A cytokine screen using CRISPR-Cas9 knock-in reporter pig iPS cells reveals that Activin A regulates NANOG

NANOG functions as the gateway for the generation of pluripotent stem cells (PSCs) in mice and humans. NANOG protein is highly expressed in pig pre-implantation embryos, indicating NANOG is a conserved pluripotency-associated factor. However, pig NANOG reporter PSCs have yet to be established, and the regulation of pluripotency by NANOG is not completely understood in this animal. In this study, pig NANOG tdTomato knock-in reporter PSCs were established using CRISPR/Cas9. The resulting cell line was treated with several cytokines and inhibitors to identify the key pathway that regulates NANOG expression and the development of pluripotency. In order to determine how NANOG expression affects RNA expression, we performed high-throughput RNA sequencing on knock-in and WT cells and then identified genes that were differentially expressed. Nanog knock-in reporter PC-iPS, WT PC-iPS and pig pre-implantation embryos were analysed together for determine the pluripotency of Nanog PC-iPS; We also analysed the Activin A treatment groups (Act) and SB 431542 treatment groups (SB), three replicates per group.