulted in a tighter epithelial colony morphology and fewer differentiated cells. PI3K inhibition could not substitute for FGF or TGFB and did not affect phosphorylation of extracellular signal-regulated kinase, and thus acts independently of these pathways. Upon removal of PI3K inhibition, TSC transcription factor levels reverted to normal TSC levels, indicating that murine TSCs can reversibly switch between these two states. In summary, PI3K inhibition reduces the heterogeneity and seemingly heightens the stem cell state of TSCs as indicated by the simultaneous upregulation of multiple key marker genes and cell morphology. Stem Cells 2019;37:1307-1318.

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Stem cells (Dayton, Ohio), 37, 1549-4918, 1307-1318, 2019

PMID: 31233251

Trophoblast organoids as a model for maternal-fetal interactions during human placentation.
Turco MY, Gardner L, Kay RG, Hamilton RS, Prater M, Hollinshead MS, McWhinnie A, Esposito L, Fernando R, Skelton H, Reimann F, Gribble FM, Sharkey A, Marsh SGE, O'Rahilly S, Hemberger M, Burton GJ, Moffett A

The placenta is the extraembryonic organ that supports the fetus during intrauterine life. Although placental dysfunction results in major disorders of pregnancy with immediate and lifelong consequences for the mother and child, our knowledge of the human placenta is limited owing to a lack of functional experimental models. After implantation, the trophectoderm of the blastocyst rapidly proliferates and generates the trophoblast, the unique cell type of the placenta. In vivo, proliferative villous cytotrophoblast cells differentiate into two main sub-populations: syncytiotrophoblast, the multinucleated epithelium of the villi responsible for nutrient exchange and hormone production, and extravillous trophoblast cells, which anchor the placenta to the maternal decidua and transform the maternal spiral arteries. Here we describe the generation of long-term, genetically stable organoid cultures of trophoblast that can differentiate into both syncytiotrophoblast and extravillous trophoblast. We used human leukocyte antigen (HLA) typing to confirm that the organoids were derived from the fetus, and verified their identities against four trophoblast-specific criteria. The cultures organize into villous-like structures, and we detected the secretion of placental-specific peptides and hormones, including human chorionic gonadotropin (hCG), growth differentiation factor 15 (GDF15) and pregnancy-specific glycoprotein (PSG) by mass spectrometry. The organoids also differentiate into HLA-G extravillous trophoblast cells, which vigorously invade in three-dimensional cultures. Analysis of the methylome reveals that the organoids closely resemble normal first trimester placentas. This organoid model will be transformative for studying human placental development and for investigating trophoblast interactions with the local and systemic maternal environment.

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Nature, , 1476-4687, , 2018

PMID: 30487605

Regulation of Placental Development and Its Impact on Fetal Growth-New Insights From Mouse Models.
Woods L, Perez-Garcia V, Hemberger M

The placenta is the chief regulator of nutrient supply to the growing embryo during gestation. As such, adequate placental function is instrumental for developmental progression throughout intrauterine development. One of the most common complications during pregnancy is insufficient growth of the fetus, a problem termed intrauterine growth restriction (IUGR) that is most frequently rooted in a malfunctional placenta. Together with conventional gene targeting approaches, recent advances in screening mouse mutants for placental defects, combined with the ability to rapidly induce mutations and by CRISPR-Cas9 technology, has provided new insights into the contribution of the genome to normal placental development. Most importantly, these data have demonstrated that far more genes are required for normal placentation than previously appreciated. Here, we provide a summary of common types of placental defects in established mouse mutants, which will help us gain a better understanding of the genes impacting on human placentation. Based on a recent mouse mutant screen, we then provide examples on how these data can be mined to identify novel molecular hubs that may be critical for placental development. Given the close association between placental defects and abnormal cardiovascular and brain development, these functional nodes may also shed light onto the etiology of birth defects that co-occur with placental malformations. Taken together, recent insights into the regulation of mouse placental development have opened up new avenues for research that will promote the study of human pregnancy conditions, notably those based on defects in placentation that underlie the most common pregnancy pathologies such as IUGR and pre-eclampsia.

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Frontiers in endocrinology, 9, 1664-2392, 570, 2018

PMID: 30319550

Open Access

Divergent wiring of repressive and active chromatin interactions between mouse embryonic and trophoblast lineages.
Schoenfelder S, Mifsud B, Senner CE, Todd CD, Chrysanthou S, Darbo E, Hemberger M, Branco MR

The establishment of the embryonic and trophoblast lineages is a developmental decision underpinned by dramatic differences in the epigenetic landscape of the two compartments. However, it remains unknown how epigenetic information and transcription factor networks map to the 3D arrangement of the genome, which in turn may mediate transcriptional divergence between the two cell lineages. Here, we perform promoter capture Hi-C experiments in mouse trophoblast (TSC) and embryonic (ESC) stem cells to understand how chromatin conformation relates to cell-specific transcriptional programmes. We find that key TSC genes that are kept repressed in ESCs exhibit interactions between H3K27me3-marked regions in ESCs that depend on Polycomb repressive complex 1. Interactions that are prominent in TSCs are enriched for enhancer-gene contacts involving key TSC transcription factors, as well as TET1, which helps to maintain the expression of TSC-relevant genes. Our work shows that the first developmental cell fate decision results in distinct chromatin conformation patterns establishing lineage-specific contexts involving both repressive and active interactions.

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Nature communications, 9, 2041-1723, 4189, 2018

PMID: 30305613

Open Access

A Critical Role of TET1/2 Proteins in Cell-Cycle Progression of Trophoblast Stem Cells.
Chrysanthou S, Senner CE, Woods L, Fineberg E, Okkenhaug H, Burge S, Perez-Garcia V, Hemberger M

The ten-eleven translocation (TET) proteins are well known for their role in maintaining naive pluripotency of embryonic stem cells. Here, we demonstrate that, jointly, TET1 and TET2 also safeguard the self-renewal potential of trophoblast stem cells (TSCs) and have partially redundant roles in maintaining the epithelial integrity of TSCs. For the more abundantly expressed TET1, we show that this is achieved by binding to critical epithelial genes, notably E-cadherin, which becomes hyper-methylated and downregulated in the absence of TET1. The epithelial-to-mesenchymal transition phenotype of mutant TSCs is accompanied by centrosome duplication and separation defects. Moreover, we identify a role of TET1 in maintaining cyclin B1 stability, thereby acting as facilitator of mitotic cell-cycle progression. As a result, Tet1/2 mutant TSCs are prone to undergo endoreduplicative cell cycles leading to the formation of polyploid trophoblast giant cells. Taken together, our data reveal essential functions of TET proteins in the trophoblast lineage.

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Stem cell reports, , 2213-6711, , 2018

PMID: 29576538

Open Access

Integrin α2 marks a niche of trophoblast progenitor cells in first trimester human placenta
Lee CQE, Turco M, Gardner L, Simons B, Hemberger M, Moffett A

During pregnancy the trophoblast cells of the placenta are the only fetal cells in direct contact with maternal blood and decidua. Their functions include transport of nutrients and oxygen, secretion of pregnancy hormones, remodelling the uterine arteries, and communicating with maternal cells. Despite the importance of trophoblast cells in placental development and successful pregnancy, little is known about the identity, location and differentiation of human trophoblast progenitors. We identify a proliferative trophoblast niche at the base of the cytotrophoblast cell columns in first trimester placentas that is characterised by integrin α2 (ITGA2) expression. Pulse-chase experiments with 5-Iodo-2'-deoxyuridine (IdU) imply that these cells can contribute to both villous (VCT) and extravillous (EVT) lineages. These proliferating trophoblast cells can be isolated using ITGA2 as a marker by flow cytometry and express genes from both VCT and EVT. Microarray expression analysis shows that ITAG2cells display a unique transcriptional signature including NOTCH signalling and a combination of epithelial and mesenchymal characteristics. ITGA2 thus marks a niche allowing the study of pure populations of trophoblast progenitor cells.

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Development (Cambridge, England), , 1477-9129, , 2018

PMID: 29540503

Open Access

Placentation defects are highly prevalent in embryonic lethal mouse mutants.
Perez-Garcia V, Fineberg E, Wilson R, Murray A, Mazzeo CI, Tudor C, Sienerth A, White JK, Tuck E, Ryder EJ, Gleeson D, Siragher E, Wardle-Jones H, Staudt N, Wali N, Collins J, Geyer S, Busch-Nentwich EM, Galli A, Smith JC, Robertson E, Adams DJ, Weninger WJ, Mohun T, Hemberger M

Large-scale phenotyping efforts have demonstrated that approximately 25-30% of mouse gene knockouts cause intrauterine lethality. Analysis of these mutants has largely focused on the embryo and not the placenta, despite the crucial role of this extraembryonic organ for developmental progression. Here we screened 103 embryonic lethal and sub-viable mouse knockout lines from the Deciphering the Mechanisms of Developmental Disorders program for placental phenotypes. We found that 68% of knockout lines that are lethal at or after mid-gestation exhibited placental dysmorphologies. Early lethality (embryonic days 9.5-14.5) is almost always associated with severe placental malformations. Placental defects correlate strongly with abnormal brain, heart and vascular development. Analysis of mutant trophoblast stem cells and conditional knockouts suggests that a considerable number of factors that cause embryonic lethality when ablated have primary gene function in trophoblast cells. Our data highlight the hugely under-appreciated importance of placental defects in contributing to abnormal embryo development and suggest key molecular nodes that govern placenta formation.

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Nature, , 1476-4687, , 2018

PMID: 29539633

Clearance of senescent decidual cells by uterine natural killer cells in cycling human endometrium.
Brighton PJ, Maruyama Y, Fishwick K, Vrljicak P, Tewary S, Fujihara R, Muter J, Lucas ES, Yamada T, Woods L, Lucciola R, Hou Lee Y, Takeda S, Ott S, Hemberger M, Quenby S, Brosens JJ

In cycling human endometrium, menstruation is followed by rapid estrogen-dependent growth. Upon ovulation, progesterone and rising cellular cAMP levels activate the transcription factor Forkhead box O1 (FOXO1) in endometrial stromal cells (EnSCs), leading to cell cycle exit and differentiation into decidual cells that control embryo implantation. Here we show that FOXO1 also causes acute senescence of a subpopulation of decidualizing EnSCs in an IL-8 dependent manner. Selective depletion or enrichment of this subpopulation revealed that decidual senescence drives the transient inflammatory response associated with endometrial receptivity. Further, senescent cells prevent differentiation of endometrial mesenchymal stem cells in decidualizing cultures. As the cycle progresses, IL-15 activated uterine natural killer (uNK) cells selectively target and clear senescent decidual cells through granule exocytosis. Our findings reveal that acute decidual senescence governs endometrial rejuvenation and remodeling at embryo implantation, and suggest a critical role for uNK cells in maintaining homeostasis in cycling endometrium.

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eLife, 6, 2050-084X, , 2017

PMID: 29227245

Open Access

Decidualisation and placentation defects are a major cause of age-related reproductive decline.
Woods L, Perez-Garcia V, Kieckbusch J, Wang X, DeMayo F, Colucci F, Hemberger M

Mammalian reproductive performance declines rapidly with advanced maternal age. This effect is largely attributed to the exponential increase in chromosome segregation errors in the oocyte with age. Yet many pregnancy complications and birth defects that become more frequent in older mothers, in both humans and mice, occur in the absence of karyotypic abnormalities. Here, we report that abnormal embryonic development in aged female mice is associated with severe placentation defects, which result from major deficits in the decidualisation response of the uterine stroma. This problem is rooted in a blunted hormonal responsiveness of the ageing uterus. Importantly, a young uterine environment can restore normal placental as well as embryonic development. Our data highlight the pivotal, albeit under-appreciated, impact of maternal age on uterine adaptability to pregnancy as major contributor to the decline in reproductive success in older females.Advanced maternal age has been associated with lower reproductive success and higher risk of pregnancy complications. Here the authors show that maternal ageing-related embryonic abnormalities in mouse are caused by decidualisation and placentation defects that can be rescued by transferring the embryo from an old to a young uterus.

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Nature communications, 8, 2041-1723, 352, 2017

PMID: 28874785

Open Access

Long-term, hormone-responsive organoid cultures of human endometrium in a chemically defined medium.
Turco MY, Gardner L, Hughes J, Cindrova-Davies T, Gomez MJ, Farrell L, Hollinshead M, Marsh SGE, Brosens JJ, Critchley HO, Simons BD, Hemberger M, Koo BK, Moffett A, Burton GJ

In humans, the endometrium, the uterine mucosal lining, undergoes dynamic changes throughout the menstrual cycle and pregnancy. Despite the importance of the endometrium as the site of implantation and nutritional support for the conceptus, there are no long-term culture systems that recapitulate endometrial function in vitro. We adapted conditions used to establish human adult stem-cell-derived organoid cultures to generate three-dimensional cultures of normal and decidualized human endometrium. These organoids expand long-term, are genetically stable and differentiate following treatment with reproductive hormones. Single cells from both endometrium and decidua can generate a fully functional organoid. Transcript analysis confirmed great similarity between organoids and the primary tissue of origin. On exposure to pregnancy signals, endometrial organoids develop characteristics of early pregnancy. We also derived organoids from malignant endometrium, and so provide a foundation to study common diseases, such as endometriosis and endometrial cancer, as well as the physiology of early gestation.

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Nature cell biology, , 1476-4679, , 2017

PMID: 28394884

From the stem of the placental tree: trophoblast stem cells and their progeny.
Latos PA, Hemberger M

Trophoblast stem cells (TSCs) retain the capacity to self-renew indefinitely and harbour the potential to differentiate into all trophoblast subtypes of the placenta. Recent studies have shown how signalling cascades integrate with transcription factor circuits to govern the fine balance between TSC self-renewal and differentiation. In addition, breakthroughs in reprogramming strategies have enabled the generation of TSCs from fibroblasts, opening up exciting new avenues that may allow the isolation of this stem cell type from other species, notably humans. Here, we review these recent advances in light of their importance for understanding placental pathologies and developing personalised medicine approaches for pregnancy complications.

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Development (Cambridge, England), 143, 1477-9129, 3650-3660, 2016

PMID: 27802134

Plet1 is an epigenetically regulated cell surface protein that provides essential cues to direct trophoblast stem cell differentiation.
Murray A, Sienerth AR, Hemberger M

Gene loci that are hypermethylated and repressed in embryonic (ESCs) but hypomethylated and expressed in trophoblast (TSCs) stem cells are very rare and may have particularly important roles in early developmental cell fate decisions, as previously shown for Elf5. Here, we assessed another member of this small group of genes, Placenta Expressed Transcript 1 (Plet1), for its function in establishing trophoblast lineage identity and modulating trophoblast differentiation. We find that Plet1 is tightly repressed by DNA methylation in ESCs but expressed on the cell surface of TSCs and trophoblast giant cells. In hypomethylated ESCs that are prone to acquire some trophoblast characteristics, Plet1 is required to confer a trophoblast-specific gene expression pattern, including up-regulation of Elf5. Plet1 displays an unusual biphasic expression profile during TSC differentiation and thus may be pivotal in balancing trophoblast self-renewal and differentiation. Furthermore, overexpression and CRISPR/Cas9-mediated knockout in TSCs showed that high Plet1 levels favour differentiation towards the trophoblast giant cell lineage, whereas lack of Plet1 preferentially induces syncytiotrophoblast formation. Thus, the endogenous dynamics of Plet1 expression establish important patterning cues within the trophoblast compartment by promoting differentiation towards the syncytiotrophoblast or giant cell pathway in Plet1-low and Plet1-high cells, respectively.

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Scientific reports, 6, 2045-2322, 25112, 2016

PMID: 27121762

Open Access

What Is Trophoblast? A Combination of Criteria Define Human First-Trimester Trophoblast.
Lee CQ, Gardner L, Turco M, Zhao N, Murray MJ, Coleman N, Rossant J, Hemberger M, Moffett A

Controversy surrounds reports describing the derivation of human trophoblast cells from placentas and embryonic stem cells (ESC), partly due to the difficulty in identifying markers that define cells as belonging to the trophoblast lineage. We have selected criteria that are characteristic of primary first-trimester trophoblast: a set of protein markers, HLA class I profile, methylation of ELF5, and expression of microRNAs (miRNAs) from the chromosome 19 miRNA cluster (C19MC). We tested these criteria on cells previously reported to show some phenotypic characteristics of trophoblast: bone morphogenetic protein (BMP)-treated human ESC and 2102Ep, an embryonal carcinoma cell line. Both cell types only show some, but not all, of the four trophoblast criteria. Thus, BMP-treated human ESC have not fully differentiated to trophoblast. Our study identifies a robust panel, including both protein and non-protein-coding markers that, in combination, can be used to reliably define cells as characteristic of early trophoblast.

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Stem cell reports, 6, 2213-6711, 257-72, 2016

PMID: 26862703

Open Access

Maternal DNA Methylation Regulates Early Trophoblast Development.
Branco MR, King M, Perez-Garcia V, Bogutz AB, Caley M, Fineberg E, Lefebvre L, Cook SJ, Dean W, Hemberger M, Reik W

Critical roles for DNA methylation in embryonic development are well established, but less is known about its roles during trophoblast development, the extraembryonic lineage that gives rise to the placenta. We dissected the role of DNA methylation in trophoblast development by performing mRNA and DNA methylation profiling of Dnmt3a/3b mutants. We find that oocyte-derived methylation plays a major role in regulating trophoblast development but that imprinting of the key placental regulator Ascl2 is only partially responsible for these effects. We have identified several methylation-regulated genes associated with trophoblast differentiation that are involved in cell adhesion and migration, potentially affecting trophoblast invasion. Specifically, trophoblast-specific DNA methylation is linked to the silencing of Scml2, a Polycomb Repressive Complex 1 protein that drives loss of cell adhesion in methylation-deficient trophoblast. Our results reveal that maternal DNA methylation controls multiple differentiation-related and physiological processes in trophoblast via both imprinting-dependent and -independent mechanisms.

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Developmental cell, 36, 1878-1551, 152-63, 2016

PMID: 26812015

Open Access

Elf5-centered transcription factor hub controls trophoblast stem cell self-renewal and differentiation through stoichiometry-sensitive shifts in target gene networks.
Latos PA, Sienerth AR, Murray A, Senner CE, Muto M, Ikawa M, Oxley D, Burge S, Cox BJ, Hemberger M

Elf5 is a transcription factor with pivotal roles in the trophoblast compartment, where it reinforces a trophoblast stem cell (TSC)-specific transcriptional circuit. However, Elf5 is also present in differentiating trophoblast cells that have ceased to express other TSC genes such as Cdx2 and Eomes. In the present study, we aimed to elucidate the context-dependent role of Elf5 at the interface between TSC self-renewal and the onset of differentiation. We demonstrate that precise levels of Elf5 are critical for normal expansion of the TSC compartment and embryonic survival, as Elf5 overexpression triggers precocious trophoblast differentiation. Through integration of protein interactome, transcriptome, and genome-wide chromatin immunoprecipitation data, we reveal that this abundance-dependent function is mediated through a shift in preferred Elf5-binding partners; in TSCs, Elf5 interaction with Eomes recruits Tfap2c to triply occupied sites at TSC-specific genes, driving their expression. In contrast, the Elf5 and Tfap2c interaction becomes predominant as their protein levels increase. This triggers binding to double- and single-occupancy sites that harbor the cognate Tfap2c motif, causing activation of the associated differentiation-promoting genes. These data place Elf5 at the center of a stoichiometry-sensitive transcriptional network, where it acts as a molecular switch governing the balance between TSC proliferation and differentiation.

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Genes & development, , 1549-5477, , 2015

PMID: 26584622

Open Access

Direct Induction of Trophoblast Stem Cells from Murine Fibroblasts.
Kubaczka C, Senner CE, Cierlitza M, Araúzo-Bravo MJ, Kuckenberg P, Peitz M, Hemberger M, Schorle H

Trophoblast stem cells (TSCs) arise from the first cell fate decision in the developing embryo and generate extra-embryonic lineages, giving rise to the fetal portion of the placenta. Mouse embryonic and extra-embryonic lineages are strictly separated by a distinct epigenetic barrier, which is not fully overcome following expression of TSC-determining factors in embryonic stem cells. Here, we show that transient expression of Tfap2c, Gata3, Eomes, and Ets2 is sufficient to reprogram mouse embryonic fibroblasts and post-natal tail-tip-derived fibroblasts into induced TSCs (iTSCs) and surmount the epigenetic barrier separating somatic from extra-embryonic lineages. iTSCs share nearly identical morphological characteristics, gene expression profiles, and DNA methylation patterns with blastocyst-derived TSCs. Furthermore, iTSCs display transgene-independent self-renewal, differentiate along extra-embryonic lineages, and chimerize host placentas following blastocyst injection. These findings provide insights into the transcription factor networks governing TSC identity and opportunities for studying the epigenetic barriers underlying embryonic and extra-embryonic lineage segregation.

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Cell stem cell, 17, 1875-9777, 557-68, 2015

PMID: 26412560

Fgf and Esrrb integrate epigenetic and transcriptional networks that regulate self-renewal of trophoblast stem cells.
Latos PA, Goncalves A, Oxley D, Mohammed H, Turro E, Hemberger M

Esrrb (oestrogen-related receptor beta) is a transcription factor implicated in embryonic stem (ES) cell self-renewal, yet its knockout causes intrauterine lethality due to defects in trophoblast development. Here we show that in trophoblast stem (TS) cells, Esrrb is a downstream target of fibroblast growth factor (Fgf) signalling and is critical to drive TS cell self-renewal. In contrast to its occupancy of pluripotency-associated loci in ES cells, Esrrb sustains the stemness of TS cells by direct binding and regulation of TS cell-specific transcription factors including Elf5 and Eomes. To elucidate the mechanisms whereby Esrrb controls the expression of its targets, we characterized its TS cell-specific interactome using mass spectrometry. Unlike in ES cells, Esrrb interacts in TS cells with the histone demethylase Lsd1 and with the RNA Polymerase II-associated Integrator complex. Our findings provide new insights into both the general and context-dependent wiring of transcription factor networks in stem cells by master transcription factors.

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Nature communications, 6, 2041-1723, 7776, 2015

PMID: 26206133

Open Access

Epigenetic memory of the first cell fate decision prevents complete ES cell reprogramming into trophoblast.
F Cambuli, A Murray, W Dean, D Dudzinska, F Krueger, S Andrews, CE Senner, S Cook, M Hemberger

Embryonic (ES) and trophoblast (TS) stem cells reflect the first, irrevocable cell fate decision in development that is reinforced by distinct epigenetic lineage barriers. Nonetheless, ES cells can seemingly acquire TS-like characteristics upon manipulation of lineage-determining transcription factors or activation of the extracellular signal-regulated kinase 1/2 (Erk1/2) pathway. Here we have interrogated the progression of reprogramming in ES cell models with regulatable Oct4 and Cdx2 transgenes or conditional Erk1/2 activation. Although trans-differentiation into TS-like cells is initiated, lineage conversion remains incomplete in all models, underpinned by the failure to demethylate a small group of TS cell genes. Forced expression of these non-reprogrammed genes improves trans-differentiation efficiency, but still fails to confer a stable TS cell phenotype. Thus, even ES cells in ground-state pluripotency cannot fully overcome the boundaries that separate the first cell lineages but retain an epigenetic memory of their ES cell origin.

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Nat Commun., 26, 5, 5538, 2014

PMID: 25423963

Open Access

ADP-ribosyltransferases Parp1 and Parp7 safeguard pluripotency of ES cells.
Roper SJ, Chrysanthou S, Senner CE, Sienerth A, Gnan S, Murray A, Masutani M, Latos P, M Hemberger

Embryonic stem (ES) cells are in a dynamic equilibrium of distinct functional states, characterized by the heterogeneous expression of critical pluripotency factors and regulated by a spectrum of reversible histone modifications. Maintenance of this equilibrium is a hallmark of pluripotency. Here we find that the ADP-ribosyltransferases Parp1 and Parp7 play a critical role in safeguarding this state by occupying key pluripotency genes, notably Nanog, Pou5f1, Sox2, Stella, Tet1 and Zfp42, thereby protecting them from progressive epigenetic repression. In the absence of either Parp1 or Parp7, or upon inhibition of the ADP-ribosylating activity, ES cells exhibit a decrease in ground state pluripotency as they cannot maintain the typical heterogeneity characteristic of the metastable state. As a consequence, they display a higher propensity to differentiate. These findings place Parp1 and Parp7 at the genetic-epigenetic interface of pluripotency networks, fine-tuning the transcriptional heterogeneity and thereby determining the developmental plasticity of ES cells.

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Nucleic acids research, 42, 1362-4962, 8914-27, 2014

PMID: 25034692

Open Access

Derivation and maintenance of murine trophoblast stem cells under defined conditions.
Kubaczka C,Senner C,Arauzo-Bravo MJ,Sharma N,Kuckenberg P,Becker A,Zimmer A,Brustle O,Peitz M, M Hemberger,Schorle H

Trophoblast stem cells (TSCs) are in vitro equivalents to the precursor cells of the placenta. TSCs are cultured in serum-rich medium with fibroblast growth factor 4, heparin, and embryonic-fibroblast-conditioned medium. Here, we developed a simple medium consisting of ten chemically defined ingredients for culture of TSCs on Matrigel or synthetic substrates, named TX medium. Gene expression and DNA methylation profiling demonstrated the faithful propagation of expression profiles and epigenomic characteristics of TSCs cultured in TX. Further, TX medium supported the de novo derivation of TSC lines. Finally, TSCs cultured in TX differentiate into all derivatives of the trophectodermal lineage in vitro, give rise to hemorrhagic lesions in nude mice, and chimerize the placenta, indicating that they retained all hallmarks of TSCs. TX media formulation no longer requires fetal bovine serum and conditioned medium, which facilitates and standardizes the culture of this extraembryonic lineage.

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Stem cell reports, 2, 2213-6711, 232-42, 2014

PMID: 24527396

Open Access

Review: The transcriptional and signalling networks of mouse trophoblast stem cells.
PA Latos, M Hemberger

Trophoblast stem cells (TSCs) are a self-renewing stem cell population derived from the early trophoblast lineage, analogous to embryonic stem cells (ESCs) that can be generated from the inner cell mass (ICM) of the mouse blastocyst. In that sense TSCs and ESCs reflect the earliest lineage differentiation event after fertilization. TSCs are characterized by an indefinite proliferation potential and by multipotency, i.e. the ability to differentiate into all the various trophoblast cell types of the placenta. These properties are driven by specific signalling pathways orchestrating characteristic transcriptional outputs. Here we review the recent advances in studying the signalling cascades and the transcriptional regulatory networks that define specification and maintenance of TSCs, and provide a future outlook of TSC research.

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Placenta, , , , 2013

PMID: 24220516
DOI: 10.1016/j.placenta.2013.10.013