Generation and cell culture of pluripotent cells in the pig model: the search for generation and differential characterization between primed and naïve cells

Abstract

It is known that human ESCs and iPSs require bFGF but not LIF while murine ESCs and iPS require LIF but not bFGF for the maintenance of pluripotency in vitro. The "naïve" stage is represented by the pre-implantation embryo cell mass, exemplified by the murine ESCs, and the primed stage by the post-implantation epiblast and human ESCs (Nichols & Smith 2009; Ware et al., 2014). In iPS of other species, the mechanism of action of pluripotency differentiation / maintenance pathways such as JAK / STAT and MAPK, for example, is not well known. In previous experiments in our laboratory, we observed that the addition of LIF or inhibitors of the MEK and GSK3 pathways (PD0325091 and CHIR99021, or 2i) did not positively influence the production of reprogrammed colonies in cattle or porcine cells (unpublished data). In another recent study in pigs it was reported that the use of such inhibitors did not prove to be beneficial for reprogramming (Petkov et al., 2014). In this proposal the pig model will be used for the production of iPS cells with and without supplementation of bFGF, LIF and 2i, in a multifactorial design. In addition to the reprogramming efficiency, possible post-reprogramming signaling pathways in these conditions will be analyzed through the miRNA profile in search of metabolic pathways that characterize naïve or primed cells in different conditions. The generation and establishment of these cells will enable subsequent experiments with germ cell generation, gametes and analysis of their functionality. (AU)