Induction of feline fetal fibroblasts into pluripotent stem cells using cat-derived reprogramming factors

Abstract

There are few studies on the establishment of induced pluripotent stem cells (iPSCs) in cats. Although induction using heterologous reprogramming factors delivered via viral vectors has been reported, its safety and reprogramming efficiency still require improvement. In addition, the reprogramming mechanism needs further elucidation. In this study, we constructed a series of expression vectors for cat-derived reprogramming transcription factors based on the piggyBac transposon system and transfected various factor combinations into cat fetal fibroblasts (CFFs) under different electroporation conditions to generate cat iPSCs (ciPSCs). Additionally, the specific roles of these factors in reprogramming were investigated. The results showed that under the optimized electroporation conditions (DMEM/F12 buffer, 300 V, 10 ms pulse duration, 2 pulses, 25 μg plasmid DNA, and 4 mm cuvette), the survival rate and transfection efficiency of CFFs reached 64 % and 67.8 %, respectively. Based on this condition, a seven-factor combination (cOSKM + pNL + SV40 Large T) was confirmed as a better inducer for establishing ciPSCs. The obtained ciPSCs exhibit good pluripotency and passaging stability. They express stemness-related genes and proteins, and can form embryoid bodies (EBs) capable of differentiating into all three germ layers. OCT4 (O), SOX2 (S), KLF4 (K), and c-MYC (M) play important cooperative and synergistic roles in the mesenchymal-to-epithelial transition (MET) during the initial stages of reprogramming, while the supplement of NANOG (N) and LIN28 (L) can further promote MET and is important for successful reprogramming. It lays a foundation for the further breeding of cloned and genetically modified cats, and provides a tool for studying embryonic developmental diseases, screening drugs, and applying to tissue regeneration.