Cell preparation

Human umbilical cords from full-term Caesarean section patients were collected upon delivery, stored in Dulbecco’s modified Eagle medium (DMEM)/F12 (1:1) culture medium, which was supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (GIBCO, Invitrogen Inc., Carlsbad, CA, USA), and transferred immediately for cell isolation, according to a previously described protocol [14]. Briefly, the cord was cut into pieces that were 4–5 cm long, and the vessels were pulled away to isolate Wharton’s Jelly (WJ). WJ was cut into 1–2-mm³ pieces and digested with 1 mg/ml collagenase II (Millipore Sigma, St. Louis, MO, USA) with phosphate-buffered saline (PBS) at 37 °C for 45 min. The digested mixture was then passed through a 100-μm filter (BD Biosciences, Franklin Lakes, NJ, USA) to obtain cell suspensions. The cells were washed with PBS solution and then cultured in DMEM/F12 medium containing 10 % fetal bovine serum, 2 mmol/L glutamine, 1 % nonessential amino acids, and 1 % penicillin/streptomycin (GIBCO, Invitrogen Inc., Carlsbad, CA, USA) at 37 °C and 5 % CO₂. Nonadherent cells were removed by changing the medium after 3 days. Cells were expanded and identified according to the current statement of the International Society for Cellular Therapy (ISCT) [15]. Briefly, a minimal set of three standard criteria was used as the uniform definition of multipotent MSCs: adherence to plastic, specific surface antigen expression, and multipotent differentiation potential. The phenotype of multipotent MSCs is defined to be, at a minimum, the cell surface co-expression of antigens (CD105, CD73, and CD90 [≥95 % positive]) and the absence of hematopoietic lineage markers (CD45, CD34, CD14, CD19, and HLA-DR [≤2 % positive]). The surface marker was defined by the BD Stemflow hMSC Analysis Kit (BD Biosciences, Franklin Lakes, NJ, USA) containing pre-conjugated and pre-titrated cocktails of ISCT-defined positive expression markers (CD105 PerCP-Cy™5.5/CD73 APC/CD90 FITC) and negative expression markers (CD45/CD34/CD11b/CD19/HLA-DR PE). The multipotent differentiation potential of the isolated cells was identified using the Human Mesenchymal Stem Cell Functional Identification Kit (R&D, Minneapolis, MN, USA). Briefly, hUC-MSCs were seeded at 2 × 10⁴ cells/cm² in StemXVivo Osteogenic/Adipogenic Base Media. And after 24 hours, the medium was replaced with adipogenic differentiation medium to induce adipogenesis. HUC-MSCs were seeded at 4.2 × 10³ cells/cm² in StemXVivo Osteogenic/Adipogenic Base Media. When cells were to 50–70 % confluency, the medium was replaced by osteogenic differentiation medium. Differentiation medium was replaced every 3 days, and after 3 weeks cells were fixed in 10 % formalin and processed for histochemical analysis. Adipogenic differentiation was detected by oil red staining, and osteogenic differentiation was analyzed by alizarin red staining. This project was approved by the Human Ethics Committee of The First Affiliated Hospital at Sun Yat-sen University, and written informed consent was obtained for umbilical cord collections.