228 Wnt SIGNALLING PATHWAY ACTIVATION IN CAT EMBRYONIC STEM LIKE-CELLS AND ITS ROLE IN MAINTAINING PLURIPOTENCY

Abstract

Stem cells from domestic animals are important for deriving therapeutic applications, generating models for human diseases, and developing alternative methods for conservation and preservation of endangered species. Cat embryonic stem like-cells (cESC) have been derived from in vivo and in vitro-produced blastocysts (Gómez et al. 2010 Theriogenology 74, 498). Although cESC colonies can be cultured in an undifferentiated state for several passages, they gradually lose their capacity to maintain pluripotency. Therefore, to maintain pluripotency of cat ESC during in vitro culture, it is necessary to develop a better understanding of the mechanisms involved in self-renewal and differentiation, as well as to enhance in vitro culture conditions. In mouse ESC, the Wnt/β-catenin signalling pathway has been identified as an essential pathway for maintenance of pluripotency and avoidance of differentiation (Kirby et al. 2012). Nonetheless, activation of the Wnt signalling and its role in human ESC remains controversial. Wnt activation is mediated by the cytoplasmic protein – Disheveled – that inactivates a multi-protein complex, including glycogen synthase kinase 3 (GSK3β) and inhibits β-catenin degradation. In the present study, we evaluated the role of Wnt/β-catenin signalling in self-renewal and maintenance of an undifferentiated state of cat ESC. Cat ESC were cultured on mitotically inactivated cat embryonic fibroblasts (CEF) in modified-ESC medium (DMEM-F12, 200 mM l-glutamine + 0.14% β-mercaptoethanol, 1.25% nonessential amino acids, 15% knockout replacement serum, 5% fetal bovine serum, 1000 U mL^–1 leukemia inhibitory factor (LIF), and 10 ng mL^–1 basic fibroblast growth factor, bFGF) and supplemented with GSK3β inhibitor -SB216763 (10 μM v. 20 μM v. 0 μM). The concentrations of β-catenin and GSK3β in cat ESC colonies were measured by ELISA, and the effect of GSK3β on cat ESC was measured by their cell size, morphology, expression of pluripotent markers at the mRNA and protein level (POU5F1, NANOG, SOX-2), and their ability to differentiate into ectoderm cell lineage. Our results indicated that GSK3β inhibitor inactivates GSK3β, leading to an increase in total β-catenin in cat ESC. Moreover, colonies cultured in the presence of GSK3β inhibitor showed flattened shape and irregular borders (compared with the dome shape and marked borders in nontreated colonies), and both the concentration and the passage significantly reduced the colony cell size, the expression of POU5F1 and SOX-2 at the mRNA and protein level, and lowered their ability to differentiate into neurogenic-like cells compared with that of colonies cultured without the GSK3β inhibitor. Even though we demonstrated that the Wnt/β-catenin signalling pathway influenced the expression of POU5F1 and SOX-2 of cat ESC, it is not clear why the accumulation of β-catenin did not enhance self-renewal. Further studies are required to evaluate the influence of GSK3β inhibitor and other small molecules on self-renewal of cat ESC cultured without the presence of a feeder cell layer.