FIG. 2.

Cdx2 staining of rat embryonic stem cells and rat blastocysts. (A) RT-PCR of Cdx2. From left to right: (L) 100 bp Ladder, Lane 1: ESC line 2 at passage 6, Lane 2: ESC line 3 at passage 6, Lane 3: ESC Control from Qilong Ying, Lane 4: Positive Control TS #1, Lane 5: Positive Control TS #2 supplied by Dr. Michael Soares and Mohammed Rumi at University of Kansas Medical Center, Lane 6: ESC line 1 at passage 10, Lane 7: Negative Control mouse D3 ESCs obtained from ATCC, Lane 8: Negative Control (water). (B) Semi-quantitative RT-PCR for Cdx2. 18 rat ESC lines derived here and two rat ESC control supplied by Dr. Qilong Ying express Cdx2 gene at levels higher than negative control cells lines: rat adult tissues and rat embryonic fibroblasts (REFs) and rat umbilical cord mesenchymal stromal cells (RUCs). The positive control cells, rat trophoblast stem cells (TS) express Cdx2 at a higher level than all rat ESCs lines except one euploid rat ESC line that was 60% tetraploid. The delta threshold count (delta Ct) was averaged from two technical replicates and normalized using the Ct value of PBGD. The data from all lines is shown in Table 3. (C) Immunocytochemistry for Cdx2 and Nanog. Left panels are DAPI staining of nuclei. Middle panels are immunofluorescence for Cdx2. Right panels are immunofluorescence for Nanog. As seen in the middle panels, while the staining intensity of Cdx2 varies throughout the colonies, it is more intense at the edges of the colony. While Cdx2 staining varies all Nanog staining cells also had some degree of Cdx2 staining. There was consistent overlap of Cdx2 and Nanog staining. (D) Immunocytochemistry for Cdx2 and Nanog in 4.5 days post coitus rat blastocysts. As seen in the bottom left panel, the inner cell mass contains Cdx2 positively stained nuclei (arrow), albeit at lower staining intensity than in trophectoderm cells. Additionally, Cdx2 staining was observed in the cytoplasm of inner cell mass cells (arrowhead). XEN and TS cell pellets were supplied by Drs. Michael Soares and Mohammed Rumi, University of Kansas Medical Center; rat ESC control cell pellet was supplied by Dr. Qilong Ying, University of Southern California (USC). In C the calibration bar is 50 μm; in D the calibration bar is 25 μm. Color images available online at www.liebertonline.com/scd