Clonal isolation of an intermediate pluripotent stem cell state

Pluripotent stem cells of different embryonic origin respond to distinct signaling pathways. Embryonic stem cells (ESCs), which are derived from the inner cell mass of preimplantation embryos, are dependent on LIF-Stat3 signaling, while epiblast stem cells (EpiSCs), which are established from postimplantation embryos, require acti-vin-Smad2/3 signaling. Recent studies have revealed heterogeneity of ESCs and the presence of intermediate pluripotent stem cell populations, whose responsiveness to growth factors, gene expression patterns, and associated chromatic signatures are compatible to a state in between ESCs and EpiSCs. However, it remains unknown whether such cell populations represent a stable entity at single-cell level. Here, we describe the identification of clonal stem cells from mouse ESCs with global gene expression profiles representing such a state. These pluripotent stem cells display dual responsiveness to LIF-Stat3 and activin-Smad2/3 at single-cell level and thus named as intermediate epiblast stem cells (IESCs). Furthermore, these cells show accelerated temporal gene expression kinetics during embryoid body differentiation in vitro consistent with a more advanced differentiation stage than that of ESCs. The successful isolation of IESCs supports the notion that traverse from naïve ground state toward lineage commitment occurs gradually in which transition milestones can be captured as clonogenic entity. Our finding provides a new model to better understand the multiple pluripotent states.