Isolation and Characterization of hUCMSCs

Fresh umbilical cords were obtained from informed, consenting mothers at the First People’s Hospital of Nanjing (China) and rapidly processed. The cords were rinsed twice with phosphate-buffered saline (PBS) supplemented with penicillin and streptomycin (pen/strep; Gibco, Carlsbad, CA), and the cord vessels were removed. The washed cords were subsequently cut into small fragments that were individually attached to the substrate of culture plates. This was followed by the addition of stem cell culture medium (Cyagen, Guangzhou, China) and incubation at 37 ℃ with 5% CO₂. The medium was replaced every 3 days after the initial culture, and well-developed colonies of fibroblast-like cells appeared ~10 days later. The colonies were then trypsinized (Gibco) and passaged into new plastic plates for further expansion. The human umbilical cord MSCs (hUCMSCs) from passages 3–7 were used for all the experiments. The experimental protocol was approved by the Nanjing Medical University Ethics Committee.
The osteogenic, adipogenic, and chondrogenic differentiation identified by Alizarin Red staining, Oil Red O staining, and Alcian Blue staining, respectively. To accomplish the differentiation, passages 3–7 MSCs were cultured in OriCell™ hUCMSCs osteogenic, adipogenic, or chondrogenic differentiation media (all from Cyagen) as described by manufacturer.
After the third passage, the hUCMSCs were trypsinized and washed twice with PBS; they were then stained with human anti-CD105, anti-CD73, anti-CD90, anti-CD45, anti-CD34, anti-CD14, anti-CD19, or anti-HLA-DR. Identical concentrations of FITC- or PE-conjugated mouse IgG isotype antibodies were used as negative controls (all from BD Biosciences Pharmingen, San Jose, CA). At least 10,000 events were acquired on a FACSVerse instrument (BD Bioscience), and the results were analyzed using FlowJo software (Tree Star, Ashland, OR).