What Is Trophoblast? A Combination of Criteria Define Human First-Trimester Trophoblast

摘要

Highlights ? Criteria to identify human trophoblast cells in?vitro are controversial ? HLA class I profile and methylation of ELF5 confirmed as good trophoblast markers ? Levels of C19MC miRNAs are a novel trophoblast marker ? Using these criteria, BMP4-, A83-01-, and PD173074-treated hESC are not trophoblast Summary 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. prs.rt("abs_end"); Introduction One of the key early events in the establishment of pregnancy is the development of trophoblast subpopulations from the trophectoderm (TE) of the implanting blastocyst ( Rossant, 2001 ). Ethical and logistical difficulties limit our knowledge of these earliest stages of placentation in humans. In the mouse, trophoblast stem cells (TSC) have been isolated, but there is still no reliable source of such cells for humans. While it is possible to isolate primary trophoblast from human first-trimester placentas, they rapidly differentiate and do not proliferate in?vitro. Attempts have been made to overcome this problem by obtaining trophoblast cell lines from early placentas by transformation, or by driving human embryonic stem cells (hESC) along the trophoblast differentiation pathway ( Xu et?al., 2002 , Nagamatsu et?al., 2004 , Harun et?al., 2006 , James et?al., 2007 , Genbacev et?al., 2011 , Marchand et?al., 2011 , Takao et?al., 2011 , Udayashankar et?al., 2011 and Amita et?al., 2013 ). However, all these strategies have been plagued with difficulties in identifying the cells as “trophoblast” in culture ( Roberts et?al., 2014 ). In?vivo, trophoblast cells can be identified by their anatomical location and the?expression of particular markers. In?vitro, there is a lack of consensus about the best criteria to use to define trophoblast. A distinctive feature of trophoblast is its profile of human leukocyte antigen (HLA) class I expression. There are two main differentiation pathways in human placentas, villous (VCT) and extravillous (EVT) cytotrophoblast. VCTs fuse to form an overlying syncytiotrophoblast (ST), and EVTs form multinucleated placental bed giant cells deep in the decidua and myometrium. ST and VCT are HLA class I null, whereas EVT express HLA-C and -E molecules, and HLA-G, which is uniquely expressed by trophoblast ( Apps et?al., 2009 ). In contrast, most normal somatic cells are HLA class I positive and express HLA-A, -B, -C, and -E ( Wei and Orr, 1990 ). Only neurons, glial cells, germ cells, hepatocytes, and exocrine pancreas are negative ( Fleming et?al., 1981 , Anderson et?al., 1984 , Lampson and Hickey, 1986 and Jalleh et?al., 1993 ). Thus, human first-trimester trophoblast cells never express HLA-A and -B, and are the only cells that normally express HLA-G. 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 cells 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 and 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-p