Deriving Human Naïve Embryonic Stem Cell Lines from Donated Supernumerary Embryos Using Physical Distancing and...

Until recently, naïve pluripotent stem cell lines were not captured from human embryos because protocols were based upon those devised for murine embryonic stem cells. In contrast with early lineage segregation in mouse embryos, human hypoblast specification is not solely dependent upon FGF signaling; consequently, its maturation during embryo explant culture may provide inductive signals to drive differentiation of the epiblast. To overcome this potential risk, here we describe how cells of the immature inner cell mass of human embryos can be physically separated during derivation, achieved via "immunosurgery", to eliminate the trophectoderm, followed by disaggregation of the remaining inner cell mass cells. A modification of a culture regime developed for propagation of human pluripotent stem cells reset to the naïve state is used, which comprises serum-free medium supplemented with various inhibitors of signaling pathways, polarization, and differentiation. Colonies arising from the first plating of an inner cell mass may be pooled for ease of handling, or propagated separately to allow establishment of clonal human naïve embryonic stem cell lines.