FIG. 1.

Generation and characterization of rat embryonic stem cells (ESCs) from transgenic F344 and nontransgenic Dark Agouti (DA) rats. (A, B) Rat ESCs and primitive endoderm-like (XEN-like) cells in a mixed culture. After collapse of the blastocyst, the inner cell mass starts expanding. Phase-bright colonies with ESC morphology are found on or adjacent to XEN-like cells (A). The ESCs are positive for alkaline phosphatase (AP, shown in A) and Oct4 staining (B), and the XEN-like cells are negative for both Oct4 and AP. (C) Both XEN and ESCs can be expanded, frozen/thawed, and maintained in the 2i medium. To enrich ESCs, the colony is manually selected, dissociated, and expanded (see C). (D) RT-PCR characterization of pluripotency transcription factors expression by ESCs. Lanes from left to right: Oct4, Sox2, Nanog, PBGD, and water only (control). Block 1 (left): Positive-control ESCs (supplied by Dr. Qilong Ying, University of Southern California). Block 2: ESCs derived from a β-galactosidase (β-gal) transgenic rat (line 3). At left: Dppa3/Stella expression by ESCs. Lanes labeled by rat ESC lines (lines are listed in Table 1). Lines 2 and 3 were derived using 2i medium with 15% heat-inactivated fetal bovine serum (CHIR99021, GSK-3β inhibitor and PD0325901, MEK inhibitor) and leukemia inhibitory factor (LIF). Line 1 was derived using human basic fibroblast growth factor (bFGF) and LIF and 15% heat-inactivated fetal bovine serum. Lane labeled USC represents positive control rat ESCs from Qilong Ying (University of Southern California). Lane labeled W, water only (control). Lane labeled L is a 100 bp ladder. (E) Oct4 and Nanog immunocytochemical staining. Top: ESCs derived from a F344 β-gal transgenic rat (line 3) at passage 4 stained for Oct4 (green), or Nanog (red). Bottom: Secondary antibody only controls. Panels from left to right: Normarski illumination shows colony size and morphology, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, blue) staining for DNA, immunofluorescence micrograph for Oct4 (green), and at the far right, immunofluorescent staining for Nanog (red). Lower panel shows ESCs stained with secondary antibody only. (F) SSEA1 immunocytochemical staining. ESC derived from a F344 β-gal transgenic rat (line 2) at passage 12 stained for SSEA1 (red). From LEFT to RIGHT: Normarski illumination, DAPI nuclear stain (blue), SSEA1 (red), and at the far right, merged DAPI and SSEA1 staining. In all images, scale bar is 100 μm. PCR, polymerase chain reaction; SSEA1, stage-specific embryonic antigen-1; GSK, glycogen synthase kinase; MEK, mitogen-activated protein kinase; F344, Fischer 344; USC, University of Southern California; 2i, 2 inhibitor. Color images available online at www.liebertonline.com/scd