Marker analysis was carried out on RF ESC line at passages 10, 49, and 117, on RP01 at passages 25, and 58, on RP02 at passage 27, and on RP03 at passage 30. All lines shared similar marker expression, including alkaline phosphatase activity (Fig. 2A, 2I), OCT-4 (Fig. 2B, 2J), SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 (Fig. 2D–2G). SSEA-1, which is present in mouse but not primate ESCs, was also detected (Fig. 2C) [22, –24]. Karyotype analysis was performed on RF at passages 25, 47, and 102, RP01 at passage 46, RP02 at passage 39, and RP03 at passage 35. All expressed normal 44XY or 44XX karyotypes (Fig. 2H, 2P).
Figure Figure 2.. The characteristics of undifferentiated rabbit RF (A–H) and RP01 (I–P) embryonic stem cells (ESCs). (A, I): Alkaline phosphatase activity. Shown is immunostaining for (B, J) Oct-4, (C, K) stage-specific embryonic antigen (SSEA)-1, (D, L) SSEA-3, (E, M) SSEA-4, (F, N) tumor-related antigen (TRA)-1-60, and (G, O) TRA-1-81. Normal 44XY and 44XX, G-banded karyotypes for rabbit RF (H) and RP (P) ESCs, respectively. Cell nuclei were counterstained with Hoechst 33342 (blue). Similar results were obtained for RP02 and RP03 ESCs. Scale bars = 100 μm (B, J), 50 (A, C–G, I, K–O).
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All rabbit ESC lines expressed the pluripotency genes OCT-4, NANOG, SOX-2, and UTF-1 (Table 3; supplemental online Table S1A) [22, 25, , –28]. OCT-4 protein expression was also confirmed in these cell lines with immunostaining (Fig. 2B, 2J). Signal transduction involving the fibroblast growth factor (FGF), transforming growth factor-β (TGF-β)/bone morphogenetic protein (BMP), and WNT pathways has been implicated in the maintenance of ESC pluripotency [29, , , , , , –36]. Therefore, we compared the transcriptional status of these major signaling pathways in rabbit, human, and mouse ESCs. The ESCs from all three species expressed FGF signaling pathway genes, including FGF1, FGF2, four FGF receptors (FGFR1, FGFR2, FGFR3, and FGFR4), and the components of downstream activation cascades (SOS1 and PTPN11) (Table 3; supplemental online Table S1B). WNT signaling pathway genes, the coreceptor LRP6, the key signal transducer β-catenin, and inhibitors Dkk1 and the transmembrane protein Kremen1 were expressed in all three species, whereas WNT3A was not detected in any of these species [37, 38]. However, inhibitors Dkk2 and Gsk3-β were not expressed in the rabbit ESCs but were expressed in human and mouse ESCs, and ligands WNT1, WNT2, WNT4, and WNT5A were expressed in rabbit ESCs, whereas human and mouse ESCs expressed only WNT4 (Table 3; supplemental online Table S1C). In TGF signaling, ligand genes (TGF-β1 and BMP4), LeftyA, as an inhibitor of Nodal, and the important regulator factors SMAD 1, 2, and 4 were detected in all three species, but ligand gene Nodal was not detected in rabbit ESCs but did express in human and mouse ESCs (Table 3; supplemental online Table S1D) . On the other hand, expression levels of FGF1, SOS1, and SMAD 1, 2, and 4, and LeftyA were lower in the rabbit ESCs than in human and mouse ESCs (supplemental online data).