Transcriptomic Signature of Trophoblast Differentiation in a Human Embryonic Stem Cell Model

Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, USA.
Biology of Reproduction (Impact Factor: 3.32). 03/2011; 84(6):1258-71. DOI: 10.1095/biolreprod.110.086413
Source: PubMed


Identification of genes involved in trophoblast differentiation is of great interest in understanding cellular and molecular mechanisms involved in placental development and is relevant clinically to fetal development, fertility, and maternal health. Herein, we investigated differentiation of human embryonic stem cells (hESCs) down the trophoblast lineage by culture with bone morphogenetic protein 4 (BMP4) over a 10-day period. Within 2 days, the stemness markers POU5F1 and NANOG were markedly down-regulated, followed temporally by up-regulation of the CDX2, KRT7, HLA-G, ID2, CGA, and CGB trophoblast markers. To understand, on a global scale, changes in the transcriptome during the differentiation of hESCs down the trophoblast lineage, a large-scale microarray analysis was performed. Through whole-genome analysis, more than 3800 genes displayed statistically significant and 2-fold or greater changes in expression during the time course. Of those genes that showed the largest increases, many were involved in processes associated with trophoblast biology; however, novel genes were also identified. Some of them are hypothesized to be associated mainly with extracellular matrix remodeling (e.g., NID2) and cell migration and invasion (e.g., RAB25). Using Ingenuity pathways analysis software to identify signaling pathways involved in trophoblast differentiation or function, we discovered that many genes are involved in WNT/beta-catenin, ERK/MAPK, NFKB, and calcium signaling pathways, suggesting potential roles for these families in trophoblast development. This work provides an in vitro functional genomic model with which to identify genes involved in trophoblast development.

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    • "For instance, supplementation with Activin A and FGF forces endoderm specification (D'Amour et al., 2005; Sui et al., 2012). FGF, in the presence of BMP4 (another member of TGFb family, which antagonizes Activin A), is essential for the specialization of hESC toward the trophoblast (TB) lineage (Xu et al., 2002; Marchand et al., 2011). TGFb signaling simultaneously induces the expression of NANOG and the suppression of SOX2, key genes for pluripotency (Greber et al., 2008). "
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    ABSTRACT: WNT/β-catenin signaling has been described as a crucial regulator of embryonic stem cells and embryogenesis. However, little is known on its role during human preimplantation embryo development, besides the RNA expression of its multiple players. In this study, we performed β-catenin loss- and gain-of-function studies on human preimplantation embryos by adding either Cardamonin or GSK3-inhibitor, 1-Azakenpaullone, to the embryo culture medium from the cleavage until blastocyst stages (day 3 - day 5/6). β-catenin was displayed in the cortical region underneath the membrane during all stages, but it only showed nuclear localization at cleavage stages after stabilization with 1-Azakenpaullone. We did not observe any effects on the inner cell mass markers NANOG, POU5F1, SOX2 and SALL4 in these functional experiments. However, both β-catenin degradation and stabilization caused inhibition of the trophectoderm (TE) fate, illustrated by KRT18 and GATA3 RNA, and CDX2 protein expression. Based on the TE-specific WNT3 protein expression in blastocysts, we postulated that this protein may be an upstream regulator for the observed membrane β-catenin function. The addition of either WNT3 or 1-Azakenpaullone to the culture medium promoted EOMES expression specific for trophoblast development. In both studies, the canonical WNT pathway target gene, TCF1, was not affected. Therefore, we conclude that WNT3 and membrane-associated β-catenin promote progenitor trophoblast development in human blastocysts. These results have important implications in assisted reproduction and stem cell biology. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email:
    Molecular Human Reproduction 06/2015; 21(9). DOI:10.1093/molehr/gav036 · 3.75 Impact Factor
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    • "We compared the change in transcript expression during trophoblast differentiation of the LIN28-associated transcripts identified in this study, the significant LIN28 targets from the Peng study (21), known trophectoderm and pluripotency genes (45), and all expressed genes (Figure 3A). As expected, transcript levels of trophectoderm marker genes significantly increased upon TE differentiation, while transcript levels of pluripotency-related genes significantly decreased. "
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    ABSTRACT: LIN28 function is fundamental to the activity and behavior of human embryonic stem cells (hESCs) and induced pluripotent stem cells. Its main roles in these cell types are the regulation of translational efficiency and let-7 miRNA maturation. However, LIN28-associated mRNA cargo shifting and resultant regulation of translational efficiency upon the initiation of differentiation remain unknown. An RNA-immunoprecipitation and microarray analysis protocol, eRIP, that has high specificity and sensitivity was developed to test endogenous LIN28-associated mRNA cargo shifting. A combined eRIP and polysome analysis of early stage differentiation of hESCs with two distinct differentiation cues revealed close similarities between the dynamics of LIN28 association and translational modulation of genes involved in the Wnt signaling, cell cycle, RNA metabolism and proteasomal pathways. Our data demonstrate that change in translational efficiency is a major contributor to early stages of differentiation of hESCs, in which LIN28 plays a central role. This implies that eRIP analysis of LIN28-associated RNA cargoes may be used for rapid functional quality control of pluripotent stem cells under manufacture for therapeutic applications.
    Nucleic Acids Research 05/2014; 42(12). DOI:10.1093/nar/gku430 · 9.11 Impact Factor
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    • "IGF2, CDH1 or E-cadherin) and hormones (e.g. b-hCG) (Marchand et al., 2011). "
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    ABSTRACT: During human pre-implantation development the totipotent zygote divides and undergoes a number of changes that lead to the first lineage differentiation in the blastocyst displaying trophectoderm and inner cell mass on day 5. The trophectoderm is a differentiated epithelium needed for implantation and the inner cell mass (ICM) forms the embryo proper and serves as a source for pluripotent embryonic stem cells. The blastocyst implants around day 7. The second lineage differentiation occurs in the ICM after implantation resulting in specification of primitive endoderm and epiblast. Knowledge on human pre-implantation development is limited due to ethical and legal restrictions on embryo research and scarcity of materials. Studies in the human are mainly descriptive and lack functional evidence. Most information on embryo development is obtained from animal models and embryonic stem cell cultures and should be extrapolated with caution. This paper reviews totipotency and the molecular determinants and pathways involved in lineage segregation in the human embryo, as well as the role of embryonic genome activation, cell cycle features and epigenetic modifications.
    Molecular Human Reproduction 04/2014; 20(7). DOI:10.1093/molehr/gau027 · 3.75 Impact Factor
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