Asian Research Thesis Index

Abstract

Embryonic stem cells are the pluripotent cells that act as a root of embryonic development to engender the specialized cells of the body. This property can be captured in vitro to cultivate indefinitely, providing a remarkable model to study early development and diseases. In vitro, mouse pluripotent embryonic stem cells (mESCs) exist in two different states, naïve and primed. The naïve state mimics in vivo inner cell mass of blastocyst with the pluripotency genes expressed more uniformly and have more developmental potential than the primed state. The primed state corresponds to a later embryonic developmental stage than the naïve state. Both states of cells express similar level of some key pluripotency genes but the signaling pathways that support their self-renewal and pluripotency are strikingly different. Mitochondrial metabolism, among others, is a major difference between the two states. The primed state has more mature mitochondria but doesn’t use them for ATP generation, which is generated solely through glycolysis. The naïve state however, has a bivalent metabolic state, i.e. uses both glycolysis and mitochondria to generate ATP. What role mitochondria play in these two states remains a very interesting and relevant question. Here in this study, we investigated the function of mitochondrial respiration in naïve mESCs by blocking the electron transport chain (ETC) with specific pharmacological inhibitors, Rotenone and Antimycin, and genetically with shRNAs against mitochondrial specific transcription factor A (TFAM) and a helicase, Twinkle. Both inhibitors and shRNAs resulted in blockade of cell proliferation and when released, the cells resumed self-renewal without affecting the pluripotency. The paused phenotype by ETC inhibition also appeared in the blastocysts cultured in vitro, which naturally die within 2-3 days of the development while Rotenone treatment extends survival of blastocysts for 3 more days. Moreover, the treated blastocysts were able to give live pups when injected back into surrogate mother, confirming a normal developmental potential. Upon ETC inhibition with either compounds or shTFAM, the mitochondrial mass and membrane potential increased and level of reactive oxygen species (ROS) decreased. The shRNAs against TFAM and Twinkle greatly reduced the mitochondrial DNA (mtDNA) copy number and expression of mtDNA encoded mRNA, leading to complete blockade of mitochondrial respiration. Paused mESCs stopped oxygen consumption and enhanced glycolysis instead to maintain a steady level of ATP generation, which is essential for the cells to be alive. Mechanistically, ETC inhibition induced pause is distinct from the pause induced through either mTOR or Myc inhibition. Neither pyruvate, aspartate nor nucleosides supplementation could rescue the self-renewal of mESCs which were shown to have the ability in other cell types upon ETC inhibition. The total non-targeted metabolomics showed that the ETC regulates the carbohydrates, proteins, lipid and nucleic acid metabolism. Specifically, lysosomal-related pathways are found to be significantly and commonly changed from both metabolomics and proteomic analyses. We could confirm the blockade of autophagy and lysosomal pathways in ETC inhibition-induced paused cells. These analyses suggest that an intimate link exists between mitochondria and lysosome in mESCs and highlights the potential important function of lysosomes for mESCs self-renewal. This work reported a new type of pause by ETC inhibition in mESCs and identified some unique metabolic roles of mitochondrial ETC for mESCs self-renewal. Furthermore this study first time shows the effect of Ursolic acid on mouse embryonic stem cells.

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