BM was obtained from 55 healthy volunteer donors (2–45 years of age) after obtaining informed consent per the guidelines of the University of Minnesota Committee on the Use of Human Subjects in Research. MAPCs were generated as previously described (3
). Briefly, BM mononuclear cells were depleted of CD45+
and glycophorin A+
cells using micromagnetic beads (Miltenyi Biotec, Sunnyvale, California, USA). Cells (5 × 103
) that were negative for CD45 and glycophorin A were diluted in 200 μl expansion medium consisting of 58% low-glucose DMEM (Invitrogen Corp., Grand Island, New York, USA) and 40% MCDB-201 (Sigma Chemical Co., St. Louis, Missouri, USA), supplemented with 1× insulin-transferrin-selenium, 1× linoleic acid–BSA, 10–8
M dexamethasone, 10–4
M ascorbic acid 2-phosphate (all from Sigma Chemical Co.), 100 U penicillin, and 1,000 U streptomycin (Invitrogen Corp.); along with 0–10% FCS (HyClone Laboratories, Logan, Utah, USA), 10 ng/ml EGF (Sigma Chemical Co.), and 10 ng/ml PDGF-BB (R&D Systems Inc., Minneapolis, Minnesota, USA). Cells were then plated in wells of 96-well plates that had been coated with 5 ng/ml of fibronectin (Sigma Chemical Co.). Cells were fed every 4–6 days. Once cells were more than 40–50% confluent, adherent cells were detached with 0.25% trypsin-EDTA (Sigma Chemical Co.) and replated at 1:4 dilution, in bigger culture vessels coated with 5 ng/ml fibronectin and MAPC expansion medium, to maintain cell densities between 2 × 103
and 8 × 103
Differentiation conditions and characterization.
To induce differentiation into endothelial cells, MAPCs were replated at 1 × 104 to 2 × 104 cells/cm2 in fibronectin-coated culture vessels or chamber slides, in 60% low-glucose DMEM (Invitrogen Corp.) and 40% MCDB-201 (Sigma Chemical Co.), supplemented with 1× insulin-transferrin-selenium, 1× linoleic acid–BSA, 10–8 M dexamethasone, 10–4 M ascorbic acid 2-phosphate (all from Sigma Chemical Co.), 100 U penicillin, and 1,000 U streptomycin (Invitrogen Corp.), plus 10 ng/ml VEGF (a kind gift from S. Ramakrishna, University of Minnesota). In some instances, FCS (HyClone Laboratories) was added. Cultures were maintained by media exchange every 4–5 days. In some instances, cells were subcultured after day 9 at a 1:4 dilution under the same culture conditions for more than 20 population doublings.
A breeding colony of NOD/SCID mice was established using mice obtained from The Jackson Laboratory (Bar Harbor, Maine, USA). Mice were kept in specific pathogen–free conditions, and maintained on acidified water and autoclaved food. Trimethoprim (60 mg/ml water) and sulfamethoxazole (300 mg/ml water) (Hoffmann–La Roche Inc., Nutley, New Jersey, USA) were given twice weekly.
Transplantation of MAPC-derived endothelial cells to an animal model for tumor and wound neoangiogenesis.
Three Lewis lung carcinoma spheroids were implanted subcutaneously in the shoulder of each mouse. Three days and 5 days after tumor implantation, mice were injected with 0.25 × 106 human MAPC–derived endothelial cells or human foreskin fibroblasts via tail vein injection. After 14 days, animals were sacrificed, and tumors were removed and cryopreserved using OCT compound (Sakura Finetek USA Inc., Torrance, California, USA) at –80°C. In addition, we removed the ears that had been clipped to tag each mouse; these were cryopreserved using OC compound at –80°C. Sections of the tissues (5 μm thick) were mounted on glass slides, and were fixed and stained as described below.
Undifferentiated MAPCs (106) were injected intravenously into 6-week-old NOD-SCID mice. Animals were maintained for 12 weeks and then sacrificed. In one animal, a thymic tumor was found. The thymus was removed and cryopreserved in OCT compound at –80°C. Tissue sections (10 μm thick) were mounted on glass slides, and were fixed and stained as described below.
MAPCs, or MAPCs induced to differentiate into endothelial cells for 2–21 days, were detached using 0.25% trypsin-EDTA (Sigma Chemical Co.). Cells were stained with Ab’s against Tie, Tek, Flk1, Flt1, vWF (all from Santa Cruz Biotechnology Inc., Santa Cruz, California, USA), CD13, CD31, CD34, CD36, CD44, CD49b, AC133, HLA class I, HLA class II, β2-microglobulin, cKit (all from Becton Dickinson Immunocytometry Systems, Mountain View, California, USA), VCAM, ICAM-1, VE-cadherin, CD62E, CD62L, CD62P, or MUC18, recognized by the Ab H1P12, (all from Chemicon International USA Inc., Temecula, California) at saturating concentrations. Cells were washed and labeled with FITC, phycoerythrin (PE), or APC-conjugated secondary goat anti-mouse, goat anti-rabbit, or sheep anti-goat Ab’s (Sigma Chemical Co.), then washed and analyzed using a FACSCalibur flow cytometry system (Becton Dickinson Immunocytometry Systems).
For in vitro cultures, undifferentiated MAPCs, or MAPCs induced to differentiate into endothelium by plating in fibronectin-coated chamber slides with VEGF for 2–18 days, were fixed with 4% paraformaldehyde (Sigma Chemical Co.) for 4 minutes at room temperature and stained for surface markers. Alternatively, for cytoskeleton staining, chamber slides were fixed with methanol for 2 minutes at –20°C. For nuclear ligands, cells were further permeabilized with 0.1% Triton X-100 (Sigma Chemical Co.) for 10 minutes. Slides were incubated for 30 minutes with primary Ab, then incubated with FITC-, PE-, or Cy5-coupled anti-mouse IgG, anti-goat IgG, or anti-rabbit IgG Ab. After each incubation step, slides were washed with PBS containing 1% BSA. Primary Ab’s against CD31, CD34, CD36, CD44, HLA class I, HLA class II, and β2-microglobulin were used at a 1:50 dilution. Primary Ab’s against VCAM, ICAM-1, VE-cadherin, H1P12, ZO-1, MUC18, αvβ3, αvβ5, β-catenin, γ-catenin (all from Chemicon International USA Inc.), Tek, Tie, and vWF (all from Santa Cruz Biotechnology Inc.) were used at a 1:50 dilution. Stress fibers were stained with Ab’s (used at 1:200 dilution) against the 20 k-Da myosin light chain (Sigma Chemical Co., clone no. MY-21). Secondary Ab’s were purchased from Sigma Chemical Co., and were used at the following dilutions: anti-goat IgG–Cy3 (1:40), anti-goat IgG–FITC (1:160), anti-mouse IgG–Cy3 (1:150), anti-mouse IgG–FITC (1:320), anti-rabbit FITC (1:160), and anti-rabbit Cy3 (1:30). TOPRO-3 was purchased from Sigma Chemical Co. Cells were examined by fluorescence microscopy using a Zeiss Axiovert scope (Carl Zeiss Inc., Thornwood, New York, USA) and by confocal fluorescence microscopy using a confocal microscope (Olympus AX70; Olympus Optical Co. Ltd., Tokyo, Japan).
Samples of normal tissue or tumor were sectioned using a cryostat, into sections 5–10 μm thick. Sections were fixed with acetone for 10 minutes at room temperature and permeabilized with 0.1% Triton X-100 for 5 minutes. Slides to be analyzed for vWF, Tie, or Tek were incubated overnight, followed by secondary incubation with FITC-, PE-, or Cy5-coupled anti-mouse IgG, anti-goat IgG, or anti-rabbit IgG Ab’s, and sequential incubation with Ab’s against mouse CD45–PE, human CD45–FITC, human β2-microglobulin–FITC, mouse CD31–FITC, and TOPRO-3 for 60 minutes. After each step, slides were washed with PBS containing 1% BSA. Slides were examined by fluorescence microscopy using a Zeiss Axiovert scope, and by confocal fluorescence microscopy using a confocal microscope. Three-dimensional reconstruction consisted of the acquisition of sequential 0.5-μm confocal photos from 35 slides of 5-μm-thick sections to a total of 350 photos. Slides were stained with vWF-Cy3, and alternately double-stained with human β2-microglobulin–FITC or mouse CD31–FITC Ab’s. The photos from each slide were aligned with the next slide in MetaMorph software (Universal Imaging Corp., West Chester, Pennsylvania, USA), and the 3D reconstruction was made in 3D Doctor software (Able Software Corp., Lexington, Massachusetts, USA). The volume of the relative contributions of human (green) and murine endothelial cells (false colored as blue) to the 3D vessel were calculated as cubic pixels of each color. The area of each color was also calculated as square pixels in four tumor vessels appearing in each of the 35 slides to obtain an accurate percentage of contribution. The area of each color was also calculated in alternate slides of four different tumors.
MAPCs and MAPC-derived endothelial cells were incubated at 37°C in 20% or 10% O2 for 24 hours. Cells were stained with Ab’s against Flk1, Flt1, Tek, and IgG control, fixed in 2% paraformaldehyde, and analyzed by flow cytometry. In addition, VEGF concentrations in the culture supernatants were measured using an ELISA kit (Amersham Biotech, Piscataway, New Jersey, USA).
Histamine-mediated release of vWF.
MAPCs and MAPC-derived endothelial cells were incubated with 10 μM histamine (Sigma Chemical Co.) in serum-free medium for 25 minutes. Untreated and treated cells were fixed with methanol at –20°C for 2 minutes, stained with Ab’s against vWF and myosin, and analyzed using fluorescence and/or confocal microscopy.
MAPCs (106) and MAPC-derived endothelial cells (106) were incubated with 75 ng/ml IL-1α (R&D Systems Inc.) in serum-free medium for 24 hours. Cells were fixed in 2% paraformaldehyde and stained with Ab’s against HLA class I, HLA class II, β2-microglobulin, vWF, CD31, VCAM, CD62E, and CD62P, or with control Ab’s, and analyzed using FACS.
In vitro vascular tube formation: Extracellular matrix (Sigma Chemical Co.) was added to a 24-well plate at 0.5 ml per well, and incubated for 3 hours at 37°C. MAPCs and MAPC-derived endothelial cells (104 per well) were added in 0.5 ml of serum-free medium containing 10 ng/ml VEGF and incubated at 37°C.
The DiI-Ac-LDL staining kit was purchased from Biomedical Technologies (Stoughton, Massachusetts, USA). The assay was performed per the manufacturer’s recommendations.