230 GENERATION OF PUTATIVE RABBIT EMBRYONIC STEM CELLS

Abstract

The rabbit, as a laboratory animal model, has several advantages in the study of human physiological disorders. In this study, stable putative pluripotent rabbit embryonic stem cells (rESCs) were derived from in vivo-fertilized and in vitro-cultured blastocysts. The rabbit ICMs were obtained by 0.05% trypsin–0.008% EDTA treatment and mechanical separation; the ES-like cell colonies seen several days later. ICM-derived outgrowths which were treated with 5 mg/mL^-1 dispase, followed by 0.05% trypsin–0.008% EDTA, were mechanically disaggregated into small clumps and reseeded on MEFs. The putative ES cell lines maintained expression of pluripotent cells markers and normal XY karyotype for long periods of culture (>1 month). The putative rESCs expressed alkaline phosphatase, transcription factor Oct-4, stage-specific embryonic antigens (SSEA-1, SSEA-3, and SSEA-4), and tumor-related antigens (TRA-1-60 and TRA-1-81). The morphological characteristics of the putative ESCs are closer to those of human ESCs; their high speed of proliferation, however, is closer to that of mouse ESCs. Putative rabbit ESCs were induced to differentiate into many cell types including trophoblast cells, similar to primate ESCs, in vitro, and formed teratomas with derivatives of the 3 major germ layers in vivo when injected into SCID mice. Using RT-PCR measurement, but with some differences in ligands and inhibitors, and comparing with human and mouse ESCs, the putative rabbit ESCs expressed similar genes related to pluripotency (Oct-4, Nanog, SOX2, and UTF-1) and similar genes of FGF, WNT, and TGF signaling pathways related to the proliferation and self-renewal. Our further research work showed that TGF beta and FGF pathways cooperate to maintain pluripotency of rabbit ESCs similar to those of human ES cells.