A Simple and Robust Method for Establishing Homogeneous Mouse Epiblast Stem Cell Lines by Wnt Inhibition

摘要

Introduction

Epiblast stem cells (EpiSCs) are pluripotent stem cells (PSCs) derived from the epiblasts of early postimplantation mouse embryos (). EpiSCs can proliferate indefinitely in culture and differentiate into derivatives of all three germ layers in vitro and in teratomas. However, EpiSCs possess several different characteristics compared with other PSCs such as mouse embryonic stem cells (mESCs). Whereas mESCs show dome-shaped colonies, EpiSCs show a flatter colony morphology. Other characteristics that differ between EpiSCs and mESCs are the status of X chromosome inactivation in female cells, the culture conditions needed, the expression of several genes/markers (e.g., mESC-specific Pecam1 and EpiSC-specific Fgf5), and their clonogenicity and contribution to chimeras when injected into blastocysts (). Most of these cellular characteristics of EpiSCs are shared by human PSCs such as human ESCs (hESCs) and human induced PSCs (hiPSCs). EpiSCs and human PSCs are considered “primed” PSCs, in contrast to “naïve”-type stem cells such as mESCs (). Therefore, comparisons between EpiSCs and hESCs/hiPSCs should contribute to our understanding of the nature of the primed state and provide insights into the processes underlying the naive-to-primed state transition.One difficulty in deriving EpiSCs is the need for microdissection of small postimplantation embryos. The original protocols used only the epiblast layer separated from the surrounding visceral endoderm (VE) tissue as a source of the cell lines. The overall efficiency of EpiSC derivation from epiblast explants varies from <10% to 40% (; this study). reported that EpiSCs carrying the Oct4-GFP transgene contained both GFP-positive and GFP-negative populations in culture, and that the Oct4-GFP-positive minor population could contribute to the tissues of chimeras constructed by blastocyst injection, whereas the GFP-negative population could not. It is also known that EpiSCs express marker genes for the mesoderm, endoderm, or primitive streak (albeit at low levels and in conjunction with pluripotency markers), and that some of these lineage markers show heterogeneous expression among cells within the same culture (). It has been suggested that this heterogeneous expression of lineage markers might predispose or prime EpiSCs toward particular cell lineages even while the cells remain pluripotent (). Alternatively, the heterogeneous expression could reflect some degree of spontaneous differentiation in cultures of EpiSCs.Wnt signaling has been described as a positive regulator of self-renewal in mESCs (); however, the involvement of the canonical Wnt signaling pathway (i.e., β-catenin-dependent Wnt signaling, as opposed to non-canonical Wnt signaling, which is β-catenin independent) in pluripotency remains controversial (reviewed by ). In fact, β-catenin null mESCs have been generated successfully in naive conditions (). Interestingly, showed that mESCs can be maintained in the ground state when cultured in medium containing leukemia inhibitory factor (LIF) plus two inhibitors (2i) for ERK signaling and GSK3 activity. However, although the GSK3 inhibitor they used was an effective agonist of the Wnt/β-catenin signaling pathway, the maintenance of ground-state mESCs requires dual inhibition. Blocking the secretion of all WNT proteins in ground-state mESCs by deleting the Porcupine gene also proved to be compatible with pluripotency, demonstrating that the WNT protein secretion may not be necessary for pluripotency ().Given that the retention of β-catenin in the cytoplasm has been implicated in the self-renewal of both EpiSC and hESCs (), we reasoned that inhibition of Wnt signaling might favor the growth of primed-state PSCs, i.e., EpiSCs. Here, we devised a simple and robust technique to derive high-quality EpiSCs using the small-molecule Wnt-inhibitor IWP-2, which acts on the protein Porcupine, blocking the secretion of WNT proteins and consequently pharmacologically inhibiting downstream Wnt signaling. Here, we show that the use of IWP-2 allows one to derive EpiSCs from epiblast cells without having to remove the VE, and to stably maintain EpiSCs by blocking the endogenous WNT-mediated spontaneous differentiation that otherwise would arise in culture. The homogeneous properties of the resulting EpiSCs at the cellular level, regarding morphology and expression of lineage markers, should facilitate studies on establishing and maintaining stable culture of cells in the primed pluripotent state, and directing differentiation from that state.