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Abstract

Pluripotency in the early embryo is defined as the capacity of a single cell to generate all lineages of the adult organism. In vivo this property is possessed only transiently by the cells of the epiblast, but it can be indefinitely “captured” in vitro by deriving embryonic stem (ES) cells from the inner cell mass of the blastocyst. A second property that characterizes ES cells is self-renewal, the capacity to generate more stem cells. Self-renewal requires the coordination of cell proliferation and cell-fate choice (Orford and Scadden, 2008). Originally, mouse ES cells were expanded on mitotically inactivated fibroblasts, in a culture medium containing fetal bovine serum and the leukemia inhibitory factor (LIF). Under these conditions ES cells are metastable, since they show heterogeneity both in morphology and expression of the core pluripotency factors Oct4 and Nanog due to differentiation signals coming from the serum components (Nichols and Smith, 2009). In serum + LIF medium, ES cells depend on the transcription factor and proto‐oncogene Myc for pluripotency and self‐renewal (Smith et al., 2010). Myc proteins have been shown to repress the expression of the primitive endoderm master regulator Gata6, and control the cell cycle by regulating the mir-17-92 miRNA cluster. The laboratory of Austin Smith has recently shown that the culture in the absence of serum but in the presence of two inhibitors (2i + LIF) of the Erk and glycogen synthase kinase-3 (GSK3) pathways is sufficient to stabilize ES cells in a more naïve, so-called ground state of pluripotency, which more closely resembles the status of the inner cell mass of the blastocyst (Ying et al., 2008). Since different culture environments impose distinctive transcriptional and epigenetic properties on mouse ES cells (Marks et al., 2012), we re-evaluated the expression and requirement of the Myc proteins in the naïve state of pluripotency. Making use of ES cells expressing a Myc-GFP reporter from the endogenous c-myc locus, we found that c-Myc protein expression is significantly lower in naïve ES cells compared to cells cultured in serum + LIF. When both c- and N-myc genes are conditionally deleted (Myc dKO) using the Cre-loxP system, naïve mouse ES cells undergo cell cycle arrest. Although this type of cell cycle behavior with a decreased rate of cell division is often associated with differentiation, Myc dKO ES cells form smaller but undifferentiated colonies. Most surprisingly, Myc dKO ES cells maintain the expression of the core stem cell factor network including Oct4, Nanog and Sox2 both at the RNA and the protein level. To determine the molecular properties of Myc dKO ES cells, we performed RNA‐seq analysis 24 and 96 hours after Cre induction. Our results show that Myc is not exclusively regulating the cell cycle machinery but is directly and indirectly involved in multiple aspects of ES cell metabolism. In the absence of both Myc proteins, expression of genes controlling and executing ribosomal biogenesis as well as protein and DNA synthesis is strongly down-regulated, leading to a state of “metabolic dormancy”. The state of “metabolic dormancy” of ES cells in vitro resembled the state of embryonic diapause in vivo. Diapause is a poorly understood phenomenon of reversible arrest of embryonic development prior to implantation. In mice, facultative diapause occurs to delay implantation of newly formed embryos when the mother is feeding a previous litter. When we compared our data to reported global gene expression profiling of diapause embryos (Hamatani et al., 2004), we observed that dKO Myc ES cells possess surprising similarity with the dormant blastocyst, characterized by reduced DNA synthesis and cell division, low metabolic activity and activation of the insulin pathway (Given, 1988; Hamatani et al., 2004). These data suggest that, in the absence of differentiation cues, c-Myc and N-Myc control the cell cycle of ES cells and the transcriptional networks responsible for the entire metabolism and biosynthesis pathways while the pluripotency network is controlled by other means. Loss of Myc activity has dramatic consequences on the metabolic status of ES cells promoting their entry into a state of likely reversible dormancy closely resembling arrested diapause embryos.