Transient inhibition of mTOR in human pluripotent stem cells enables robust formation of mouse-human chimeric embryos

Abstract

It has not been possible to generate naïve human pluripotent stem cells (hPSCs) that substantially contribute to mouse embryos. We found that a brief inhibition of mTOR with Torin1 converted hPSCs from primed to naïve pluripotency. The naïve hPSCs were maintained in the same condition as mouse embryonic stem cells and exhibited high clonogenicity, rapid proliferation, mitochondrial respiration, X chromosome reactivation, DNA hypomethylation, and transcriptomes sharing similarities to those of human blastocysts. When transferred to mouse blastocysts, naïve hPSCs generated 0.1 to 4% human cells, of all three germ layers, including large amounts of enucleated red blood cells, suggesting a marked acceleration of hPSC development in mouse embryos. Torin1 induced nuclear translocation of TFE3; TFE3 with mutated nuclear localization signal blocked the primed-to-naïve conversion. The generation of chimera-competent naïve hPSCs unifies some common features of naïve pluripotency in mammals and may enable applications such as human organ generation in animals.

Interspecies chimeric conditions affect the developmental rate of human pluripotent stem cells

“…Previous reports of successful human cell contributions to chimeric mammalian embryos [ 82 , 85 , 86 ], including a recent report of the highest contribution (4%) of human cells in mouse-human chimeric embryos [ 87 ], could imply that human pluripotent stem cells may be induced to accelerate their developmental rate to match that of their embryonic host species. However, maturation rates of human cells in interspecies chimeras have not been well characterized.…”

MiT/TFE Family of Transcription Factors: An Evolutionary Perspective

“…In addition, embryonic stem cell transition to differentiated states is negatively regulated by TFE3 activation, which is controlled by the FLCN/folliculin-interacting protein (Fnip)1/Fnip2 complex ( Betschinger et al, 2013 ). Furthermore, TFE3 activation is responsible for the conversion of primed human pluripotent stem cells to the naive state when mTOR is transiently inhibited ( Hu et al, 2020 ). These findings suggest a role for TFE3 as a repressor of the genes required for the fate specification of pluripotent embryonic stem cells, a process that is prevented by pro-differentiation signaling pathway regulated by FLCN and a non-canonical RagGTPases mechanism during normal development.…”

Reprogramming: Emerging Strategies to Rejuvenate Aging Cells and Tissues

“…They also display important functional differences, notably in their ability to integrate into other species embryos [ 163 ]. Numerous research projects aim at developing and optimizing cell culture processes to increase the ‘naivety’ of hPSCs to approach that of murine naive cells and to increase their capacity to integrate into blastocysts [ 164 , 165 , 166 ].…”