Culture of Mouse Endothelial Cells
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The MAEC line was a kind gift from Dr Hiroko Inoue, Tsurumi University School of Dental Medicine, Japan.
- The cell line was established from p53-deficient mouse aortas in vitro. Cells were grown in Medium 199 with 5% FBS, 1 U/ml of heparin sodium and 5 ng/ml of murine VEGF , at 37°C, 5% CO2.
- Media was changed every 3–4 days.
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Culture of Mouse Endothelial Cells
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The MAEC line was a kind gift from Dr Hiroko Inoue, Tsurumi University School of Dental Medicine, Japan.
- The cell line was established from p53-deficient mouse aortas in vitro. Cells were grown in Medium 199 with 5% FBS, 1 U/ml of heparin sodium and 5 ng/ml of murine VEGF , at 37°C, 5% CO2.
- Media was changed every 3–4 days.
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Induction of 2nd iPSCs
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All isolated somatic cells were cultured in the presence of Dox (2 ug/ml) for induction of 2nd iPSCs.
- Fibroblasts and hematopoietic cells were cultured in ES/iPSC medium.
- FLCD45 were cultured in the presence of 10 ng/ml human TPO, 10 ng/ml mouse EPO, 10 ng/ml mouse IL-3, 10 ng/ml mouse IL-6, 10 ng/ml mouse Flt3 ligand, 10 ng/ml mouse GM-CSF, 10 ng/ml mouse VEGF and 50 ng/ml mouse SCF .
- HSCs, HPCs and MPs were cultured in the presence of 10 ng/ml human TPO, 10 ng/ml mouse IL-3, 10 ng/ml mouse IL-6 and 10 ng/ml mouse Flt3 ligand .
- Macrophages were cultured in the presence of 5 ng/ml M-CSF .
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Neural progenitor cell-secreted VEGF is necessary and sufficient for the regulation of microglia (a–b) VEGF regulates microglia in vitro.
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Microglia treated with recombinant VEGF show increased proliferation (a), chemotaxis (b), and phagocytosis (c) (n = 3–6 wells/condition).
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Primary Culture of Epicardial Cells and Cardiomyocytes
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Primary epicardial cells were cultured in 10% FBS/MEM containing recombinant soluble factors, such as TGFβ1 (10 ng/ml&systems), FGF2 (100 ng/mloche), , , , , PGF-BB (100 ng/ml) or retinoic acid (1 µmol/l), for 3 days.
- The soluble factors were added on the day of heart removal.
- The medium was replaced daily.
- For western blot analysis, cells were treated with TGFβ1 for 2 days, beginning on the day following heart removal, at a final concentration of 1 ng/ml.
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Cells
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Human umbilical vein ECs (Type Tissue Collection) were seeded in sparse (5,000 cells per cm2) or confluent (50,000 cells per cm2) conditions.
- HUVECs starved in medium'>MV2 medium with 1% FCS for 2 h-overnight were stimulated with 20 ng ml−1 of mouse VEGF-A164 , 200 ng ml−1 of COMP-Angiopoietin-1 (COMP-Ang1)−1.
- PAE cells expressing human VEGFR2 (PAE/VEGFR2 (ref.
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Matrigel angiogenesis assay in vivo
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C57BL/6 mice background (8 weeks old) were subcutaneously (s.c.) flank-injected with 600 µl of matrigel supplemented with VEGF (100 ng/ml) and heparin .
- The negative controls contained heparin alone.
- Each group consisted of four animals.
- After seven days, mice were sacrificed and matrigel plugs were extracted.
- The angiogenic response was evaluated by macroscopic analysis of the plug at autopsy and by measurement of the hemoglobin content within the pellet of matrigel.
- Hb was mechanically extracted from pellets reconstituted in water and measured using the Drabkin method by spectrophotometric analysis at 540 nm.
- The values were expressed as optical density (OD)/100 mg of matrigel.
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Human ES cell culture and differentiation
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Human H1, H9 and UCLA1-6 (UCLA stem cell core) [–1 bFGF .
- EBs were differentiated according to previous reports with minor modification [μM Y27632 (Rho kinase inhibitor IV, EMD Chemicals), 3 ng ml−1 Activin A , 5 ng ml−1 bFGF, 10 ng ml−1 BMP4 and 5 μM IWR-1 (EMD Chemicals), for the first 4 days.
- EBs were then cultured in StemPro 34 supplemented with 10 ng ml−1 bFGF, 5 ng ml–1 vascular endothelial growth factor (VEGF) and 5 μM IWR-1 for 5 additional days before plating onto elastic substrates.
- Usage of all human ES cell lines is approved by the UCLA Embryonic Stem Cell Research Oversight (ESCRO) Committee and the Institutional Review Boards (IRB, approval #2009-006-04).
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Derivation and characterization of iECs
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iECs were derived using a three-dimensional approach with modifications .
- Briefly, to initiate differentiation, iPSCs were cultured in ultra-low, non-adhesive dishes to form embryoid body (EB) aggregates in EBM2 media in the absence of leukemia inhibitor factor (LIF).
- After 4 days of suspension culture, the EBs were reattached onto 0.2% gelatin-coated dishes and cultured in medium'>EBM2 medium supplemented with VEGF-A165 (50 ng/mL).
- After 3 weeks of differentiation, single cell suspensions were obtained using a cell dissociation buffer and labeled with APC-conjugated CD31 (e) and PE-conjugated CD144 anti-mouse antibodies.
- iECs were purified by fluorescence activated cell sorting (FACS) of CD31+CD144+ population.
- iECs were maintained in EBM2 media supplemented with recombinant murine vascular endothelial growth factor (50 ng/ml).
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Angiogenic potential of AGT+/− EPCs is decreased in vitro.
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The migration assay was performed using EGM-2 basal media containing VEGF (10 ng/ml), EGF (50 ng/ml), or SDF-1α (100 ng/ml), which served as chemoattractants.
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