earticle

영어

Canine induced pluripotent stem cells (ciPSCs) have great potential for veterinary regenerative medicine. To generate integration-free iPSCs from aged dog’s fibroblasts, canine adult fibroblasts (CAFs) were reprogrammed using an RNA-based strategy involving the non-integrating, self-replicating Venezuelan equine encephalitis (VEE) virus. Using this RNA system, CAFs were co-transfected with the T7-VEE-OKS-iG RNA replicon plus B18R mRNA for 4 h. Subsequently, the transfection medium was replaced with Advanced-Dulbecco’s modified Eagle’s medium (Advanced-DMEM) containing 10% fetal bovine serum and 200 ng/mL of the B18R protein. One day after the final transfection, cells were grown in stage 1 medium and selected with puromycin (0.5 μg/mL) until Day 10. On Day 11, the stage 1 medium was changed to mouse embryonic fibroblast- conditioned medium containing 20% KnockOut Serum Replacement (KSR) medium. Putative ciPSC colonies first appeared between Days 15 and 25. Interestingly, two distinct types of ciPSC colonies were identified, which showed TRA-1-60 expression or alkaline phosphatase activity. Furthermore, analysis of the gene-expression levels suggested that these putative ciPSCs were in a transition state. Although further studies will be needed, this study will contribute to the development of cell-based therapeutics for aged dogs.