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    • The most commonly used trophoblast markers reported in the l

    2018-10-20

    The most commonly used “trophoblast” markers reported in the literature are cytokeratin 7 (KRT7), HLA-G, and human chorionic gonadotropin (hCG), but these are either not specific to all trophoblast O4I1 or are expressed in other cell types. Several of the transcription factors (TF) that define the transcriptional network of mouse TSC have also been used (e.g. CDX2 and EOMES) (Senner and Hemberger, 2010). However, it is not known whether the same network operates in humans or what the pattern of expression is in normal first-trimester trophoblast populations (Table S1). ELF5 is a TF that is expressed in mouse TSC to sustain their potential for self-renewal and commitment to the extraembryonic lineage (Donnison et al., 2005; Ng et al., 2008). In mice, the promoter of Elf5 is hypermethylated in ESC and hypomethylated in TSC (Ng et al., 2008). In human early placental tissue, the ELF5 promoter is mostly hypomethylated (Hemberger et al., 2010). Thus, the lack of methylation of the ELF5 promoter could potentially be an additional marker to define trophoblast, although it is still unknown whether ELF5 hypomethylation is present specifically in trophoblast or in other placental cell types. Another possible candidate for defining trophoblast is the expression of specific non-protein-coding microRNAs (miRNAs), in particular the chromosome 19 miRNA cluster (C19MC) that is located in the leukocyte receptor complex on chromosome 19q13.41 (Bentwich et al., 2005). C19MC miRNAs are primate specific and maternally imprinted, with expression normally restricted only to the placenta and hESC (Bentwich et al., 2005; Laurent et al., 2008; Bortolin-Cavaillé et al., 2009; Noguer-Dance et al., 2010). C19MC is the largest cluster of miRNAs in humans and is highly expressed in human trophoblast cells (Bortolin-Cavaillé et al., 2009; Donker et al., 2012). In this study we test these four criteria, which include both protein and non-protein-coding markers, using primary human trophoblast. We focused on the first trimester, as this is when placental development occurs. We show that, by using these criteria in combination, reliable identification of genuine trophoblast is possible. As proof of principle, we then tested these four diverse characteristics (expression of trophoblast protein markers and C19MC miRNAs, HLA class I profile, and methylation status of ELF5 promoter) on two cell types: 2102Ep, an embryonal carcinoma (EC) cell line, and trophoblast-like cells induced from BMP4-treated hESC. Here, we show that both cell types show some properties typical of trophoblast, but neither displays all four characteristics. We propose that this classification system will provide a stringent method to define human trophoblast cells in vitro.
    Results
    Discussion The main obstacle in defining trophoblast cell fate in cell lines in vitro has been that there is no marker exclusive to trophoblast cells that could serve as an unambiguous readout of cell lineage allocation. Therefore, our aim in this study was to identify a set of criteria that would allow cells to be rigorously assigned to the trophoblast lineage. These criteria have been defined using first-trimester primary trophoblast, the period of gestation when exuberant trophoblast proliferation and development of the placenta occurs. Furthermore, obstetric outcome is affected by placental dysfunction before 10 weeks’ gestational age (Smith, 2010). In future, analysis of trophectoderm and trophoblast later in gestation can be done to confirm that these criteria define trophoblast throughout pregnancy. Our tables illustrate that many of the markers currently in use are either only present in some trophoblast subtypes (e.g. CDX2, ELF5, HLA-G), and/or are not specific to trophoblast (e.g. KRT7, CDX2, EOMES). Therefore, using information from our previous microarray data of fluorescence-activated cell-sorted trophoblast cells, we selected genes involved in the transcriptional network that drive murine TSC, and show that TFAP2C and GATA3 are expressed in all mononuclear trophoblast cells, providing useful additional markers (Biadasiewicz et al., 2011; Kuckenberg et al., 2012).