Human umbilical cord (hUC) specimens were obtained using protocols approved by the Ethical Committee of Faculty of Medicine, Cairo University.

After obtaining each patient’s own informed consent, 20 fresh UC samples of women with healthy pregnancies were retrieved at the end of gestation during caesarean deliveries from Department of Obstetrics and Gynecology. hUC Wharton jelly was harvested from term deliveries at the time of birth with the mother’s consent. hUCMSC was isolated by 3 mg collagenase II enzyme (IgG, C. histolyticum, US Biological, Life Science) digestion and maintained in 2% fetal bovine serum and 1x Pen/Strep (Invitrogen, CA). Cells were incubated at 37°C in an incubator with 5% CO2 at saturating humidity till they reach 70%~80% confluency; cultures were washed twice with phosphate buffer saline (PBS) and cells were trypsinized with 0.25% trypsin for 5 minutes at 37°C. After centrifugation, cell pellets were resuspended with serum-supplemented medium and incubated in 50 cm² culture flask (GIBCO/BRL). The resulting cultures were referred to as first-passage cultures (11).

We further characterized and quantified expressions of several Wharton Jelly mesenchymal stem cell markers (at the end of the 4th passage). Cells were trypsinized and adjusted to 1×10⁶ cells/ml. Then 1×10⁵ cells were incubated with 10μl of monoclonal antibodies: CD29 PE and CD44 PE, at 4°C in the dark; the same species isotypes served as a negative control. After 20 min incubation, 2 ml of PBS containing 2% FCS solution was added to each tube of monoclonal treated cells. The mixtures were then centrifuged for 5 min at 2500 rpm followed by discarding the supernatant and resuspending cells in 500 μl PBS containing 2% FCS. Cell analysis was performed using CYTOMICS FC 500 Flow Cytometer (Beckman Coulter, FL, USA) and using CXP software version 2.2.

The technique used for collecting UCB from the umbilical vein is the in utero technique. With the in utero technique, the blood is collected in the delivery room while the placenta is still inside the uterus. The in utero technique is more preferred than any other technique, because it is faster and simpler. Only one person is necessary and there is a lower risk of blood contamination, as it is not necessary to move the placenta before the blood is collected. Thirty seconds after delivery, the umbilical cord is clamped and cleaned with betadine solution. The UCB is collected from the umbilical vein with a 12.5-gauge needle connected to a sterile 50 ml Falcon tissue culture tube, containing 10 ml of phosphate-citrate-dextrose anticoagulant. The umbilical cord is “milked” and the uterus is gently massaged during the last period of the collection in order to increase the blood flow (12). The hUCB mononuclear cell fraction was isolated from the buffy coats through density gradient centrifugation with Ficoll-Paque (Gibco-Invitrogen, Grand Island, NY). Anticoagulated blood sample was carefully layered on 20 ml Ficoll and then centrifuged for 35 minutes at 400 xg. The upper layer was aspirated leaving the hUCB mononuclear cell layer undisturbed at the interphase. The interphase layer, the hUCB mono-nuclear cell layer, was carefully aspirated and washed twice in PBS containing 2 mM EDTA and centrifuged for 10 minutes at 200 xg at 20ºC. The cell pellet was re-suspended in a final volume of 300 μl of buffer. 12×10⁶ MNCs (isolated from twenty hUCB samples) were layered onto fibronectin coated 96-well plates and cultured in M199 medium supplemented with 20% fetal calf serum (FCS), 0.1% human vascular endothelial growth factor-1 (VEGF1), and 0.1% insulin-like growth factor (IGF-1) at 37°C for 48 hours, after which the supernatant was removed (13). The purity of the CD34⁺ of MNCs was evaluated by flow cytometry. Mononuclear cells were incubated with Dil-Ac-LDL (6 μg/ml) for 1 h at 37°C in the dark. Thereafter, the cells were washed twice with PBS at 37°C and incubated with FITC Conjugated Ulex Lectin (10 μg/ml) for one hour at 37°C in the dark. After two final washing steps with PBS at 37°C, the cells were fixed and subsequently analyzed for uptake of Dil-Ac-LDL and binding of FITC-Ulex lectin using a Leica inverted fluorescence microscope.

UC was diluted to 1:4 with phosphate buffered saline (PBS) containing 2 mM EDTA (Gibco-Invitrogen, Grand Island, NY). MNCs were separated by centrifugation over a Ficoll-Paque (Gibco-Invitrogen, Grand Island, NY) density gradient at 400 xg rpm for 35 minutes at 20°C. The MNC fraction was collected and washed first in PBS and then with MACS (magnetic cell sorting) buffer (PBS supplemented with 0.5% bovine serum albumin and 2 mM EDTA, pH 7.2). CD34⁺ cells were isolated from MNCs, using the CD34⁺ positive cell selection kit (MiniMacs; Miltenyi Biotec, Bergisch Gladbach, Germany). Isolation of CD34⁺ cells was confirmed by flow cytometry.

After the seven-day expansion period, the identified cells were collected and counted. Thereafter the conditioned medium was removed and the cells were cultured (3×10⁵~1×10⁶/1.5 ml/9.6 cm²) in endothelial cell growth medium-2 (EGM-2) containing FBS (2%), hydrocortisone, hFGF, VEGF, R3-IGF-1, ascorbic acid, hEGF, gentamycin, amphotericin-B, and heparin (Lonza, Basel, Switzerland). After three days of culture, the cells were collected and transferred to plates coated with fibronectin (10 mg/ml) (Sigma-Aldrich, St. Louis, USA) at a density of 1×10⁶ cells/1.5 ml/9.6 cm² and cultured for an additional three days in fresh EGM-2 medium. Real-time PCR was done for VEGFR2 and von Willebrand genes to ensure the presence of different types of stem cells.

We further characterized and quantified expression of several endothelial cell markers. 1×10⁵ cells were incubated with 10 μl of monoclonal antibodies: CD31 FITC and VE-cadherin PE (Beckman coulter, USA) at 4°C in the dark; the same species isotypes served as a negative control. After 20 minutes incubation, 2 ml of PBS containing 2% FCS solution was added to each tube of monoclonal treated cells. The mixtures were then centrifuged for 5 minutes at 2500 rpm followed by discarding the supernatant and resuspending cells in 500 μl PBS containing 2% FCS. Cell analysis was performed using CYTOMICS FC 500 Flow Cytometer (Beckman coulter, FL, USA) and using CXP software version 2.2.

Real-time qPCR amplification and analysis were performed using an Applied Biosystems software version 3.1 (StepOne™, USA). The qPCR assay and the primer sets were optimized at the annealing temperature. All cDNA, including previously prepared samples (for VEGFR2 and von Willebrand), internal control (for GAPDH gene expression as housekeeping gene), and non-template control (water to confirm the absence of DNA contamination in the reaction mixture), was in duplicate. Primers sequences of all assessed genes were illustrated in Table 1.

Statistical analyses were done using Statistical Package for Social Studies (SPSS) software, version 11, for Windows. The clinical scores were shown as the mean±standard deviation. Data were analyzed statistically by Friedman’s test and the Wilcoxon signed-rank test. p values of less than 0.05 were accepted as significant.