American Society of Hematology

Figure 4.

$Figure 4. Endothelial phenotype and function of CD34+CD43–KDR+ cells isolated after 6 days of H1/OP9 coculture. (A) FACS analysis of KDR+ and KDR– fractions of CD34+CD43– cells isolated on day 9 of H1/OP9 coculture. Phenotype of CD34+CD43– KDR– cells was compared with phenotype of CD34–CD43– cells obtained after depletion of CD34+ and CD43+ cells. Plots show isotype control (open) and specific mAb (tinted) histograms. Values within plots indicate specific mean fluorescence intensity (ΔMFI) calculated by formula: linear-scaled MFI of specific mAb-stained cells – linear-scaled MFI of isotype control mAb-treated cells. The representative experiment is shown. Similar results were obtained in 5 independent experiments with H1- and H9-derived CD34+CD43–KDR+/– cells isolated on day 6 (n = 2) and day 9 (n = 3) of differentiation. (B) CD34+CD43–KDR+ cells were cultured 7 days in endothelial expansion conditions and examined for markers of mature endothelial cells. Immunofluorescent staining was performed with primary antibodies against VE-cadherin (goat IgG; R&D Systems), von Willebrand factor (VWF; rabbit IgG; Sigma) and endothelial NO synthetase (eNOS; mouse IgG1; BD Pharmingen) followed by respective secondary antibody against goat IgG-Alexa Fluor-555 (red fluorescence), rabbit IgG-Alexa Fluor-488 (green fluorescence), and mouse IgG-Alexa Fluor-488 (Molecular Probes). Negative controls were done using appropriate primary IgG controls (Sigma). Cell nuclei were visualized by DAPI staining (blue fluorescence). Images were captured with an inverted DMIRB microscope (Leica Microsystems) equipped with a 20×/0.3 objective lens, and were acquired with a MagnaFire camera and software (Optronics). Fluorescent images were composed using Adobe Photoshop software. Ac-LDL uptake was assessed by incubation with DiI-Ac-LDL conjugate as described in “Materials and methods.” Scale bar represents 50 μm. Insets show FACS analysis of respective surface (VE-cadherin) and intracellular (eNOS, VWF) markers in parallel cultures, or instant FACS profiles of cells incubated with DiI-Ac-LDL at 37°C (Ac-LDL uptake) versus 4°C (control Ac-LDL binding). (C) Vascular tubes formation by CD34+CD43– KDR+ cells (scale bar represents 200 μm, left panel; and 50 μm, right panel). Images were captured with an inverted DMIRB micrscope (Leica Microsystems) equipped with a 5×/0.12 (left) or 20×/0.3 (right) objective lens, and were acquired through a MagnaFire camera and software (Optronics). (D) TNF-induced up-regulation of ICAM-1 and induction of VCAM-1 expression in CD34+CD43–KDR+ endothelial cultures. Numbers within plots indicate ΔMFI values for untreated (blue) and TNF-treated (red) cells. VLA-4 staining was used as a control. A representative example of 3 independent experiments is shown.$

Endothelial phenotype and function of CD34⁺CD43^–KDR⁺ cells isolated after 6 days of H1/OP9 coculture. (A) FACS analysis of KDR⁺ and KDR^– fractions of CD34⁺CD43^– cells isolated on day 9 of H1/OP9 coculture. Phenotype of CD34⁺CD43^– KDR^– cells was compared with phenotype of CD34^–CD43^– cells obtained after depletion of CD34⁺ and CD43⁺ cells. Plots show isotype control (open) and specific mAb (tinted) histograms. Values within plots indicate specific mean fluorescence intensity (ΔMFI) calculated by formula: linear-scaled MFI of specific mAb-stained cells – linear-scaled MFI of isotype control mAb-treated cells. The representative experiment is shown. Similar results were obtained in 5 independent experiments with H1- and H9-derived CD34⁺CD43^–KDR^+/– cells isolated on day 6 (n = 2) and day 9 (n = 3) of differentiation. (B) CD34⁺CD43^–KDR⁺ cells were cultured 7 days in endothelial expansion conditions and examined for markers of mature endothelial cells. Immunofluorescent staining was performed with primary antibodies against VE-cadherin (goat IgG; R&D Systems), von Willebrand factor (VWF; rabbit IgG; Sigma) and endothelial NO synthetase (eNOS; mouse IgG1; BD Pharmingen) followed by respective secondary antibody against goat IgG-Alexa Fluor-555 (red fluorescence), rabbit IgG-Alexa Fluor-488 (green fluorescence), and mouse IgG-Alexa Fluor-488 (Molecular Probes). Negative controls were done using appropriate primary IgG controls (Sigma). Cell nuclei were visualized by DAPI staining (blue fluorescence). Images were captured with an inverted DMIRB microscope (Leica Microsystems) equipped with a 20×/0.3 objective lens, and were acquired with a MagnaFire camera and software (Optronics). Fluorescent images were composed using Adobe Photoshop software. Ac-LDL uptake was assessed by incubation with DiI-Ac-LDL conjugate as described in “Materials and methods.” Scale bar represents 50 μm. Insets show FACS analysis of respective surface (VE-cadherin) and intracellular (eNOS, VWF) markers in parallel cultures, or instant FACS profiles of cells incubated with DiI-Ac-LDL at 37°C (Ac-LDL uptake) versus 4°C (control Ac-LDL binding). (C) Vascular tubes formation by CD34⁺CD43^– KDR⁺ cells (scale bar represents 200 μm, left panel; and 50 μm, right panel). Images were captured with an inverted DMIRB micrscope (Leica Microsystems) equipped with a 5×/0.12 (left) or 20×/0.3 (right) objective lens, and were acquired through a MagnaFire camera and software (Optronics). (D) TNF-induced up-regulation of ICAM-1 and induction of VCAM-1 expression in CD34⁺CD43^–KDR⁺ endothelial cultures. Numbers within plots indicate ΔMFI values for untreated (blue) and TNF-treated (red) cells. VLA-4 staining was used as a control. A representative example of 3 independent experiments is shown.

This Feature Is Available To Subscribers Only