title: Inhibition of mTOR induces a paused pluripotent state.
abstract: Cultured pluripotent stem cells are a cornerstone of regenerative medicine owing to their ability to give rise to all cell types of the body. Although pluripotent stem cells can be propagated indefinitely in vitro, pluripotency is paradoxically a transient state in vivo, lasting 2-3 days around the time of blastocyst implantation. The exception to this rule is embryonic diapause, a reversible state of suspended development triggered by unfavourable conditions. Diapause is a physiological reproductive strategy widely employed across the animal kingdom, including in mammals, but its regulation remains poorly understood. Here we report that the partial inhibition of mechanistic target of rapamycin (mTOR), a major nutrient sensor and promoter of growth, induces reversible pausing of mouse blastocyst development and allows their prolonged culture ex vivo. Paused blastocysts remain pluripotent and competent-able to give rise to embryonic stem (ES) cells and live, fertile mice. We show that both naturally diapaused blastocysts in vivo and paused blastocysts ex vivo display pronounced reductions in mTOR activity, translation, histone modifications associated with gene activity and transcription. Pausing can be induced directly in cultured ES cells and sustained for weeks without appreciable cell death or deviations from cell cycle distributions. We show that paused ES cells display a remarkable global suppression of transcription, maintain a gene expression signature of diapaused blastocysts and remain pluripotent. These results uncover a new pluripotent stem cell state corresponding to the epiblast of the diapaused blastocyst and indicate that mTOR regulates developmental timing at the peri-implantation stage. Our findings have implications in the fields of assisted reproduction, regenerative medicine, cancer, metabolic disorders and ageing.
authors.affiliation: Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences and Diabetes Center, University of California, San Francisco, San Francisco, California 94143, USA.
authors.forename: Aydan
authors.initials: A
authors.lastname: Bulut-Karslioglu
authors.affiliation: Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences and Diabetes Center, University of California, San Francisco, San Francisco, California 94143, USA.
authors.forename: Steffen
authors.initials: S
authors.lastname: Biechele
authors.affiliation: Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801, USA.|Departments of Bioengineering and Physics, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801, USA.
authors.forename: Hu
authors.initials: H
authors.lastname: Jin
authors.affiliation: Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences and Diabetes Center, University of California, San Francisco, San Francisco, California 94143, USA.
authors.forename: Trisha A
authors.initials: TA
authors.lastname: Macrae
authors.affiliation: Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801, USA.|Departments of Bioengineering and Physics, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801, USA.
authors.forename: Miroslav
authors.initials: M
authors.lastname: Hejna
authors.affiliation: The Centre for Phenogenomics (TCP), Toronto, Ontario M5T 3H7, Canada.
authors.forename: Marina
authors.initials: M
authors.lastname: Gertsenstein
authors.affiliation: Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801, USA.|Departments of Bioengineering and Physics, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801, USA.
authors.forename: Jun S
authors.initials: JS
authors.lastname: Song
authors.affiliation: Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences and Diabetes Center, University of California, San Francisco, San Francisco, California 94143, USA.
authors.forename: Miguel
authors.initials: M
authors.lastname: Ramalho-Santos
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology*; D002478:Cells, Cultured; D015536:Down-Regulation; D047108:Embryonic Development; D005260:Female; D005855:Germ Layers / Q000166:cytology; D006657:Histones / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology; D039904:Pluripotent Stem Cells / Q000166:cytology*; D014176:Protein Biosynthesis; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D006657:Histones; C546843:mTOR protein, mouse; D058570:TOR Serine-Threonine Kinases