| Embryonic stem cell (ESCs) is a type of pluripotent cell which can be derived from inner cell mass (ICM) of the pre-implantation blastocysts. The first mouse ESCs line was successfully established in1981and has been widely used in gene function study as well as regenerative medicine, since it possesses the capacities of self-renewal and differentiation to all three germ layers somatic cells. The laboratory rat (Rattus norvegicus) is the first animal model in scientific studies and has been served for varieties of researching fields including physiology, pathology and pharmacology in the past150years. Given its physiological similarities to human, rat is currently the most commonly used animal model for many pre-clinical tests, especially those related to cardiovascular disease, diabetes, breast cancer, chronic inflammatory diseases and age-related diseases. Mouse embryonic stem cells have been abundantly derived and studied in the past two dacades. However, relative studies on rat models have been lagged far behind since the lack of authentic rat embryonic stem cell with the capability of germline transmission. In2008, Ying et.al developed a chemically-defined basal culture system that contains N2B27medium supplemented with several small molecule inhibitors (3i:CHIR99021, PD184352and SU5402or2i:CHIR99021and PD0325901)and successfully derived germline competent rat ES cell lines which could be maintained in undifferentiated self-renewing status in long-term in vitro cultures. These rat ES cells can be genetically modified and propagated in vitro, while retain their ability of germline contribution in chimeras, which is ambiguously demonstrated by the generation of TP53gene knockout rats through homologous recombination in the rat ESCs. The development of rat ESCs-based gene targeting technologies bring a bright future for genetic manipulations in rat models.In this research, we derived rat ESCs from pre-implantation blastocysts of Dark Agouti (DA) rats by using N2B27-2i culture system with high efficiency of derivation. The rat ESCs we derived displayed compacted colonly morphologies which showed high similarity to mouse ESCs. Subsequently, RT-PCR, immunostaining and western blot further confirmed the expressions of pluripotency markers such as Oct4, Sox2, Nanog, Fgf4, Rex1, and Eras in rat ESCs. Moreover, chimeric rats have been generated by injection of ESCs derived from DA rat (brown coat) into diploid receptor blastocysts of F344rat (white coat). Finally, the germline transmission has been proved with obtaining of brown coat F2pups through mating adult F1chimeric rats with white coat SD rats. It indicated that the rat ESCs we derived are authentic pluripotent stem cells with capacity of germline contribution.Subsequently, we found that a modified culture medium serum-free N2B27-2i medium supplemented with two additional small molecule inhibitors of ROCK inhibitor Y-27632and TGF-β receptor Alk5inhibitor A-83-01(Termed N2B27-4i medium) can further enhance the proliferation and germline contribution ability of rat ESCs, even after long-term culture. Furthermore, the rat ESCs have been used as donor cells in nuclear transfer. These results suggested that after activation, the reconstructed rat embryos could only develop to2-cell stage cultured in vitro, whereas they possessed the developmental potency to blastocyst cultured in vivo. Nuclear transferred embryonic stem (ntES) cells derived from these reconstructed blastocysts displayed the typical morphology of rat ESCs and developed up to E11.5, but not to full-term.In summary, this is the first case of successful derivation of rat ES cell lines in China. A series of identification in molecular and developmental levels demonstrated rat ESCs we derive are authentic pluripotent stem cells, which contribute to germline. These findings will facilitate the explorations on the study of underlying pathological mechanisms of human diseases and will promote the progress of regenerative medicine by means of generation o frat ESCs-based disease models... |
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