299 Epigenetic Reprogramming of Porcine Fibroblast Cells Induced by Sturgeon's Oocyte Extract

Abstract

The direct conversion of differentiated cells into undifferentiated or pluripotent cells would present scientific and medical benefits because of the potential for customized transplantation therapy. Although somatic cell nuclear transfer is one powerful way to fully reprogram somatic cells into a pluripotent state with the aid of oocyte or egg cytoplasm, the therapeutic applications of this approach have been hindered by technical complications as well as ethical objections. An alternative strategy for epigenetic reprogramming of differentiated cells into pluripotent status is desperately required. We have developed a reversible permeabilization protocol with digitonin to deliver sturgeon oocyte extract to porcine fibroblast cells ex ovo. Porcine fibroblasts were permeabilized by 4 μg mL–1 of digitonin for 2 min at 4°C and then incubated in sturgeon’s oocyte extract for 5 h at 15 to 18°C followed by resealing of the cell membrane. We found that the sturgeon’s oocyte extract induced the reduction of overall levels of tri-methylation at lysine 9 of histone H3 (H3K9Me3), which might be related to preservation of DNA methylation in fully differentiated cells, of porcine fibroblast cells. However, permeabilized porcine fibroblasts after treatment with the extract were increasingly acetylated at lysine 9 on histone 3 (H3K9Ac), which might be associated with expression of pluripotency genes. In addition, the cells treated with the extract showed up-regulation of Oct3/4, Sox2, and Nanog gene expression. When somatic cell nuclear transfer embryos reconstructed by using the treated donor cells were transferred into surrogates, the pregnancy rate was slightly high. These results showed that sturgeon’s oocyte extract can reprogram porcine somatic cells into undifferentiated status. Further work needs to exploit the epigenetic reprogramming of differentiated cells into the undifferentiated state using different species oocyte extract.