Figure 1
Derivation of Epiblast stem cells (EpiSCs) from haploid mouse embryos. (A) Experimental design schematic. Superovulated oocytes were activated in SrCl2-containing media and (B) activated oocytes were cultured to the (C) two-cell stage and subsequently to blastocyst-stage embryos. Blastocysts were cultured on iMEFs in medium containing FGF4 and EpiSC-like colonies were picked and expanded. (D) Bright-field microscopy of blastocyst outgrowths after 96 hrs. Arrow highlights trophectoderm (TE) cells, and dotted line outlines boundary of TE outgrowth. (E) Bright-field microscopy of conventional EpiSCs (E3; left panels) cultured in the presence of bFGF and two representative maternal (parthenogenetic/gynogenetic) EpiSCs (maEpiSCs; right panels) cultured in the presence of FGF4. (F) Bright-field microscopy of TS cells, derived from normal diploid blastocysts, and cultured in the presence of FGF4. (G,H) Immunofluorescence analysis of SOX2 and OCT4 in (G) conventional EpiSCs and (H) maEpiSCs. Nuclei were stained with Dapi. (I) Dapi cell cycle staining of conventional EpiSCs and maEpiSCs. (J) Metaphase spread Giesma staining and chromosome counts show that conventional EpiSCs and maEpiSCs have a normal karyotype. (K) Clustering analysis (K-means) of RNA-Seq data. Clustering was performed according to k-means on differentially expressed (>two-fold) genes. (L) RNA-Seq expression analysis, represented on a scatter plot, of two maEpiSC biological replicates, maEpiSCs and conventional EpiSCs (E3, top middle), ES cells (top right), MEFs (bottom right), and differentiated ES cells (embryoid body (EB) day 14; bottom left). Differentially expressed genes (Log2 adjusted) are shown (RPKM > 1, FC > two-fold). (M) UCSC browser views of RNA-Seq data.
