Journal: JVS-Vascular Science

Article Title: Hyperoxia impairs induced pluripotent stem cell-derived endothelial cells and drives an atherosclerosis-like transcriptional phenotype

doi: 10.1016/j.jvssci.2024.100193

Figure Lengend Snippet: Physiological oxygen (physoxia) enhances mitochondrial respiration and glycolysis in late passage induced pluripotent stem cells directed to endothelial identity (iPSC-ECs). Y6 iPSC-derived endothelial cells on passage 3 (early) or 8 (late) were subjected to a Seahorse metabolic analysis with mitochondrial stress test (MST) or glycolysis stress test ( GST ). (A) MST for cells grown under physoxia (4% oxygen) or hyperoxia (21% oxygen), with or without addition of pyruvate transport inhibitor UK5099. (B) Glycolytic stress test curve for the samples in (A) . (C) MST for late passage cells grown under physoxia or hyperoxia, with or without UK5099 treatment. (D) GST curve for cells in (C) . ∗ P < .05 by two-way analysis of variance testing with correction for multiple comparisons. Error bars in all frames show standard error of three technical replicates. The graph images are one of two independently conducted experiments. ECAR , extracellular acidification rate; OCR , oxygen consumption rate.

Article Snippet: Physiological oxygen samples were maintained in 4% O 2 in a hypoxia chamber (Stem Cell Technologies, Vancouver, Canada; #27310). iPSC-ECs were treated with 1 μM of UK5099 (SigmaPZ0160; Sigma-Aldrich, St. Louis, MO) or vehicle control for 24 hours.

Techniques: Derivative Assay

Journal: JVS-Vascular Science

Article Title: Hyperoxia impairs induced pluripotent stem cell-derived endothelial cells and drives an atherosclerosis-like transcriptional phenotype

doi: 10.1016/j.jvssci.2024.100193

Figure Lengend Snippet: Induced pluripotent stem cells directed to endothelial identity (iPSC-ECs)grown at physoxia show enhanced endothelial marker retention in late passages. (A) Early passage iPSC-ECs grown under hyperoxia or physoxia with and without addition of UK5099 were immunostained for CD144 and DAPI and imaged with confocal microscopy. Representative images from each group are shown along with all channels alone or merged. Original magnification 20× with scale bars showing 100 μm. DAPI is blue, CD144 is green. (B) Quantification of CD144 + cells/DAPI + cells from two independent replicates. (C) Late passage Y6 iPSC-EC grown under physoxia or hyperoxia with or without addition of UK5099 were fixed, immunostained for CD31 and underwent flow cytometry. A mixture of all cells unstained were used as a negative control. (B) Representative ridge plots showing physoxia grown samples retaining CD144. (D) Quantification of CD144-positive cells in experiment in (C) . (E) Cells from (B) were also stained with CD31 and assessed by flow cytometry. Ridge plot showing expression of CD31, which was retained in late passage cells grown under physoxia. (F) Quantification of CD31-positive cells from experiment in (E) . Error bars show ± SE among three technical replicates. ∗ P < .05 with Student's t test.

Article Snippet: Physiological oxygen samples were maintained in 4% O 2 in a hypoxia chamber (Stem Cell Technologies, Vancouver, Canada; #27310). iPSC-ECs were treated with 1 μM of UK5099 (SigmaPZ0160; Sigma-Aldrich, St. Louis, MO) or vehicle control for 24 hours.

Techniques: Marker, Confocal Microscopy, Flow Cytometry, Negative Control, Staining, Expressing

Journal: JVS-Vascular Science

Article Title: Hyperoxia impairs induced pluripotent stem cell-derived endothelial cells and drives an atherosclerosis-like transcriptional phenotype

doi: 10.1016/j.jvssci.2024.100193

Figure Lengend Snippet: Induced pluripotent stem cells directed to endothelial identity (iPSC-ECs) sub-culturing results in senescence and functional loss not ameliorated by physoxia. (A) iPSC-ECs grown to early or late passage under hyperoxia or physoxia was treated (+) with UK5099 or untreated (−) and subjected to beta-galactosidase assay to assess cell senescence. Late passage cells showed greater senescence in both physoxia and hyperoxia groups. Graph shows beta galactosidase normalized to total protein per well. (B) Cells from experiment in (A) were also treated with fluorescent EdU to assess proliferation. Graph shows the number of cells proliferating and not proliferating in each group (C) . Cells from the experiment in (A) were tested for total nitric oxide synthase (NOS) activation, quantified as DAF normalized to Hoechst staining. (D) Cell lysates from the experiment in (C) were probed with specific antibody to phosphorylated endothelial NOS and quantified by Western blotting. (D) Cell lysates from the experiment in ( C ) were probed with specific antibody to phosphorylated eNOS and quantified by Western blotting. (E) Cells from experiment in (A) were exposed to Matrigel matrix for 16 hours and analyzed for endothelial tube formation. Representative images from each condition imaged with fluorescence microscopy are shown. Lens: 20x; zoom: ×1. (F) Representative images from Western blot quantified in (D) . (G) Experiment in (E) was quantified for the number of junctions between tube structures in ImageJ. Error bars show ± SE in three independent experiments. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .005; ∗∗∗∗ P < .001.

Article Snippet: Physiological oxygen samples were maintained in 4% O 2 in a hypoxia chamber (Stem Cell Technologies, Vancouver, Canada; #27310). iPSC-ECs were treated with 1 μM of UK5099 (SigmaPZ0160; Sigma-Aldrich, St. Louis, MO) or vehicle control for 24 hours.

Techniques: Functional Assay, β-Gal Assay, Activation Assay, Staining, Western Blot, Fluorescence, Microscopy