Unlike the paradigm of hypoxia at O emphasized

Unlike the paradigm of “hypoxia” at 2% O2 emphasized by studies like those of the Fisher lab, we emphasize that “hypoxic” stress for stem ARQ 197 manufacturer occurs at lower levels of O2 at ~0.5% which induce differentiation in the first 2days that favors the first lineage, but fails to support differentiation at 4–7days. Hypoxic stress creates an approximation of the higher stress levels (SAPK activation) for TSC caused by sorbitol or benzopyrene at 20% O2 (Awonuga et al., 2011; Zhong et al., 2010; Xie et al., 2010). Like other stresses at 20% O2, hypoxic stress at 0.5% O2 induces a decrease in multipotency factor and an increase of differentiation factors and induces giant cell formation. Specifically, hypoxia-induced Hand1 mediates differentiation of the first lineage, pTGC. Hand1 is necessary for PL1 expression and endoreduplication during normal differentiation (Martindill & Riley, 2008), and 0.5% O2 induces giant cell formation by day 7 despite FGF4 (Fig. 3D, panel c). O2 at 0.5% induces Hand1 at days 1–2 with or without FGF4 before mitochondria activation and PL1 also increases most at 0.5% O2 at days 1–2. But, PL1 expression is relatively low by day 7 after FGF4 removal and mitochondria activation at higher O2 levels. In addition, PL1 expression is low despite the observed differentiation of nearly all TSCs into giant cells at 0.5% O2. A rapid decrease of multipotency factors and an increase of differentiation factors are induced dominantly by stress with or without FGF4. However, 20% O2 provides for an exit from aerobic glycolysis and an increase in mitochondrial membrane charge (Δψm) that supports the most energy-requiring lineages from 4 to 7days after FGF4 removal. The average differentiation at all O2 levels is equivalent at 4days after FGF4 removal. But, differentiation increases to much higher levels only at 2–20% O2 where free ATP levels approach zero as highest differentiation is reached at day 7. The highest level of Δψm, highest level of differentiation, and lowest ATP suggest that the final increase in differentiation requires the levels of energy supplied by efficient mitochondrial OxPhos, not aerobic glycolysis. It has been suggested that proliferation requires aerobic glycolysis to preserve carbon for proliferation, but cells are confluent at 2–20% O2 by day 4 (Table 1). Lack of cell death or proliferation at day 7 suggests nearly all ATP would support differentiation and that ATP production limits differentiation as free ATP approaches zero. It is only at 20% O2 that total COX protein increases and total PKM2 protein decreases. COX activity (a product of both total COX increase and phosphorylated COX decreases) increases 13 fold with FGF4 removal at 20% O2 but only slightly >1 fold at 0.5% O2 (Table 1). Interestingly, since total PKM2 is maintained at high levels at 0.5% the removal of FGF4-maintained PKM2 phosphorylation leads to a 19 fold decrease in the block of pyruvate formation that completes glycolysis. This would significantly slow proliferation as carbon would not be shunted into anabolic pathways in steps of glycolysis prior to pyruvate formation (Hitosugi et al., 2009; Vander Heiden et al., 2012). It is only after FGF4 removal that tyrosine phosphorylation of COX and PKM2 proteins decreases. This decrease in phosphorylation of COX and PKM2 leads to increased Δψm and efficiency of ATP production, and decreased aerobic glycolysis, respectively. Thus 0.5% O2 cannot support the differentiation initiated by hypoxic stress or added hyperosmolar stress that induces robust differentiation of TSC at 20% O2. A key problem during the hypoxic stress at 0–0.5% O2 is that it induces TSC differentiation in the first 24–48h increase, marked by increases in cell size and PL1 and Plf levels (early lineage markers), but these markers are less than at 20% O2 between days 4–7 due to mitochondrial insufficiency. The tendency is that first lineage markers, which are favored early with hypoxic stress here and by all stresses at 20% O2 as reported here and previously (Awonuga et al., 2011; Rappolee et al., 2012), have a low “ceiling” of differentiation at 0.5% O2 after 7days with or without sorbitol. This suggests that energy or another mitochondrial function is rate-limiting.