Abstract
Porcine induced pluripotent stem cells (piPSCs) generated via standard reprogramming factors typically remain dependent on exogenous transgene expression to maintain pluripotency and exhibit limited differentiation potential. Improving these limitations is essential for both regenerative medicine and agricultural biotechnology. To enhance the quality of piPSCs, we introduced an episomal plasmid encoding TBX3 in addition to the standard reprogramming factors with transgene‐free piPSC lines established via WNT pathway inhibition. Furthermore, we developed optimized differentiation protocols to efficiently generate smooth muscle cells (SMCs), endothelial cells (ECs), hepatocyte‐like cells (HLCs), and cardiomyocytes from piPSCs. TBX3‐reprogrammed piPSCs (TBX3‐piPSC) expressed both SSEA‐1 and SSEA‐4 surface markers, in contrast to standard piPSCs, which expressed only SSEA‐1. TBX3‐piPSC rapidly silenced exogenous reprogramming factors and formed teratomas containing well‐differentiated tissues from all three germ layers. In contrast, standard piPSCs formed poorly differentiated teratomas composed mainly of mesenchymal and neuroectodermal tissues, consistent with previous reports of transgene‐dependent piPSCs. TBX3‐piPSC also formed embryoid bodies that demonstrated broader differentiation capacity. Under directed differentiation conditions, TBX3‐piPSC efficiently gave rise to SMCs, ECs, HLCs, and beating cardiomyocytes. Thus, we conclude that the addition of TBX3 as a reprogramming factor promotes the generation of transgene‐free piPSCs with enhanced differentiation potential. These findings provide a significant advancement in porcine stem cell biology and expand the potential applications of piPSCs.