Microenvironment-mediated reversion of epiblast stem cells by reactivation of repressed JAK–STAT signaling

Embryonic stem cells (ESC) and epiblast stem cells (EpiSC) are distinct pluripotent stem cell states that require different signaling pathways for their self-renewal. Forward transitions between ESC and EpiSC can be accomplished by changing culture conditions; however reverse transitions between EpiSC and ESC are rare events that require transgene insertion or culture on feeders. We demonstrate that transgene-free reversion of EpiSCs to ESCs can be enhanced by local microenvironmental control and the subsequent reactivation of dormant LIF-STAT3 signaling. Reactivation of LIF responsiveness occurs in regions of colony constraint (high local cell density) typical of culture on feeders, a condition that can be recapitulated using micropatterned (μP) colonies under defined conditions. This increased LIF responsiveness results in a subsequent increase in the frequency of EpiSC reversion. Importantly, the resulting revertant EpiSCs are functionally indistinguishable from naïve mESC. Our findings demonstrate that signaling pathway activation and repression create barriers to cell fate transitions that can be overcome by microenvironmental control.