Abstract
Reactive oxygen species (ROS) have a crucial role in stem-cell differentiation; however, the mechanisms by which ROS regulate the differentiation of stem cells into endothelial cells (ECs) are unknown. Here, we determine the role of ROS produced by NADPH oxidase 2 (Nox2) in the endothelial-lineage specification of mouse induced-pluripotent stem cells (miPSCs). When wild-type (WT) and Nox2-knockout (Nox2−/−) miPSCs were differentiated into ECs (miPSC-ECs), the expression of endothelial markers, arterial endothelial markers, pro-angiogenic cytokines, and Notch pathway components was suppressed in the Nox2−/− cells but increased in both WT and Nox2−/− miPSCs when Nox2 expression was upregulated. Higher levels of Nox2 expression increased Notch signaling and arterial EC differentiation, and this increase was abolished by the inhibition of ROS generation or by the silencing of Notch1 expression. Nox2 deficiency was associated with declines in the survival and angiogenic potency of miPSC-ECs, and capillary and arterial density were lower in the ischemic limbs of mice after treatment with Nox2−/− miPSC-ECs than WT miPSC-EC treatment. Taken together, these observations indicate that Nox2-mediated ROS production promotes arterial EC specification in differentiating miPSCs by activating the Notch signaling pathway and contributes to the angiogenic potency of transplanted miPSC-derived ECs.
Highlights
Reactive oxygen species (ROS) such as hydrogen peroxide and superoxide, as well as the balance between ROS generation and elimination are important regulators of cell survival and proliferation[13,14,15]
The expression of stemness markers (Oct[4] and Nanog) in WT mouse iPSCs (miPSCs) and Nox2−/− miPSCs declined at similar rates during the first six days of differentiation (Supplementary Figure 3B,C), which suggests that the impaired endothelial specification of Nox2−/− miPSCs cannot be attributed to greater persistence of the undifferentiated state
Nox[2] deficiency was associated with declines in endothelial-marker expression after just five days of differentiation, so we investigated the effect of exogenous ROS on the expression of endothelial markers and angiogenic proteins in WT miPSCs at an earlier stage of endothelial differentiation by exposing the cells to low levels of hydrogen peroxide (H2O2) or to diphenylene iodonium (DPI), which inhibits ROS production. mRNA measurements of CD31, CD144, vascular endothelial growth factor (VEGF), and Ang-1 expression increased significantly when WT miPSCs were cultured in the presence of 10 μM H2O2 (Fig. 3A) and declined significantly when Ad-GFP– or adenoviruses coding for Nox2 (Ad-Nox2)–transfected miPSCs were cultured with DPI (Fig. 3B,C)
Summary
Mouse embryonic fibroblasts (MEFs) were isolated from embryos on embryonic day 13.5 and cultured in Dulbecco’s modified Eagle medium with 10% fetal bovine serum[26]. Mouse iPSCs were grown on mitomycin C-treated MEF feeders in standard ESC medium (Dulbecco’s modified Eagle medium supplemented with 2 mM l-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 0.1 mM β-mercaptoethanol, 50 U/mL penicillin, 50 μg/mL streptomycin, and 0.1 μg/mL leukemia inhibitory factor) with 10% knockout serum replacement. All culture reagents were from Invitrogen (Carlsbad, CA) unless otherwise indicated
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