UC tissue fragments collected in the Concordia Program (http://www.bancsang.net/professionals/en_concordia/) with appropriate donor informed consent were used for sourcing UC-MSC. UC samples were washed twice with sterile Phosphate Buffer Solution (PBS, Gibco) for removing any traces of blood, transferred to a Petri dish and cut into two fragments for testing either one of the two protocols for UC-MSC derivation: mechanical or enzymatic, respectively. In both cases, the fragments were weighted, cut longitudinally and split open to expose the inner surface for then removing the two arteries and the vein (Figure 1, top panel). For the mechanical method, a surgical scalpel was used for scraping the Wharton's Jelly, which was then spread uniformly onto the plastic surface of a 100 mm cell culture plate (Corning) and incubated 30 min at 37ºC (Figure 1, middle panel). In the enzymatic digestion, the tissue was minced and digested with 0.5 mg/mL collagenase I (Sigma-Aldrich) for 2 h and 5 additional minutes with 0.05% trypsin (Gibco) at 37ºC in DMEM (Gibco). Digestion was stopped by adding a final concentration of 5% human Serum B (hSerB, Banc de Sang i Teixits) and passed through 100 µm filters (Millipore) in order to discard undigested tissue fragments (Figure 1, bottom panel). The flow-through was centrifuged at 300g for 10 min. In both cases, subsequent cell expansion was performed using 20 mL of expansion medium consisting of DMEM supplemented with 20% hSerB, 2x104 IU/mL penicillin, 20 mg/mL streptomycin, and 120 µg/mL amphotericin B (Invitrogen).
Cell number and viability were determined by the haemocytometer-based Trypan Blue dye exclusion assay, and the MSC nature of expanded cells was determined according to the criteria established by the International Society for Cellular Therapy [4], including the evaluation of characteristic features such as adherence to plastic and morphology (assessed by bright field microscopy in Leica DMIL LED), immunophenotype by flow cytometry in a FACSCalibur (BD Biosciences). FACS analysis was performed to evaluate expression of surface markers CD31 (555445, BD), CD45 (HI30, BD Biosciences), CD90 (5E10, BD Biosciences), CD73 (AD2, BD Biosciences), CD105 (43A4E1 clone, Miltenyi) and HLA-DR (TU36, BD Biosciences) in a FACS Calibur flow cytometer (BD Biosciences). PE-conjugated IgG1 (X40, BD Biosciences) and FITC-conjugated IgG2bk (27-35, BD Biosciences) antibodies were used as isotype controls. Multipotentiality of UC-MSC was assessed in vitro using StemPro differentiation media (Gibco) and specific stainings for the detection of cell fate into either osteogenic, chondrogenic or adipogenic lineages, were performed as described elsewhere [5].