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

•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

SummaryControversy 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.