Induction Of Bovine Pluripotent Stem Cells With Defined Factors

Induced pluripotent stem cell (iPSC)by defined factors is of great importance to producing transgenic animals and studying the reprogramming mechanism of cell. So, in this study, we attempted to derive induced pluripotent cells from bovine fetal skin fibroblast cells (bFFC) with four defined factors.Four lentivirus plasmids carried pSox2, hOct4, pMyc and pKlf4 gene respectively were transfected into 293T cells, and the produced lentivirus that could generate the defined factors were colleced and mixed, then the mixture was added into cell culture medium to infect bFFCs. We continued to observe the cell morphology and GFP protein expression. Once the shape of fibroblasts cells began to change, they would be seeded onto mouse embryonic fibroblast feeders. After several passages, clonies with clear-edege were selected and identified. Results as follow:After infected with lentivirus for 15 days, some bFF cells began to exhibit a progressive morphological change from spindle-shaped to globular. When seeded onto feeder, some flat colonies with cutting edege formed, Clonies cells had big caryon and exhibited a high nucleus/cytoplasm ratio. After a dozen passages, the intensity of green fluorescence gradually decreased in those clonies cells, and the number of clonies which express green fluorescent protein gradually decreased.Most clonies cells exhibited normal karyotype, and were positive for alkaline phosphatase test. When detected the mRNA by RT-PCR, all selected clonies were positive for Oct4, Nanog, c-Myc and Klf4 but negtive for Sox2. By contrast, bFF cells were negative for all these genes mRNA detection. As a positive control, the mouse embryonic stem cells transcripted all these five pluripotent-related genes mRNA. Immunocytoche- mical analysis showed that Oct4 and Nanog protein could be expressed in those induced clonies cells, but the other pluripotency markers ( Sox2,SSEA-1,SSEA-3,SSEA-4,TRA-1-60 and TRA-1-80 ) could not be expressed. When suspended and cultured in ordinary culture medium, some embryoid bodies formed 10 days later, and these embryoid bodies were apt to aggregation. When injected the induced clonies cells bovine iPS cells into the dorsal skin of nude mice, a lot of teratomas containning tissues from ectoderm, mesoderm, and endoderm formed 6 weeks later. The teratoma was irregular spherical shape, soft, strong and aggressive. The surface of teratomas distribute many blood vessels which iconnected with nude mice subcutaneous tissue.Conclusion: We successfully induced the bovine iPS cells from fetal bovine primitive fibroblast cell by four defined factors pSox2, hOct4, pMyc and pKlf4, and founded the bovine iPS cell line.