Our data suggest that the two main properties of stemness, pluripotency and self-renewal, are differentially regulated in PGC reprogramming induced by hypoxia.

Hypoxia is defined as a reduction in oxygen supply to a tissue below physiological levels. However, physiological hypoxic conditions occur during early embryonic development; and in adult organisms, many cells such as bone marrow stem cells are located within hypoxic niches. Thus, certain processes take place in hypoxia, and recent studies highlight the relevance of hypoxia in stem cell cancer physiology. Cellular response to hypoxia depends on hypoxia-inducible factors (HIFs), which are stabilized under low oxygen conditions. In a hypoxic context, various inducible HIF alpha subunits are able to form dimers with constant beta subunits and bind the hypoxia response elements (HRE) in the genome, acting as transcription factors, inducing a wide variety of gene expression. Typically, the HIF pathway has been shown to enhance vascular endothelial growth factor (VEGF) expression, which would be responsible for angiogenesis and, therefore, re-oxygenation of the hypoxic sites. Embryonic stem cells inhibit a severely hypoxic environment, which dictates their glycolytic metabolism, whereas differentiated cells shift toward the more efficient aerobic respiration for their metabolic demands. Accordingly, low oxygen tension levels have been reported to enhance induced pluripotent stem cell (iPS) generation. HIFs have also been shown to enhance pluripotency-related gene expression, including Oct4 (Octamer-binding transcription factor 4), Nanog and Wnt. Therefore, cell metabolism might play a role in stemness maintenance, proliferation and cell reprogramming. Moreover, in the hypoxic microenvironment of cancer cells, metabolism shifts from oxidative phosphorylation to anaerobic glycolysis, a process known as the Warburg effect, which is involved in cancer progression and malignancy.

Summary Aging causes stem cell dysfunction as a result of extrinsic and intrinsic changes. Decreased function of the stem cell niche is an important contributor to this dysfunction. We use the Drosophila testis to investigate what factors maintain niche cells. The testis niche comprises quiescent “hub” cells and supports two mitotic stem cell pools: germline stem cells and somatic cyst stem cells (CySCs). We identify the cell-cycle-responsive Dp/E2f1 transcription factor as a crucial non-autonomous regulator required in CySCs to maintain hub cell quiescence. Dp/E2f1 inhibits local Activin ligands through production of the Activin antagonist Follistatin (Fs). Inactivation of Dp/E2f1 or Fs in CySCs or promoting Activin receptor signaling in hub cells causes transdifferentiation of hub cells into fully functional CySCs. This Activin-dependent communication between CySCs and hub regulates the physiological decay of the niche with age and demonstrates that hub cell quiescence results from signals from surrounding stem cells.

Cellular reprogramming is accompanied by a metabolic shift from oxidative phosphorylation (OXPHOS) toward glycolysis. Previous results from our laboratory showed that hypoxia alone is able to reprogram primordial germ cells (PGCs) into pluripotency and that this action is mediated by hypoxia-inducible factor 1 (HIF1). As HIF1 exerts a myriad of actions by upregulating several hundred genes, to ascertain whether the metabolic switch toward glycolysis is solely responsible for reprogramming, PGCs were cultured in the presence of a pyruvate kinase M2 isoform (PKM2) activator, or glycolysis was promoted by manipulating PPARγ. Conversely, OXPHOS was stimulated by inhibiting PDK1 activity in normoxic or in hypoxic conditions. Inhibition or promotion of autophagy and reactive oxygen species (ROS) production was performed to ascertain their role in cell reprogramming. Our results show that a metabolic shift toward glycolysis, autophagy, and mitochondrial inactivation and an early rise in ROS levels are necessary for PGC reprogramming. All of these processes are governed by HIF1/HIF2 balance and strict intermediate Oct4 levels. Histone acetylation plays a role in reprogramming and is observed under all reprogramming conditions. The pluripotent cells thus generated were unable to self-renew, probably due to insufficient Blimp1 downregulation and a lack of Klf4 and cMyc expression.

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