Stemness of Human Pluripotent Cells: Hypoxia-Like Response Induced by Low Nitric Oxide

Abstract

The optimization of conditions to promote the stemness of pluripotent cells in vitro is instrumental for their use in advanced therapies. We show here that exposure of human iPSCs and human ESCs to low concentrations of the chemical NO donor DETA/NO leads to stabilization of hypoxia-inducible factors (HIF-1α and HIF-2α) under normoxia, with this effect being dependent on diminished Pro 402 hydroxylation and decreased degradation by the proteasome. Moreover, the master genes of pluripotency, NANOG and OCT-4, were upregulated. NO also induces a shift in the metabolic profile of PSCs, with an increased expression of hypoxia response genes in glycolysis. Furthermore, a reduction in the mitochondrial membrane potential with lower oxygen consumption and increased expression of mitochondrial fusion regulators, such as DRP1, was observed. The results reported here indicate that NO mimics hypoxia response in human PSCs and enhances their stemness properties when cultured under normoxic conditions.

The Role of Nitric Oxide in Stem Cell Biology

“…In this line, for the first time, our group studied the effect of low NO on the hypoxia response in human PSCs (hPSCs). We found that the treatment with low NO doses promotes HIF-1α and HIF-2α protein accumulation in hPSCs cultured under normoxic conditions (21% O 2 ), mimicking the hypoxia response in cells cultured at 5% of O 2 in a hypoxia incubator [14] (Figure 2B). We also revealed an increase in the expression of NANOG and OCT-4 mRNA and protein levels in human iPSCs exposed to a low dose of DETA-NO in normoxia when compared with untreated cells.…”

“…Considering these preliminary results, we proposed low doses of NO under normoxic conditions could activate a response similar to hypoxia; and that this activation may be responsible for the mechanism by which NO promotes pluripotency and delays differentiation into ESCs and iPSCs. In this line, we recently described the essential role of NO inducing a cellular response to hypoxia in PSCs [14].…”

“…and that this activation may be responsible for the mechanism by which NO promotes pluripotency and delays differentiation into ESCs and iPSCs. In this line, we recently described the essential role of NO inducing a cellular response to hypoxia in PSCs [14].…”

“…This evidence indicates that NO could be a physiological regulator of cellular respiration and metabolism (Figure 1B). Furthermore, the potential role of NO in regulating the cellular response to hypoxia in vitro has been described [10,[12][13][14].…”

“…These actions are not dependent on changes in mROS levels while GPX1 mRNA expression increase, which is compatible with the maintenance of pluripotency as it is described in the review. All in all, these outcomes indicate that NO induces a hypoxia-like response, regulating mitochondrial functionality and metabolic parameters in human PSCs, thus maintaining pluripotency [14]. In this context, the use of NO is proposed to supplement culture media for human PSCs (hPSCs) expansion.…”