290 GENERATION OF INDUCED PLURIPOTENT STEM CELLS FROM BOVINE FETAL FIBROBLAST CELLS BY DEFINED FACTORS

Abstract

Induced pluripotent stem cells (iPS) have broad potential applications in drug screening, regenerative medicine, and basic biology, and using iPS as starting materials that have identical properties to embryonic stem cell would probably revolutionize the production of gene-targeted livestock. However, iPS in livestock is still lacking except for in pigs. Instructed by Yamanaka’s idea, in the current study we attempted to generate bovine iPS from fetal fibroblast cells (bFF; from a fetus 2.5 months old after gestation) by using 4 defined transcriptional factors: Oct-4, Sox2, Klf4, and c-Myc. A lentivirus haboring the 4 factors acted as a vehicle to transfect the bFF. One bFF cell line was infected for at least 3 replicates. After transfection, the treated bFF were then cultured in DMEM supplemented with 4 ng mL^–1 of basic fibroblast growth factor and 1000 IU mL^–1 of leukemia inhibitory factor at 37.5°C, 5% CO₂, on the mouse embryonic feeder cells pretreatd with mitomycin C. From Day 16 after the onset of induction, morphology of a few typical spindle-like bFF gradually changed into round, ball-like cells and grew into colonies. Afterward, when these colonies were harvested and subcultured in stem cell medium supplemented with 1000 IU mL^–1 of leukemia inhibitory factor and 4 ng mL^–1 of basic fibroblast growth factor, new colonies with clear-cut, round edge emerged, and the cells in the colonies had increased nuclear:cytoplasm ratio, kept normal karyotype up to 10 passages, and were alkaline phosphatase staining positive. We also found that the cells exhibited part of the stem cell markers, as evidenced by being Nanog, SSEA1 positive but SSEA3 and TRA-60 negative. Moreover, embryoid bodies could be formed in vitro, and terotoma formation after injection into nude mice also displayed 3 layers. Taken together, we found that 4 lentivirus-mediated, defined transcriptional factors could successfully induce bFF into iPS-like cells.