Stem cells in large animals are an excellent model for cell therapy research and fine resources for producing transgenic animals. However, there are only few reports of stem cells in large animals because of technical differences between species. In this report, we successfully generate bovine induced pluripotent stem cells (iPSC) using 4 human reprogramming factors (Oct4, Sox2, Klf4, and c-myc) under control of PiggyBac transposition vector. Fibroblasts derived from bovine fetuses were transfected using FugeneHD agent. After 21 days, colony-shaped structures on the culture plates were mechanically detached and then seeded on a mouse embryonic fibroblast (MEF) feeder layer pretreated with mitomycin C. The culture medium was DMEM/F12 supplemented with 20% serum replacement, 5 ng mL–1 basic fibroblast growth factor (bFGF), 0.1 mM β-mercaptoethanol, 1% NEAA, and 1% penicillin-streptomycin antibiotics. The iPSC colonies showed alkaline phosphatase activity and expressed several pluripotency markers (Oct4, Sox2, SSEA1, and SSEA4). To confirm differentiation potential, the iPSC were cultured as embryoid bodies and then plated again. βIII-tubulin (ectoderm) and GFAP or α-SMA (mesoderm) were well expressed on the attached cells. The results revealed that the bovine fibroblasts were well inducted to iPSC that had potential of multilineage differentiation. We hope this technology contributes to improving transgenic cattle production.
This study was financially supported by IPET (grant # 109023-05-3-CG000, 111078-03-1-CG000) and the BK21 program for Veterinary Science.
Signalling Through Retinoic Acid Receptors is Required for Reprogramming of Both Mouse Embryonic Fibroblast Cells and Epiblast Stem Cells to Induced Pluripotent Stem Cells