Activin-SMAD signaling is required for maintenance of porcine iPS cell self-renewal through upregulation of NANOG and OCT4 expression

Porcine induced pluripotent stem cells (piPSCs) retain the enormous potential for farm animal reproduction and translational medicine, and have been reported by many laboratories worldwide. Some piPSC lines were bFGF-dependence and showed mouse EpiSC-like morphology; other lines were LIF-dependence and showed mouse ESC-like morphology. Metastable state of piPSC line that required both LIF and bFGF was also reported. Because bona fide pig embryonic stem cells were not available, uncovering piPSC state-specific regulatory circuitries was the most important task. In this study, we explored the function of Activin-SMAD signaling pathway and its downstream activated target genes in piPSCs. Transcriptome analysis showed that genes involved in Activin-SMAD signaling pathway were evidently activated during porcine somatic cell reprogramming, regardless piPSCs were LIF- or bFGF-dependent. Addition of Activin A and overexpression of SMAD2/3 significantly promoted expressions of porcine NANOG and OCT4, whereas inhibition of Activin-SMAD signaling by SB431542 and SMAD7 reduced NANOG and OCT4 expressions, and induced piPSCs differentiation exiting from pluripotent state. Our data demonstrate that activation of Activin-SMAD signaling pathway by addition of Activin A in culture medium is necessary for maintenance of self-renewal in porcine pluripotent stem cells.