Intracellular Ca2+ Homeostasis and Nuclear Export Mediate Exit from Naive Pluripotency

• Published in: Cell stem cell 2019-08, Vol.25 (2), p.210-224.e6
• Main Authors: MacDougall, Matthew S., Clarke, Ryan, Merrill, Bradley J.
• Format: Article
• Language: English
• Subjects: Atp2b1; calcitonin; calcium; CRISPR; embryonic stem cell; nuclear export; pluripotency; Ranbp3; self-renewal; Tcf7l1
• ISSN: 1934-5909; 1875-9777
• DOI: 10.1016/j.stem.2019.04.015

Progression through states of pluripotency is required for cells in early mammalian embryos to transition away from heightened self-renewal and toward competency for lineage specification. Here, we use a CRISPR mutagenesis screen in mouse embryonic stem cells (ESCs) to identify unexpected roles for nuclear export and intracellular Ca2+ homeostasis during the exit out of the naive state of pluripotency. Mutation of a plasma membrane Ca2+ pump encoded by Atp2b1 increased intracellular Ca2+ such that it overcame effects of intracellular Ca2+ reduction, which is required for naive exit. Persistent self-renewal of ESCs was supported both in Atp2b1−/−Tcf7l1−/− double-knockout ESCs passaged in defined media alone (no LIF or inhibitors) and in wild-type cells passaged in media containing only calcitonin and a GSK3 inhibitor. These new findings suggest a central role for intracellular Ca2+ in safeguarding naive pluripotency. [Display omitted] •A CRISPR screen identifies Ranbp3 and Atp2b1 as regulators of naive pluripotency•Nuclear export of β-catenin and phospo-Stat3 is reduced in Ranbp3−/− cells•Atp2b1−/−Tcf7l1−/− double-knockout ESCs sustain self-renewal in N2B27 alone•Wild-type ESCs self-renew in N2B27 with the combination of calcitonin and GSK3i MacDougall et al. identify Ranbp3 and Atp2b1 as regulators of naive pluripotency exit. Intracellular Ca2+ levels decrease during exit, and increasing Ca2+ levels in combination with GSK3i or Tcf7l1−/− sustains self-renewal and pluripotency in the absence of inhibitors or LIF.