β-catenin perturbations control differentiation programs in mouse embryonic stem cells

Abstract

12The Wnt/β-catenin pathway is involved in development, cancer and embryonic stem 13 cell (ESC) maintenance; its dual role in stem cell self-renewal and differentiation is still 14 controversial. Here, we applied an elegant in vitro system enabling conditional β-15 catenin control in β-catenin null mouse ESCs. We report that moderate induction of 16 exogenous β-catenin enhances epiblast stem cell (EpiSC) derivation in vitro. Using a 17 different genetic model and β-catenin chemical modulation, we derived a new protocol 18 for ESCs to EpiSCs differentiation, based on NDiff227 and the GSK3α/β inhibitor 19Chiron, that is more efficient than standard ActivinA/Fgf2-based protocols. Finally, we 20 report that moderate β-catenin levels favour early stem cell commitment towards 21 mesoderm if the protein is overexpressed only in the 'ground state' of pluripotency 22 conditions, or endoderm if the overexpression is maintained during the differentiation, 23unravelling the controversial role of this signalling pathway at the exit from 24 pluripotency. 25 26 Introduction 27Pluripotent Cells (PCs) are characterized by indefinite proliferative and differentiation 28 potential and their identity is determined by the balance between signals promoting 29 self-renewal and differentiation. The first step for stem cell differentiation is the exit 30 from the pluripotent state, tightly controlled by underlying gene regulatory network 31 dynamics which can drive specific lineage commitment. During murine development 32 in vivo, embryonic stem cells (ESCs), that represent the naïve pluripotent state of the 33 early epiblast 1-3 , convert into the late epiblast and finally in terminally differentiated somatic cells. ESCs can be derived from the pre-implantation epiblast and used to 35 study cell pluripotency and differentiation, providing an excellent in vitro system for 36 understanding signalling pathway interplay in cell fate decision making. 37In serum-based cultures, mouse ESCs (hereafter called ESCs) are heterogeneous for 38 the expression of pluripotency genes 1,2,4-10 , while, when cultured in serum-free media 39 supplemented with inhibitors of MEK1/2 (PD) and GSK3α/β (Chiron) and in presence 40 or not of the Leukaemia Inhibitory Factor-LIF (2i or 2i+LIF) 3 , a uniform self-renewal 41 condition known as 'ground state' of pluripotency is established; it is characterized by 42 homogenous gene expression 11-14 , genome demethylation 15-17 and stable naïve 43 pluripotency 18,19 . Mouse epiblast stem cells (hereafter called EpiSCs) also represent 44 a relevant in vitro model because of their similarities with embryonic stem cells of 45 human origin 20 . EpiSCs, derived from the post-implantation epiblast, are also capable 46 of differentiating in all the germ-layers 20,21 ; however, they differ from ESCs in 47 morphology, clonogenicity, gene expression, epigenome status and, most importantly, 48 ability to contribute to chimaeras 14,[20][21][22] . EpiSCs require ActivinA and the fibroblast 49 growth factor 2 (FGF2) 20,21 for in vitr...