Importantly because none of these markers are

Importantly, because none of these markers are specific for trophoblast or trophoblast populations in general, they must be used in combination. For example, SWAN-71 and HTR8/SVneo, two widely used “trophoblast” cell lines, are hypermethylated at the ELF5 promoter, HTR8/SVneo does not express C19MC miRNAs (Hemberger et al., 2010; Donker et al., 2012; Morales-Prieto et al., 2012), and the HLA profile is unlike either VCT or EVT (King et al., 2000). Similarly, we show that BAP-treated hESC display only some of our trophoblast markers (aggregates of cells with TFAP2C and GATA3 expression and partial hypomethylation of ELF5 promoter). In contrast, their HLA class I expression pattern and the decreased C19MC expression are not typical of primary trophoblast. The Exendin-4 appearing in BAP-treated cultures are epithelial cells, but their actual identity requires further work. Nonetheless, we can conclude that BAP-treated hESC do not fully differentiate into cells with all the characteristics of first-trimester trophoblast cells.
Experimental Procedures
Acknowledgments C.L. is in receipt of a scholarship from Agency of Science, Technology and Research (A∗STAR) and the Centre for Trophoblast Research (Cambridge). The Wellcome Trust (090108/Z/09/Z, 085992/Z/08/Z), British Heart Foundation (PG/09/077/27964), and Canadian Institutes of Health Research (CIHR MOP 77803) supported this work. We thank Jodi Garner, Steve Marsh, Richard Saffery, Boris Novakovic, and Maria Caffarel for useful advice. We are grateful to Diane Moore for helping recruit donors, and acknowledge and thank all donors for participating in this study. We are grateful to Nigel Miller for help with flow cytometric sorting.