Fig. 1
Epiblast cells were committed to neural lineage in 2i/LIF culture condition. a Epiblasts isolated from mouse E5.75 embryos. b Small domed colonies appeared after culturing epiblast cell clumps on MEF feeder in 2i/LIF medium for 3 days. c All clones exhibited neural-like morphology after two passages in 2i/LIF medium. d Real-time PCR showed the mRNA expression pattern of neural-like clones (NLC) was similar to neural stem cells (NSC) other than ESCs. Pluripotent markers, Oct4 and Sox2; neuroectoderm markers, Sox2 and Pax6; neural stem cell marker, Nestin. RNA collected on day 3 after replating the domed colonies. e Morphology of EpiSCs isolated from mouse E5.75 epiblasts. f NLC emerged by passaging EpiSCs in 2i/LIF medium twice. g NLC expressed the neural stem cell marker NESTIN (green) and the neuron marker TuJ-1 (red). Nuclei counterstained by Hoechst 33342 (blue). h Embryos were obtained from ROSAmT/mG × Nes-cre mouse strains. ROSAmT/mG × Nes-cre E13.5 embryos expressed GFP in the neural system. Only domed ESC clones cultured in 2i/LIF medium and flattened EpiSCs maintained in AFX medium (10 ng/ml Activin A, 5 ng/ml FGF2, and 2 μM XAV939) expressed red tomato; however, domed colonies expressing GFP were observed after EpiSCs were cultured in 2i/LIF for 3 days, indicating CRE activity driven by the Nestin promoter. Bar, 100 μm. E embryonic day, ESC embryonic stem cell, MEF mouse embryonic fibroblasts, EpiSC epiblast stem cell, OCT4 octamer-binding transcription factor 4, Pax6 paired box 6, SOX2 sex determining region Y-box 2