(A) Derivation of mouse Col2.3GFP ESC line. (a) The isolated blastocyst on feeder cells. (b) Day 2 at passage 0 with blastocyst outgrowths. (c) Day 5 at passage 0, with formation of a stem cell cluster ready for passage. (d) Day 2 at passage 1. (e) Representative image of mouse Col2.3GFP ESC line maintained on feeder cells. (f) Representative image of mouse Col2.3GFP ESC line maintained in feeder-free and serum-free conditions. Scale bar: 200 µm. (B) A representative chromosome spread of mouse Col2.3GFP ESC line (karyotype 40, XX). (C) Immunofluorescence staining for pluripotency markers Oct4 and SSEA1. Nuclei were stained with DAPI. Scale bars: 400 µm. (D) Mouse Col2.3GFP ESCs differentiated into three germ layers cells in vitro. qRT-PCR analysis with lineage specific markers in undifferentiated mouse Col2.3GFP ESCs and differentiated Col2.3GFP ESC embryonic bodies. *, p<.05; **, p<.005 (relative to ESC). (E) Hematoxylin-eosin stained sections of teratoma formation with endoderm (respiratory), mesoderm (cartilage) and ectoderm (neural rosettes) tissues. Scale bar: 200 µm.