Porcine induced pluripotent stem cells (piPSCs) had been reported during the past 5 years, but there were few reports on how the cell signaling works in piPSCs. In order to clarify the signaling work that dominated the characteristic difference of two types of piPSCs which were derived from Oct4, Sox2, Klf4 and c-Myc (termed 4F piPSCs) and Oct4, Sox2, Klf4, c-Myc, Tbx3 and Nr5 alpha 2 (termed 6F piPSCs) respectively, we performed this study. 4F piPSCs and 6F piPSCs were cultured in medium with or without the ROCK inhibitor Y27632 after dissociating into single cells, the efficiency of a single cell colony and the number of AP positive colonies were assessed. The total RhoA and GTP-bind RhoA were detected in 4F piPSCs and 6F piPSCs before and after digestion into single cells. To explore the relationship between RHO-ROCK-MLC signaling pathway and the two factors Tbx3 and Nr5 alpha 2, the 4F piPSCs were infected with lenti-virus Tbx3 and Nr5 alpha 2 (termed 4F+TND). Results showed that the viability of cells could be enhanced by Y27632 and the RHO-ROCK-MLC signaling pathway was activated after dissociation into single cells in 4F piPSCs but not in 6F piPSCs. And, the 4F+TND piPSCs could be passaged and keep in high viability after dissociation into single cells, though the morphology of colonies did not change. These results indicated that the Tbx3 and Nr5 alpha 2 can improve the viability of piPSCs after dissociation into single cells by inhibiting the RHO-ROCK-MLC signaling pathway. And this provides useful information for establishing porcine pluripotent cells in future study. (C) 2014 Elsevier Inc. All rights reserved.