Abstract
Epiblast stem cells (EpiSCs) are primed pluripotent stem cells and can be derived from postimplantation mouse embryos. We now show that the absence of canonical Wnt/β-catenin signaling is essential for maintenance of the undifferentiated state in mouse EpiSCs and in the epiblast of mouse embryos. Attenuation of Wnt signaling with the small-molecule inhibitor XAV939 or deletion of the β-catenin gene blocked spontaneous differentiation of EpiSCs toward mesoderm and enhanced the expression of pluripotency factor genes, allowing propagation of EpiSCs as a homogeneous population. EpiSCs were efficiently established and propagated from single epiblast cells in the presence of both XAV939 and the Rho kinase (ROCK) inhibitor Y27632. Cell transplantation revealed that EpiSCs were able to contribute to primordial germ cells and descendants of all three germ layers in a host embryo, suggesting that they maintained pluripotency, even after prolonged culture with XAV939. Such an improvement in the homogeneity of pluripotency achieved with the use of a Wnt inhibitor should prove advantageous for manipulation of primed pluripotent stem cells.
Highlights
The canonical Wnt/b-catenin signaling pathway plays pivotal roles in early embryogenesis and in stem cell homeostasis and tumorigenesis [1]
The canonical Wnt/b-catenin signaling pathway plays a major role in maintenance of pluripotent mouse and human embryonic stem cells (ESCs) [5], [6], but it promotes the differentiation of human ESCs toward mesoderm [13], [14]
To investigate the role of canonical Wnt/bcatenin signaling in primed mouse epiblast stem cells (EpiSCs), which closely resemble human ESCs, we first examined the effects of activating such signaling in these cells
Summary
The canonical Wnt/b-catenin signaling pathway plays pivotal roles in early embryogenesis and in stem cell homeostasis and tumorigenesis [1]. Mouse epiblast stem cells (EpiSCs), which are derived from the epiblast at embryonic day (E) 5.5 to E7.5, exhibit features of pluripotency and require Nodal-Activin and fibroblast growth factor (Fgf) signaling for maintenance of this characteristic. EpiSCs have little or no ability to give rise to chimeras when injected into blastocysts, suggesting that they are in a state of primed pluripotency, which represents a developmental state later than that of naıve mouse ESCs. A recent study showed that EpiSCs that express the E-cadherin gene (Cdh1) were able to contribute to embryos after blastocyst injection if they had been treated with the Rho kinase (ROCK) inhibitor Y27632 [9]. Similar heterogeneity is evident in human ESCs, with different levels of canonical Wnt signaling possibly underlying variability in differentiation competence [12], [13]
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