Nox2 contributes to the arterial endothelial specification of mouse induced pluripotent stem cells by upregulating Notch signaling

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.


Embryoid body formation assay
Mouse iPSCs or mESCs were trypsinized and suspended as single cells. After removal of the medium, the cells were washed 3 times to remove the LIF in the medium. The cells were plated onto 6-well low-attachment plates (Corning Life Sciences) at 1× 10 5 cells per well in ESC medium without LIF.

Transfection and viral transduction of miPSCs or miPSC-ECs
miPSCs or miPSC-ECs were transfected with the Notch1 siRNA (100 nmol/L) or the non-targeting negative control siRNA (100 nmol/L), an empty (Control) vector, or vector coding for NICD1 using the Lipofectamine 2000 transfection reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). The transfection medium was replaced after six hours and the cells were incubated for another 48 hours. The recombinant adenoviruses Ad-GFP-Nox2 or Ad-GFP were used to infect miPSCs or miPSC-ECs. For the transduction, a multiplicity of infection (MOI) of 25 was used. No detectable cellular toxicity was observed. Transduction was verified via GFP expression.

Staining
Alkaline phosphatase staining was performed with an Alkaline Phosphatase Kit as directed by the manufacturer's instructions. Immunofluorescent staining was performed as described previously. 1 Briefly, cells were fixed with 4% paraformaldehyde, or tissues from EBs were fixed in ice-cold methanol, permeabilized with 0.1% Triton X-100 in PBS for 10 min, blocked in 5% donkey serum in PBS for 30 min at 37°C, and then incubated with primary antibodies (mouse anti-Oct4, mouse anti-SSEA-1, and mouse anti-EphrinB2). Bound primary antibodies were visualized with fluorescent secondary antibodies, nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; Sigma), and observations were performed with a fluorescence microscope (Leica, Hamburg, Germany).

Teratoma assay
miPSCs were harvested, washed, resuspended in ESC medium (210 6 cells/300 μL), and then injected subcutaneously into 6-week-old male NOD/SCID mice. Four weeks later, visible tumors were excised and fixed overnight with 4% paraformaldehyde solution; then, the tumor tissues were paraffin embedded, sectioned, stained with hematoxylin and eosin, and examined by a pathologist.

NADPH oxidase assay
NADPH oxidase (NOX) activity was measured via lucigenin chemiluminescence. 2 Cells were washed five times with ice-cold PBS and centrifuged at 1000 g and 4°C for 10 min; then, the cell-containing pellets were resuspended in lysis buffer (20 mM KH2PO4, pH 7.0, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 g/mL aprotinin, and 0.5 g/mL leupeptin), and the suspended cells were homogenized with 100 strokes in a Dounce homogenizer on ice. The homogenate (100 L) was added to 100 L of phosphate buffer (50 mM, pH 7.0) containing 1 mM EGTA, 150 mM sucrose, 5 M lucigenin, and 100 M NADPH, and photon emissions were measured every 15 s for 5 min in a luminometer; sample measurements were blanked by subtracting measurements obtained with the buffer alone before the results were calculated. NOX activity was presented as relative chemiluminescence (light) units per second per milligram of protein. Each experiment was performed three times in three replicate wells.

Measurement of secreted VEGF via ELISA
miPSC-ECs were seeded in a 6-well plate, and the supernatants were collected from the cells 48 hours later. Total cellular protein was measured with a protein assay kit (Bio-Rad Laboratories, Hercules, CA), and VEGF levels were measured with an ELISA assay kit (Neobioscience Technology, Shengzhen, China) as directed by the manufacturer's instructions. Duplicate assays were performed for each of three independent experiments.

Cell migration assay
Migration was evaluated with transwell chambers (24-well plates, 8-μm pore size, Corning, Inc.) as described previously. 3 Cells (1×10 5 ) were loaded with ECM2 media into the upper chamber and incubated for 10 hours; then, the non-migrated cells were removed, and the cells that had migrated to the lower compartment were fixed, stained with 0.1% crystal violet, and viewed under a microscope. Cells were counted in five randomly chosen fields per chamber, and three chambers were evaluated for each sample.

Cell proliferation assay
Cell proliferation was evaluated via the incorporation of bromodeoxyuridine (BrdU) into newly synthesized DNA. Cells were seeded into 96-well plates and cultured for the indicated time periods; then, BrdU was added, the cells were cultured for 6 hours, and BrdU incorporation was measured as directed by the manufacture's instructions. (Calbiochem, San Diego, CA). Three replicates were evaluated per sample.

In vitro tube formation assay
The wells of a 48-well plate were filled with 200-μL Matrigel (10 mg/mL, Matrigel Basement Membrane Matrix); then, cell suspensions (1×10 5 cells/well) were added to the Matrigel surface, and the formation of capillary-like structures was evaluated 12-18 hours later. Tube length was measured in five randomly chosen high-power fields in each well under a microscope. Each experiment was performed three times in three replicate wells. Toxicol Appl Pharmacol 244, 291-9 (2010).

Supplementary Figure 1. The mRNA expression profile of NOX subunits in miPSCs and Nox2
deficiency reduces NADPH oxidae activity and ROS production in miPSCs.
A, Quantitative PCR analyses show that Nox2 and Nox4 were highly expressed, and Nox1 was expressed at low levels in WT miPSCs (n=3; **P<0.01 vs Nox1). B, Quantitative PCR analyses showed that Nox2 expression was knocked out, and that the mRNA levels of Nox1, Nox4, p22phox, p47phox, and p67phox between Nox2 -/-miPSCs and WT miPSCs were similar. WT miPSCs and Nox2 -/-miPSCs were assessed for NADPH oxidase activity (C) and ROS production (