Establishment of primitive endoderm stem cell lines in mice [coculture]

The mammalian blastocyst consists of three distinct cell types: epiblast, trophoblast, and primitive endoderm (PrE). Although stem cell lines that retain functional properties of epiblasts or trophoblasts have been established, establishment of stem cell lines that fully retain developmental potential of PrE has long been awaited. Here we report derivation of primitive endoderm stem (PrES) cells, which are capable to give rise functional PrE derived tissues and support fetal development of PrE-depleted blastocysts in chimeras, in mice. ES (#1M), TS cell (#1F), PrES (#6-5), and XEN (#6-5) cells were dissociated to single cells by incubation with 0.05% trypsin-EDTA at 37 °C for 3 min. The cells were resuspended in DMEM (Thermo Fisher Scientific) with 10% FBS, 1 × glutamine-penicillin-streptomycin, 0.1mM ß-ME. The cells were replated alone or in combinations of each at a concentration of 20 cells per microwell (1.3×103 cells/dish) on 60mm EZSPHERE® SP dishes (Iwaki) and were placed at 37 °C and 5% CO2 for 1 day or 3 days. Overall design: Gene expression of cultured cells were observed using RNA-seq.