Putting mammalian early embryonic cells into dormancy

Mammalian development starts at fertilization and continually progresses until birth, except in cases in which an interruption is favorable to the embryo and the mother. Many mammals have the ability to pause development in case of suboptimal resources or routinely as part of their reproductive cycle-a phenomenon called 'embryonic diapause'. Diapause can be mimicked in vivo in mice via surgical removal of the ovaries or hormone injections. This procedure is laborious and invasive, ruling out its use across species. We have developed in vitro protocols through which mouse blastocysts, human blastoids and pluripotent stem cells from both species can be induced to enter a diapause-like dormant state via pharmacological inhibition of mTOR. Here, we describe in detail how embryos, blastoids and stem cells can be transitioned into and out of dormancy under different culture conditions. We further explain critical parameters to ensure success and propose experimental readouts. These in vitro embryonic dormancy setups can be used to uncover molecular mechanisms of dormancy, to test environmental or pharmacological effectors and to further innovate culture systems for species in which in vitro reproductive technologies are limited. We anticipate that researchers with ~1 year of embryo- and stem cell-handling experience should be able to achieve consistent results and evaluate outcomes. Altogether, inducing dormancy in vitro offers the possibility to slow down embryonic development for exploratory investigations of molecular mechanisms and eventually to expand the time window before implantation for clinical assays.