PROJECT SUMMARY Murine trophoblast stem cells (TSCs) are cells isolated from blastocyst stage mouse embryos that are capable of differentiation into multiple trophoblast cell types in vitro and can colonize the placenta in utero. These cells have greatly advanced the study of trophoblast development, due to the ability to expand TSCs and differentiate them in vitro, facilitating cytological, biochemical, and epigenomic studies. However, no well-validated human TSCs have been described, limiting these studies to mouse models. One potential solution that has been pursued by multiple groups is the trans-differentiation of pluripotent stem cells (PSCs), which encompass both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), into trophoblast. Although PSCs can trans-differentiate into cells with many features of TSCs, these cells, called trophoblast stem-like cells (TS-LCs), do not have the developmental potential or self-renewal capacity of TSCs. Consequently, TS-LCs are limited as models for trophoblast function. These findings reveal there is an epigenetic block to trans-differentiation that must be overcome to produce bona fide induced trophoblast sem cells (iTSCs) from PSCs. We propose three Aims, two of which will characterize epigenetic features in PSCs that contribute to the trans-differentiation barrier. In the third Aim, we propose to use a combination of genome engineering and a novel hybrid differentiation/trans-differentiation protocol to overcome this barrier and produce bona fide iTSCs. Successful completion of these studies will provide considerable insight into the epigenetic barriers to trans-differentiation, and potentially the first bona fide iTSC lines derived from human PSCs.