Characterization of transgenic ES cell line. Normal morphologic ES clones were visible under the phase-contrast microscope and stained positive for AP (A). Using the handing drop method, the ES cells formed the embryo-like aggregates and outgrowths after plating them onto tissue culture plates (B). Teratoma formation in immunodeficiency mice from injected ES cells. Hematoxylin and eosin staining was performed on the teratomas. The resulted teratomas contained tissues representing all three germ layers: endoderm, gut-like epithelium; mesoderm, cartilage; exoderm, epidermal tissues (C). Immunofluorescence analysis of ES cells for pluripotency markers. The clones express the embryonic markers Oct4 and Sox-2. Nuclei were stained with hoechst 33342. Underlying fibroblasts provide a negative control (D). RT-PCR analysis of ES cell marker genes Oct4, Nanog, Sox-2, and Rex-1; β-actin was used as the loading control. M: DL 2000 DNA Marker (E).