ESCs were first discovered in

ESCs were first discovered in 1981 independently by two groups (Martin, 1981; Evans and Kaufmann, 1981) and, while they were initially cultivated in medium containing physiological glucose (Glc) concentrations of 5.5 mM, descriptions of their routine cultivation in 25 mM Glc can be traced back to reports published as early as 1987 (Doetschman et al., 1987). It is unclear why this switch in culture conditions occurred, especially since newer reports suggest that ESC derivation is more successful in physiological Glc by increasing the pool of proliferating more hints (Wang et al., 2006). The mechanism by which normoglycemia supports proliferation is currently unknown, although the same study illustrating that exposure to hyperglycemia leads to an increase in reactive oxygen species (ROS) may provide an important first step in elucidating the connection between hyperglycemia and complications during ESC establishment and growth. The beneficial effect of physiological Glc concentrations on ESC proliferation is in line with in vivo findings from diabetic pregnancies, in which poorly controlled blood Glc levels result in embryos that are small in size, possibly due to a decrease in proliferation mediated by hyperglycemia-induced overactivation of p21 (Zanetti et al., 2001; Varma et al., 2005; Scott-Drechsel et al., 2013). These in vivo data and the newer studies suggesting a beneficial effect of physiological Glc on ESC growth are in contrast to a previous study, in which a high Glc environment resulted in an increase in ESC proliferation (Kim et al., 2006). Molecularly, Glc may increase proliferation of cells through various mechanisms, including increased phosphorylation and inhibition of forkhead box O transcription factors (FOXOs). As FOXO proteins have been suggested to transcriptionally regulate the cell-cycle inhibitory genes p21 and p27 (Dijkers et al., 2000; Hauck et al., 2007), the inhibition of these transcription factors and the consequent inhibition of cell-cycle inhibitors would contribute to extensive cellular proliferation. In line with this notion, Kim et al. (2006) found that hyperglycemia activated the serine/threonine kinase AKT in ESCs, a known upstream regulator of FOXO nuclear exclusion (Dickson et al., 2001). Due to this apparent controversy, we sought to examine exposure of ESCs to high Glc levels for longer than 12 hr, as was done by Kim et al. (2006), to investigate whether this longer exposure would be able to mimic the in vivo effects of Glc on the early embryo and represent the conditions found during ESC derivation. Indeed, in acute exposures (5 days), we found decreased proliferation. We further suggest that a molecular cascade involving oxidative stress, inhibition of AKT, activation of c-jun NH2-terminal kinase (JNK), and transcriptional regulation of p21 and p27 through FOXO1, FOXO3a, and β-catenin (βcat) produces the proliferation inhibition caused by hyperglycemia.
Results
Discussion Molecularly, we suggest FOXO transcription factors as the mediators for the noted reduction in proliferation and the upregulation of ROS-removal enzymes (Figure 6). Previous studies have identified FOXO3a as a key regulator of Sod2 (Kops et al., 2002), p27 (Dijkers et al., 2000), and p21 expression (Hauck et al., 2007). Here, an shRNA-mediated knockdown of FoxO1/3 led to a major decline in FOXO3a binding to these promoters. Interestingly, there was still FOXO3a binding observed on the promoters of these genes, which may be due to the fact that the knockdown was not 100% effective. The combined regulation of p27 and Sod2 by FOXO4 and CTNNB1 has been suggested through promoter reporter studies (Essers et al., 2004), but direct evidence of FOXO/CTNNB1 binding to these promoters through ChIP has so far been lacking; however, such evidence is provided here for Sod2 and also additionally for p21. In addition, we show here that this binding is dependent on Glc-induced ROS levels, which has widespread implications not only for the routine culture of pluripotent stem cells but also for the field of diabetes and aging.