All reagents are from Life Technologies (Carlsbad, CA) unless indicated otherwise. Human umbilical vein cells (HUVECs) and Sprague–Dawley rats were purchased from ATCC (Manassas, VA) and Charles Rivers Laboratories (Wilmington, MA), respectively.
Culture of MSCs and SB623 cells and preparation of conditioned medium
Two human cell types were examined in this study; mesenchymal stromal cells (MSCs) and MSC-derived SB623 cells. Bone marrow aspirates from healthy human adults were obtained from Lonza (Walkersville, MD), rinsed, and plated in tissue culture flasks. Culture medium for the generation and maintenance of donor cells was minimum essential alpha medium (α - MEM, Mediatech, Herndon, VA) supplemented with 2mM Glutamine, 10% fetal bovine serum (Hyclone, Logan, UT) and 1% penicillin/streptomycin (referred to throughout the text as “growth medium”). Non-adherent cells were discarded, and the remaining cells were passed two times using trypsin (0.25% + 2 mM EDTA). MSCs were then either frozen for later use or plated for SB623 cell preparation. For SB623 preparation, MSCs were transfected with the pCI plasmid expressing the human Notch1 intracellular domain (NICD; Notch1 truncated at the transmembrane domain) and the neomycin-resistance gene using Fugene6 (Promega, Madison, WI) according to the manufacturer’s protocol. The next day, the medium was replaced with growth medium containing 100 μg/ml G418 and selection continued for 7 days. Selection medium was then replaced with growth medium. After removal of G418 and recovery, cells were passed two additional times. SB623 cells were harvested using trypsin-EDTA and cryopreserved for later use. Both MSCs and SB623 cells were routinely characterized by flow cytometry analysis and were found to be positive (>95%) for CD29, CD90, and CD105, and negative (<5%) for CD31, CD34, CD45, indicating their mesenchymal nature. For experiments, frozen MSCs and SB623 cells from the same human donor were thawed, re-plated and allowed to recover for approximately one week. To obtain conditioned medium, MSCs or SB623 cells were cultured in growth medium to ~90% confluence (~15,000 cells/cm2). Following rinsing in phosphate-buffered saline (PBS), the medium was replaced with Opti-MEM® medium (~150,000 cells/ml), and the conditioned medium was collected 72 hours later and stored at -80°C. At the time of collection, the number of cells was quantified (mean = 1.0 ± 0.1 million cells per flask, with no significant differences between MSCs and SB623 cells). Frozen conditioned medium samples were slowly warmed to 37°C on the day of experimentation.
Angiogenic cytokine array of MSC- and SB623-conditioned medium
To identify angiogenic trophic factors secreted by MSCs and SB623 cells, the protein levels of specific factors in donor cell-conditioned medium were measured. The Quantibody® Human Angiogenesis Array 1 (RayBiotech, Norcross, GA) was used to determine the concentrations of the following 10 cytokines in MSC or SB623 cell-conditioned media: angiogenin, angiopoietin-2, epidermal growth factor (EGF), fibroblast growth factor–2 (FGF-2/ bFGF), heparin binding-epidermal growth factor like growth factor (HB-EGF), hepatocyte growth factor (HGF), leptin, platelet derived growth factor-BB (PDGF-BB), placental growth factor (PIGF), and vascular endothelial growth factor (VEGF). To account for slight differences in actual cell numbers, the data were normalized to the cell number determined upon collecting conditioned medium, and are thus expressed as protein concentration per 1 million cells. For this assay, 4 different human donor pairs were tested.
Cell proliferation and survival of HUVECs
Two different lots of human umbilical vein endothelial cells (HUVECs) were purchased from ATCC. Experiments comparing SB623-CM and MSC-CM were performed with one lot of HUVECs; experiments assessing the effect of SU5416 on SB623-CM angiogenic activity were performed with a second lot of HUVECs. Briefly, HUVECs were plated in endothelial basal medium-2/10%FBS plus endothelial cell growth supplement (EBM-2/10%FBS plus ECGS) at 750,000 cells per 0.1% gelatin-coated T-75 flask for 24 hours. To prepare for the serum-growth factor withdrawal study, HUVEC monolayers were rinsed twice with warm PBS and incubated with fresh 12 ml EBM-2 medium for 16–24 hours at 37°C, 5% CO2. Serum-growth factor withdrawal was initiated by re-feeding each flask with 6 ml of EBM-2 medium plus 6 ml OptiMEM (control) or 6 ml of EBM-2 medium supplemented with 6 ml of either MSC- or SB623 cell-derived conditioned OptiMEM. After 7 days, non-adherent and adherent cells were collected, centrifuged at 1400 rpm for 5 minutes, and divided into three fractions for the subsequent three staining analyses.
To quantify cell death, cells were stained with 5 μg/ml of propidium iodide (PI) for 30 min at room temperature and acquisition/analysis was done on FL-2 logarithmic channel using BD FACS Calibur CellQuest program. Staining for Bcl-2 (anti-apoptotic molecule) and Ki67 (proliferation marker; BD Bioscience) was also performed. Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton-X for one hour; after staining with either fluorescein-conjugated antibody against Bcl-2 or Ki67 for one hour on ice, samples were washed, acquired, and analyzed on FL-1 channel on BD FACS Calibur. For these assays, 3 different human donor pairs were tested with 3 wells per group.
For experiments with VEGFR inhibitor, HUVECs were pre-treated with 50 nM SU5416 (EMD Millipore, Billerica, MA) in EBM-2 medium for 30 minutes. Medium was then changed to 50 nM SU5416-supplemented medium containing 6 ml of EBM-2 medium plus 6 ml of SB623 cell derived-conditioned medium. Analyses for cell proliferation and cell death were performed after 5 days in culture. For these assays, SB623 cells from 3 different human donors were tested with 3 wells per group.
HUVEC tube formation assay
HUVECs were cultured in α-MEM/0.5%FBS/2 mM glutamine/pen-strep for 24 hours and stained with 1 μM calcein dye (Life Technologies, Carlsbad, CA) prior to use in a vascular tube formation assay. In each well of a 96-well flat bottom plate, 50 μl of reduced growth factor (RGF)-Cultrex basement membrane extract (Trevigen, Gaithersburg, MD) were incubated at 37°C for 45 minutes. HUVECs were harvested using 0.25% trypsin-EDTA, rinsed, and re-suspended in α-MEM/2 mM glutamine/pen-strep at 1×105 cells/ml. 75 μl of HUVECs plus 75 μl of either MSC- or SB623-conditioned medium (from 3 different human donor pairs) was added to each well. The negative control was HUVECs plus OptiMEM medium only. Fluorescent (for calcein-stained conditions) or phase contrast photographs were taken at 16 hours, for subsequent quantification of tube formation by an experimenter blinded to the groups. A continuous, unbroken ring of cells was considered a complete vascular tube. Where indicated, 50 nM SU5416 treatment was done 30 minutes prior to changing to 50 nM SU5416-supplemented SB623 cell derived-conditioned medium (from 3 different human donors), 50 nM SU5416-supplemented OptiMEM with 10 ng/ml recombinant human VEGF (positive control), or 50 nM SU5416-supplemented OptiMEM (negative control). For this assay, 3–4 wells/ group were used and the total number of tubes per well was quantified and averaged.
Aortic ring assay
All procedures conformed to guidelines set forth in the NIH Guide for the Care and Use of Laboratory Animals. Adult Sprague–Dawley rats were euthanized prior to dissection. After opening the chest and clamping off the two ends, the aorta was removed and placed in ice-cold α-MEM/pen-strep medium prior to removal of the adipose external lining. Adipose-free aorta was rinsed twice with ice-cold EBM-2/pen-strep medium before sectioning into 1-mm-thick rings. The aortic rings were then transferred to plates containing α-MEM/2 mM glutamine/pen-strep medium and incubated at 37°C, 5% CO2 for 6 days to minimize the endogenous rat angiogenic factors; fresh medium was replaced on day 3. On day 6, the medium was replaced with α-MEM/pen-strep medium and the culture was continued for an additional 24-hour period. On day 0 of the aortic ring assay, 50 μl of RGF- basement gel was deposited per well of a 24-well plate. Each individual aortic ring was carefully placed in the middle of each well and over-layed with an additional 25 μl of RGF-Cultrex basement membrane extract. After the gel had solidified at 37°C, 5% CO2 for 30 minutes, 500 μl of α-MEM/2 mM glutamine/pen-strep was added to each well and incubated for an additional 30 minutes. To assess the angiogenic activity from MSC- and SB623-derived trophic factors, 500 μl of conditioned medium was added. As a negative control, 500 μl of unconditioned OptiMEM medium was used. Phase contrast photos were taken at 7 or 10 days and both vessel outgrowth and branching was quantified by an experimenter blinded to the experimental group. For this assay, 7 different human donor pairs were tested. To see the effect of VEGFR inhibitor on SB623-conditioned medium aniogenic activity, aortic rings were pre-treated with 50 nM SU5416 at 5% CO2/37degrees for 30 minutes; subsequently, medium was replaced with 50 nM SU5416-supplemented SB623-conditioned medium (from 1 representative donor). Each round of the assay included 3–4 wells per group (MSC-CM, SB623 CM, unconditioned medium, or SB623 CM + SU5416) that were quantified and averaged.
For each experiment (which included 3–4 wells/group), a mean value was obtained for: (1) the treatment condition for each cell type (either MSCs or SB623 cells; one value per human donor tested) and (2) the untreated group (one value for each round of testing). For statistical comparison (SigmaStat, StyStat Software, San Jose, CA) each of these values were used and comparisons were made using one way ANOVA between the following groups (1) Control (unconditioned medium; n = 3), (2) MSC-conditioned medium (n = 3-7); and (3) SB623 cell-conditioned medium (n = 3-7). Additional pair-wise comparisons were made using Tukey’s test. An alpha value of 0.05 was used to assess if the means were significantly different.