MTORC1 Regulates Self-renewal And Pluripotency Of Mouse Embryonic Stem Cells

Embryonic stem cells(ESCs)are derived from inner cell mass of the early blastocyst stage of embryonic development.They can be infinitely expanded in vitro(self-renewal),and have the potential to differentiate into various somatic cell types(pluripotency).Therefore,ESCs are a good model for in vitro research and development,and also provide important initial materials for cell therapy,in vitro simulation of disease occurrence and drug development.After years of research,the understanding of self-renewal and pluripotency of ESCs has been very rich,but mainly focused on transcriptional regulation and epigenetics,while the understanding of translation and metabolic regulation is still limited.Mouse ESCs(mESCs)are the earliest established ESCs cell lines.On the one hand,mESCs have the ability of rapid proliferation and almost unlimited self-renewal,which is helpful for large-scale expansion and continuous experiments in vitro.On the other hand,the mechanism research of mESCs is in-depth and has complete evaluation criteria(gene expression,epigenetic modification,differentiation and development ability).And mESCs have two widely used stably culture conditions in vitro:serum(serum+LIF,SL)condition and ground state(serum-free medium supplemented with LIF and two inhibitors that block both MEK/ERK and GSK3 pathways,2iL)condition.However,mESCs under the two culture conditions have their own advantages and disadvantages,and there are great differences in transcription,translation,mitochondrial metabolism and epigenetics.MEK/ERK and GSK3 pathways are involved in the regulation of many important functions in cells,and their regulation of self-renewal and pluripotency of mESCs lead to different mechanisms under same interventions between these two culture conditions.Therefore,it is necessary to carry out a comparative study about mESCs self-renewal and pluripotency under 2iL and SL conditions.The mechanistic target of rapamycin(mTOR)is an anabolic master switch that functions by forming two complexes,mTORC1(mTOR complex 1)and mTORC2(mTOR complex 2).Dysregulation of mTORC1(especially excessive activation)directly leads to developmental abnormalities,aging,multiple metabolic diseases and tumors.Recent studies have found that mTORC1 is also involved in the regulation of self-renewal and pluripotency of ESCs.The activity of mTORC1 and mTORC2 in mESCs needs to be precisely maintained at a low level:complete inhibition of mTOR activity using the mTOR kinase inhibitor INK128 will cause mESCs to enter a "pause"state,which is self-renewal arrest while maintaining pluripotency;hyperactivation of mTORC1 impedes the exit of pluripotency.Although the above studies have made many conclusions on the role and regulatory mechanism of mTOR in self-renewal and pluripotency of mESCs,there are still a series of basic and important issues to be further clarified:1,Is there a correlation between mTOR activity and pluripotency in mESCs?2,Which complex of mTOR regulates mESCs self-renewal and what is the mechanism?3,How does hyperactivation of mTORCl affect mESCs pluripotency?For above questions,in this study,we conducted research on self-renewal and pluripotency by inhibiting and activating mTOR through various means under 2iL and SL culture conditions.We have reached the following conclusions:1,The activity of mTOR showed a negative correlation with pluripotency in somatic cells and different states of mESCs,especially the mTOR activity reached the lowest under 2iL culture conditions;2,For self-renewal,under 2iL and SL culture conditions,endogenous lowlevel mTORCl controls the self-renewal of mESCs mainly by eukaryotic translation initiation factor 4F(eIF4F),which controls both cytoplasmic and mitochondrial translation,but under SL culture condition,eIF4F mediates the regulation of pluripotency by MEK/ERK pathway in a mTORC1 independent manner;3,For pluripotency,the hyperactivation of mTORC1 has opposite effects on pluripotency in 2iL and SL mESCs,the hyperactivation of mTORC1 activates mitochondrial metabolism and down-regulates pluripotency through pyruvate dehydrogenase(PDH)in 2iL culture condition,while in SL culture condition,the presence of MEK/ERK pathway blocks such an effect,thereby preventing or even partially reversing the adverse effects of hyperactivation of mTORC1 on pluripotency.Overall,we not only found that mTORC1-mitochondrial metabolic pathway is involved in the regulation of mESCs self-renewal and pluripotency,but also revealed the synergistic effect of mTORC1 and MEK/ERK signaling pathways in translation,mitochondrial metabolism and pluripotency under 2iL and SL culture conditions.These are helpful to understand the new cell biological mechanism of self-renewal and pluripotency maintenance of ESCs,to develop more culture conditions and differentiation methods,to further improve the study of embryonic stem cell fate regulation,and to promote the application of ESCs.In addition,our findings may also have broader implications.At present,adult stem cells or functional cell precursors are almost impossible to achieve long-term stable expansion in vitro like mESCs.How to activate the self-renewal of such cells while maintaining their differentiation potential is of great value and helps to promote the development of regenerative medicine.At the body level,specifically stimulating the maintenance of stem cell homeostasis in vivo,that is,maintaining its good differentiation potential during repeated regeneration of stem cells in vivo,will be of great significance for overcoming diseases and even delaying aging.